Two-Photon Fluorescence Excitation and Related Techniques in Biological

Home , Laser medicine, Optical microscope

Quarterly Reviews of Biophysics 38, 2 (2005), pp. 97–166. f 2006 Cambridge University Press 97 doi:10.1017/S0033583505004129 Printed in the United Kingdom First published online 15 February 2006

Two-photonfluorescence excitation and related techniques in biological microscopy

Alberto Diaspro1,2*, Giuseppe Chirico3,4 and Maddalena Collini3,4 1 Laboratory for Advanced Microscopy, Bioimaging and Spectroscopy (LAMBS), MicroScoBiO Research Center, University of Genoa, Italy 2 National Institute for Matter Physics (INFM) and Department of Physics, University of Genoa, Italy 3 Laboratory for Advanced Biospectroscopy (LABS), University of Milano Bicocca, Milano, Italy 4 National Institute for Matter Physics – National Council for Research (INFM-CNR), and Department of Physics, University of Milano Bicocca, Milano, Italy

Abstract. This review is concerned with two-photon excited fluorescence microscopy (2PE) and related techniques, which are probably the most important advance in optical microscopy of biological specimens since the introduction of confocal imaging. The advent of 2PE on the scene allowed the design and performance of many unimaginable biological studies from the single cell to the tissue level, and even to whole animals, at a resolution ranging from the classical hundreds of nanometres to the single molecule size. Moreover, 2PE enabled long-term imaging of in vivo biological specimens, image generation from deeper tissue depth, and higher signal-to-noise images compared to wide-field and confocal schemes. However, due to the fact that up to this time 2PE can only be considered to be in its infancy, the advantages over other techniques are still being evaluated. Here, after a brief historical introduction, we focus on the basic principles of 2PE including fluorescence correlation spectroscopy. The major advantages and drawbacks of 2PE-based experimental approaches are discussed and compared to the conventional single-photon excitation cases. In particular we deal with the fluorescence brightness of most used dyes and proteins under 2PE conditions, on the optical consequences of 2PE, and the saturation effects in 2PE that mostly limit the fluorescence output. A complete section is devoted to the discussion of 2PE of fluorescent probes. We then offer a description of the central experimental issues, namely: choice of microscope objectives, two-photon excitable dyes and fluorescent proteins, choice of laser sources, and effect of the optics on 2PE sensitivity. An inevitably partial, but vast, overview of the applications and a large and up-to-date bibliography terminate the review. As a conclusive comment, we believe that 2PE and related techniques can be considered as a mainstay of the modern biophysical research milieu and a bright perspective in optical microscopy.

1. Introduction 98

2. Historical background of two-photon effects 99 2.1 2PE 100 2.2 Harmonic generation 100 2.3 Fluorescence correlation spectroscopy 100

* Author for correspondence: Professor A. Diaspro, LAMBS-MicroScoBiO, Department of Physics, University of Genoa, Via Dodecaneso 33, 16146 Genoa, Italy. Tel.: +39 10 3536426; Fax: +39 10 314218; E-mail: diaspro@ﬁsica.unige.it 98 A. Diaspro, G. Chirico and M. Collini

3. Basic principles of two-photon excitation of fluorescent molecules and implications for microscopy and spectroscopy 101 3.1 General considerations 101 3.2 Fluorescence intensity under the 2PE condition 103 3.3 Optical consequences of 2PE 104 3.4 Saturation effects in 2PE 108 3.5 Fluorescence correlation spectroscopy 109 3.5.1 Autocorrelation analysis 110 3.5.2 Photon-counting histogram analysis 112

4. Two-photon-excited probes 115

5. Design considerations for a 2PE fluorescence microscope 119 5.1 General aspects 119 5.2 Descanned and non-descanned 2PE imaging 121 5.3 Lens objectives and pulse broadening 122 5.4 Laser sources 125 5.5 Example of a practical realization 127

6. Applications 134 6.1 Biological applications of 2PE 134 6.1.1 Brain images 134 6.1.2 Applications on the kidney 139 6.1.3 Mammalian embryos 139 6.1.4 Applications to immuno-response 141 6.1.5 Myocytes 141 6.1.6 Retina 142 6.1.7 DNA imaging 143 6.1.8 FISH applications 144 6.2 2PE imaging of single molecules 144 6.3 FCS applications 148 6.4 Signals from nonlinear interactions 151

7. Conclusions 153

8. Acknowledgements 154

9. References 155

1. Introduction Optical microscopy is the only means of seeing structure in living cells at submicron resolution (Amos, 2000; Hell, 2003; Bastiaens & Hell, 2004). In recent decades, the method has gained great chemical specificity from the development of fluorescent probes (Arndt-Jovin et al. 1985; Beltrame et al. 1985; Kasten, 1989; Shotton, 1989; Wang & Herman, 1996; Tsien, 1998, 1999; Periasamy, 2001; Haughland, 2002; Zhang et al. 2002). Imaging three-dimensional (3D) structure has been aided by the introduction of confocal optical methods (Sheppard & Choudhury, 1977; Brakenhoff et al. 1979, 1985; Sheppard & Wilson, 1980; Wilson & Sheppard, 1984; White & Amos, 1987; White et al. 1987; Minsky, 1988; Carlsson et al. 1989; Wilson, 1990; Pawley, 1995; Webb, 1996; Masters, 1996; Amos, 1998; Benedetti, 1998; Masters, 2002; Diaspro, 2PE in biological microscopy 99

2002; Amos & White, 2003; Periasamy & Diaspro, 2003). This review is concerned with two- photon excited fluorescence microscopy (2PE) (Denk et al. 1990; Pennisi, 1997; Girkin, 2003; Zipfel et al. 2003b), which is probably the most important advance in optical microscopy since the introduction of confocal imaging in the 1980s, and related nonlinear optical methods. 2PE requires scanning optics similar to those used for confocal imaging, but also ultra-fast pulsed laser sources (Curley et al. 1992; Gratton & Van de Vende, 1995; Wokosin et al. 1997; Svelto, 1998). The advantages of 2PE over confocal imaging are still being evaluated (Gu & Sheppard, 1995; Brakenhoff et al. 1996; Sako et al. 1997). In order to collect 3D data, a substantial volume of the specimen has to absorb light, with an inevitable concomitant photobleaching and phototoxicity, which may be particularly severe when ultraviolet (UV)-excited fluorochromes are used (Stelzer et al. 1994). In principle, 2PE is superior to conventional single-photon scanned imaging (e.g. confocal), in that absorption can be limited to a very small volume at the focus of the objective lens at any one time. It can be the most efficient way of collecting 3D information in the sense of using the lowest time-integrated dose of radiation to the volume of interest. Besides this efficiency, 2PE has other advantages in imaging. It uses longer wavelengths [often infrared (IR)] which are not only less scattered by the specimen but may also fail to excite background autofluorescence. This is particularly important in the imaging of single molecules (Mertz et al. 1995; Xie & Lu, 1999; Sonnleitner et al. 1999; Chirico et al. 2001a, 2003a; Cannone et al. 2003a). Finally, because of their broader and overlapping two-photon excitation (2PE) spectra, several fluorochromes can be excited simultaneously by the same IR beam (Denk, 1996; Potter, 1996; Piston, 1999; Squirrel et al. 1999; Straub et al. 2000; White & Errington, 2000; Stosiek et al. 2003). However, it has been the experience of many investigators that the intensity of the beam in 2PE has to be controlled carefully to gain the often-claimed advantage in efficiency compared with confocal laser scanning microscopy. A possible reason for this is that 2PE photobleaching in the focal plane greatly depends (according to about the third power) on the excitation intensity (Patterson & Piston, 2000; Chirico et al. 2003b). Although the introduction of 2PE appeared incremental in the sense of providing similar optical sections to those obtained with confocal optics, and using similar scanning apparatus, it was also revolutionary, in that it represented a novel application of quantum physics (Loudon, 1983; Feynman, 1985; Shih et al. 1998). 2PE has stimulated the application of other nonlinear optical processes (see below). Three-photon fluorescence excitation was first recognized in imaging in White’s laboratory in Wisconsin (Wokosin et al. 1996; Hell et al. 1996; Maiti et al. 1997). The sharply defined and tiny focal volume has proved ideal for fluorescence correlation spectroscopy (FCS) (Wiseman et al. 2000). The femtosecond pulse obtained with newly developed laser systems has been used to improve the time-resolution of lifetime imaging (Konig et al. 1996a; French et al. 1997; Sytsma et al. 1998; Straub & Hell, 1998a; Peter & Ameer-Beg, 2004). The confinement of absorption to the focus in 2PE has also been exploited in photodynamic therapy (Bhawalkar et al. 1997). Moreover, one of the most recent applications of 2PE is given by the photoactivation in confined volumes of phoactivable proteins (Post et al. 2005; Schneider et al. 2005). Therefore, 2PE has become important not only in imaging but also in medicine and biology.

2. Historical background of two-photon effects

The ﬁrst prediction of two-photon absorption was by Go¨ppert-Mayer (1931, see also Axe, 1964). She proposed, as a consequence of the Heisenberg uncertainty principle, that one atom 100 A. Diaspro, G. Chirico and M. Collini or molecule should be capable of absorbing two photons in the same quantum event within 10x16–10x17 s. Because it is a rare event at ordinary light intensities (Denk & Svoboda, 1997), it was only in the 1960s, after the development of laser sources (Svelto, 1998), that the prediction could be veriﬁed.

2.1 2PE

2+ Kaiser & Garret (1961) reported 2PE of fluorescence in CaF2 :Eu . Soon after, Singh & Bradley (1964) were able to estimate the three-photon absorption cross-section for naphthalene crystals. Rentzepis and colleagues (1970) observed three-photon excited fluorescence from organic dyes. In 1976, Berns observed microscopically what was probably a two-photon-excited fluorescence as a result of focusing an intense pulsed laser beam on to chromosomes of living cells (Berns, 1976). The first applications of 2PE to fluorescence microscopy were presented at the beginning of the 1990s by Denk and colleagues (1990), who demonstrated that images with excellent optical sectioning, could be obtained without killing cells. The development of commercially available mode-locked lasers, with high peak-power femtosecond pulses and repetition rates around 100 MHz was then the trigger for a fast uptake of the multi-photon method in biology (Spence et al. 1991; Gosnell & Taylor, 1991; Curley et al. 1992; Fisher et al. 1997; Wise, 1999; Girkin, 2001a; Courjaud et al. 2001).

2.2 Harmonic generation For many years the only practical application of two-photon absorption was in spectroscopy (Lee, 1975; Friedrich & McClain, 1980; Friedrich, 1982; Birge, 1986; Callis, 1997). Franken et al. (1961) described second-harmonic generation (SHG) in a quartz crystal using a ruby laser. SHG is the production of a scattered beam at twice the frequency of the input beam, which is propagated in phase with the input and in the original direction. Hellwarth & Christensen (1974) collected SHG signals from ZnSe polycrystals at the microscopic level. The original idea of generating 3D microscopic images by means of nonlinear optical eﬀects was ﬁrst suggested and successfully demonstrated by second-harmonic imaging in the 1970s by Sheppard and colleagues of the Oxford group (Gannaway & Sheppard, 1978; Sheppard & Kompfner, 1978). SHG has now been applied to biological specimens (Gauderon et al. 1999; Campagnola et al. 1999, 2002; Cox et al. 2003), as Gannaway and Sheppard predicted it would be. Third-harmonic generation (Barad et al. 1997; Mueller et al. 1998; Squier et al. 1998) has also shown promise.

2.3 Fluorescence correlation spectroscopy The same optics and lasers as used in imaging, led to improvements in FCS (Magde et al. 1972; Ehrenberg & Rigler, 1974; Koppel, 1974; Aragon & Pecora, 1975). Thus far, FCS has been used to determine translational and rotational diﬀusion (Ehrenberg & Rigler, 1974; Fahey et al. 1977; Borejdo, 1979), chemical reactions (Thompson & Axelrod, 1983; Icenogle & Elson, 1983; Bonnet et al. 1998; Haupts et al. 1998; Starr & Thompson, 2001), ﬂow rates (Magde et al. 1978; Gosch et al. 2000), aggregate formation (Palmer & Thompson, 1989; Qian & Elson, 1990; Petersen et al. 1993; Berland et al. 1996), and triplet state parameters (Widengren et al. 1994). 2PE in biological microscopy 101

S Internal 2 conversion 10–12 s

Absorption Inter-system –15 Fluorescence 10 s –10 10–9 s crossing 10 s Radiation less Decay <10–9 s Phosphorescence –3 S0 10 s

Fig. 1. Fluorescence pathways and transition times in a general Perrin–Jablonsky scheme for conventional excitation of ﬂuorescence. (Courtesy of Dave Jameson, University of Hawaii.)

FCS is at present a very powerful tool for the characterization of biomolecules in vitro and is now developing into an adjunct to microscopy in which it is possible to probe the molecular aggregation and dynamics inside cellular compartments (Schwille et al. 1999b; Schwille, 2001; Schwille & Heinze, 2001). The technological advances which contributed to the development of 2PE microscopy occurred in several ﬁelds, including laser scanning microscopy, ultra-fast laser sources, more sensitive detectors, fast acquisition devices, and digital electronic tools (Shotton, 1993; Piston, 1999; Tan et al. 1999; Robinson, 2001; Gratton et al. 2001; Periasamy & Diaspro, 2003; Esposito et al. 2004; Masters & So, 2004).

3. Basic principles of two-photon excitation of fluorescent molecules and implications for microscopy and spectroscopy 3.1 General considerations Conventional one-photon excited fluorescence microscopy (1PE) techniques for fluorescence excitation make use of a UV or visible photon that is able to match the energy gap (Fig. 1) between the ground and the excited state (Lakowicz, 1999). 2PE of fluorescent molecules is a nonlinear process related to the simultaneous absorption of two, typically IR, photons whose total energy is sufficient to produce a molecular transition to an excited electronic state (Birks, 1970; Denk et al. 1995; Callis, 1997; Nakamura, 1999). In general, one can use photons of wavelengths l1 and l2 within the constraint that x1 1 1 l1P ffi + ,(1) l1 l2 where l1P is the wavelength needed to prime fluorescence emission in a one-photon absorption event. For practical reasons one has l1=l2 and the starting experimental choice is l1B2l1P (Denk et al. 1990; Diaspro, 2001a; Girkin & Wokosin, 2001a), as shown in Fig. 2, where l1P=340 nm. 2PE of fluorescent molecules provides an immediate practical advantage over confocal microscopy (Denk et al. 1990; Potter, 1996; Centonze & White, 1998; Piston, 1999; Squirrel et al. 102 A. Diaspro, G. Chirico and M. Collini

1020 nm 680 nm

340 nm 1020 nm 420 nm

680 nm 1020 nm

Fig. 2. Perrin–Jablonsky simplified scheme for one-, two- and three-photon excitation. When the fluorescent molecule is brought to the excited state it relaxes back emitting the very same fluorescence as in the one-photon excitation case. In this case conventional fluorescence could be primed at 340 nm producing 420 nm emission. The rule of thumb is that one can use multiple wavelengths with respect to the one-photon excitation condition accordingly with the nth photon excitation order. Optimization can be obtained at different wavelengths.

1999; Diaspro & Robello, 2000; Wilson, 2001) since out-of-focus excitation is avoided, thereby reducing the overall photobleaching and phototoxicity of thick samples (Brakenhoff et al. 1996; Patterson & Piston, 2000; Tauer, 2002; Drummond et al. 2002). However, the observation time for a fluorophore in 2PE decreases with the third power of the excitation intensity due to photobleaching in the focal plane (Chirico et al. 2003b). These data led the authors to conclude that the fluorophore is initially excited by simultaneous two-photon absorption, and then one or more photons interact with the excited molecule to increase the probability of photobleaching similarly to a sequential absorption model previously suggested in a photobleaching study of Rhodamine 6G (Widengren & Rigler, 1996) and Rhodamine B (Sanchez et al. 1997). A simple extra photon model is not sufficient to describe the phenomenon, and possibly higher-order resonance absorption is involved. At the lowest excitation intensities, photobleaching rates between one- and two-photon excitation are comparable, but at power levels that are typically used in biological imaging (3–5 mW at the sample for 150 fs laser pulses) this difference can easily be a factor of 10. This means that the two-photon method is substantially detrimental in a subset of experiments, particularly for thin samples (<5 mm) and single-molecule studies, and cells for a particular treatment to achieve the highest collection efficiency in 2PE, thereby using the lowest possible light exposures on live cells, and minimizing photobleaching. The main advantage of 2PE lies in the high localization of the excitation and in the fact that emitted light is detected, even if strongly scattered, because the full sensitive area of the detector is used as opposed to confocal microscopy. This allows the application of 2PE to thick, turbid samples and simplifies the collection optics scheme. Further considerations are needed here to characterize 2PE in biological applications.

Since, molecular cross-sections for the two-photon absorption d2, are typically very small [of the order of 10x58 (m4 sx1)], high light ﬂuxes%1030 photons sx1 mx2 are required. 2PE in biological microscopy 103

This can be reached either by means of high-power continuous wave (CW) lasers or by means of short-pulse (%10x13 s) lasers that allow reduction of the thermal load of the sample and selectively excite a single electronic transition. In fact the time-scale of the 2PE process is

x16 x17 t2PE=10 x10 s(2)

(Louisell, 1973) as determined by the Heisenberg uncertainty principle: l t ffi 1P ffi 10x16 s, (3) 2PE 4pc for a typical visible wavelength l1P=500 nm. However, picosecond or CW IR excitation can be effectively applied to prime fluorescence (Hanninen et al. 1994; Hell, 1996; Bewersdorf & Hell, 1998; Booth & Hell, 1998; Hell et al. 1998; Leica Microsystems, 1999; Konig et al. 1999a), although with higher thermal loads on the sample. In this case, as well as direct 2PE from ground state to excited states, transitions from higher excited states also have time to occur and this brings a broadening of the 2PE cross-section. Another consideration involves the 2PE selection rules. One- photon-allowed transitions are due to opposite parity of the initial and final states, while in the two-photon case the two states have the same parity (So et al. 2000). This is due to the fact that the state reached by TPE is obtained from a two-step process through vibronic coupling. These different selection rules are responsible for the fact that, even when we divide the 2PE wavelengths by 2, a considerable shift between the 1PE and the 2PE spectra is found (Blab et al. 2001). Moreover, we must also consider that the two-photon cross- section, d2, may depend on the laser pulse width due to different vibronic coupling (He et al. 2002).

3.2 Fluorescence intensity under the 2PE condition

The 2PE fluorescence intensity, If(t), is proportional to the square of the excitation intensity, I(t), to the molecular cross-section d2=d2(lexc), and to the quantum yield g=g(lem). Here, lexc and lem, are the excitation and emission wavelengths respectively. The excitation intensity in photons cm2 sx1 is I(t)=lP(t)/(hcA), where P(t) is the instantaneous power on the % 2 illuminated area, A p[lexc/(2 NA)] (Born & Wolf, 1993), NA is the numerical aperture of the microscope objective, h and c are Planck’s constant and the speed of light in vacuum respectively. The 2PE collectable fluorescence intensity is given by: 2 2 2 2 (NA) If (t)=kd2gI (t) =kpd2gP(t) ,(4) hclexc where k is a factor that takes into account the collection efficiency of the set up. The time-averaged two-photon fluorescence intensity per molecule within an arbitrary time interval T for a CW excitation is: 2 2 2 ðÞNA nIf ,cwm ffi kpd2gPave : (5) hclexc 104 A. Diaspro, G. Chirico and M. Collini

For femtosecond lasers with pulse repetition rate fP=1/T, and pulse width, tP,the average power depends on the peak power as, Pave=tP fPPpeak(t) and Eq. (5) becomes (So et al. 2001): 2 2 2 Pave NA nIf ,Pm ﬃ d2g : (6) tPfP hclexc

The conclusion here is that CW and pulsed lasers have the very same excitation efficiency in terms of collectable fluorescence intensity when the average power of the CW laser is kept pffiffiffiffiffiffiffiffiffi higher by a factor of 1= tP ÁfP and the TPE cross-section is not changed with the laser pulse width (Xu, 2001; Konig et al. 1996b). This means that, for exciting fluorescence, 300 mW delivered by a CW laser are nearly equivalent to 1 mW of a pulsed laser with repetition rate %80 MHz and pulse width%100 fs. As an example, a 1 mW average power laser source (l=1000 nm, 80 MHz repetition rate, 100 fs pulse width) focused by a 1Á4 NA objective on molecules endowed with a typical two-photon cross-section of d2 %10 GM, gives [Eq. (6)] a fluorescence count rate %105 Hz. It must also be considered that the TPE cross-sections for picosends and CW excitations are larger than the TPE femtosecond cross-sections by at least one order of magnitude (Kirkpatrick et al. 2001). This allows for picosecond 2PE of biological samples (Bewersdorf & Hell, 1998), although local heating effects of the sample can be relevant in this case.

3.3 Optical consequences of 2PE The use of near-infrared (NIR) radiation in 2PE would imply an optical resolution lower than in conventional 1PE (Born & Wolf, 1993; Cox & Sheppard, 2004). However, since under % 2PE fluorescence is exclusively excited in a sub-femtolitre (fl) volume (Vexc 0Á1–0Á5 fl, Fig. 3), 3D optical sectioning (Weinstein & Castleman, 1971; Agard, 1984; Diaspro et al. 1990) can then be achieved with the signal-to-background ratio noticeably increased. Figure 4 shows an optical sectioning scheme along with the excitation beam shape (Born & Wolf, 1993; Bianco & Diaspro, 1989). One should consider that the laser excitation intensity spatial profile is given by the intensity point spread function (PSF), S(r/l1,exc, z/l1,exc) (Born & Wolf, 1993), where l1,exc is the wavelength that characterizes the one-photon excitation. Here we indicate positions in space with the radial [r=(x, y)] and axial (z) distances from the origin that is placed on the focal plane on the maximum laser intensity position. The PSF can be approximated with Gaussian–Lorentzian

(GL) spatial proﬁles whose widths are w0 and z0 in the radial and axial dimensions, as shown in Fig. 4. A detailed discussion of a GL propagating beam through a ﬁnite aperture is complex and can be found in Dickson (1970) and Xu (2001). Radial and axial beam waist values can be determined using the relations

2 l pw0 0Á37pl w0 ﬃ 0Á61 and z0 ﬃ = , NA l NA2 where NA=n sin(a), n is the refractive index of the medium between the lens and the sample and a is the half-aperture angle of the lens (Fig. 4).

The ﬂuorescence emission If(r, z) depends on the distribution of ﬂuorophores in the excitation volume, C(r, z) and is given by If(r, z)=C(r, z)S(r/l1,exc,z/l1,exc) for 1PE, and to 2 If(r, z)=C(r, z)S (2r/l1,exc, 2z/l1,exc) for 2PE. The second relation is due to the second-order 2PE in biological microscopy 105

(a) From source

n photons

1 Microscope

(constant) two axis α λ 1 1

δ x

δ δ y x,y,z z

Important parameters δ δ δ Resolution: x, y, z α Position: x, y, z 2 λ 2 Quantity: n2 Probability of two-photon n photons 2 absorption

To detector: n2 counts

(b)

Fig. 3. (a) Optical resolution parameters for determining the extent of the volume of two-photon excitation (TPE) event and TPE probability along the optical axis (adapted from Pawley, 1995). (b) Fluorescence emission obtained from a solution of ﬂuorescent molecules by means of one- (double cone, above) and two-photon (bright dot, pointed by the arrow) excitation. From this illustration the spatial conﬁnement of TPE is extraordinarly clear. The immediate implication is that one does not need any computation or pinhole for removing out-of-focus contributions. Out-of-focus contributions are simply absent. (Courtesy of Brad Amos, MRC, UK.) 106 A. Diaspro, G. Chirico and M. Collini

(a)

z axis

(b)

2W0 W0 0 z

2z0

Fig. 4. Optical sectioning. (a) A real 3D structured object showing a different detail plane by plane (left) can be optically sectioned (centre) to get images of the distinct features along the z-axis (right). (b) Beam waist along the x–y–z directions determining spatial resolution (see text). power dependence of the excitation probability for 2PE and to the fact that the 2PE excitation wavelength is generally taken as l1,exc=l2,exc/2. The collection of the fluorescence signal, F(r, z), with emission wavelength lem, is given by a convolution between the emission intensity PSF, S(r/lem,z/lem), and the fluorescence emission, If(r, z) (Weinstein & Castleman, 1971; Agard, 1984; Bianco & Diaspro, 1989; Diaspro et al. 1990) plus a random noise that takes into account the image collection statistics. In confocal 1PE the use of a pinhole imposes a restriction on the integration of F(r, z) over the detector area. This leads to a collection efficiency function [=the fraction of the fluorescence signal emitted from the point of coordinates (r, z) and that is actually collected by the detector] proportional to S(r/lexc, z/lexc) S(r/lem, z/lem). By comparison, assuming a 2PE scheme, all the fluorescence photons are usually collected since the pinhole is usually excluded, F(r, z) has to be averaged over the whole detector area 2PE in biological microscopy 107

z/z0 0 123

100

50 Output fluorescence rate (kHz)

0 024 6 z (µm)

Fig. 5. Confinement property of the two-photon excitation (TPE) spectroscopy. The simulation of the fluorescence output from a molecule that is moved along the optical path is performed according to Eq. (16). The simulation parameters are: Pave=1 mW, average laser power, fP=80 MHz repetition rate 2 and tP=100 fs pulse width, l=1000 nm. Two-photon cross-section, d at 10 GM, NA=1Á4. The solid line is a fit to an equation of the type A/(B2+z2/C2)2. The depth of focus is 1Á6 mm.

2 and this leads to a 2PE collection efficiency function: S (2r/lexc,2z/lexc). In confocal 1PE and in 2PE the resolution enhancement is given by the quadratic form of S due to different physical reasons. In fact, in confocal microscopy the resolution is enhanced because of a combined action of the excitation and detection PSFs. A plane of thickness z0 centred around z=0 (the focal plane as determined by the position of the objective) predominates over the other planes. In 2PE the resolution enhancement is obtained only through the excitation PSF which is squared because of the nonlinear effects of the excitation due to two statistically independent events that occur, i.e. simultaneous absorption of two photons (Andrews, 1985). It must be noted that the effect of the excitation and emission wavelengths is different in the two cases, but in both cases the fluorescence signal depends quadratically on the intensity PSF. The microscope intensity PSF which is the response of the optical system to a Dirac distribution of fluorescence, can be experimentally determined by imaging subresolution fluorescent objects, i.e. microspheres having dimensions <100 nm or thin fluorescent layers (van der Voort & Brakenhoff, 1988; Schrader et al. 1998; Diaspro et al. 1999a). The 2PE PSF is axially confined (Nakamura, 1993; Gu & Sheppard, 1995; Jonkman & Stelzer, 2001) since the integral of 2 x4 2 S(r/l1,exc, z/l1,exc) over r has a half-bell shape with a z behaviour for z4z0=pw)0/l (Fig. 5). % 2 4 2PE excitation volumes can then be estimated as Vexc pw0z0=pw0/l. As an example, for l=800 nm, NA=1Á4, the diffraction limited volume is%0Á06 mm3 (see also Table 1 for values of the half-width of the 3D intensity point spread). The axial confinement of the excitation is demonstrated in Fig. 6. In 2PE over 80% of the total intensity of fluorescence 108 A. Diaspro, G. Chirico and M. Collini

Table 1. Values of the half-width of the 3D intensity point spread function in the transverse and axial

1 1 directions, v2 and u2 respectively (values from Gu & Sheppard, 1995)

1P 2P C1P C2P

1 v2 1Á62 2Á34 1Á17 1Á34 1 u2 5Á56 8Á02 4Á01 4Á62

(a)(b)

(c)(d)

Fig. 6. One- (a, b) and two-photon (c, d) photobleaching induced within a fluorescent microsphere by means of 488 nm and 720 nm wavelengths respectively. As in Fig. 13, the better confinement of two- in respect to one-photon excitation under point-like laser scanning conditions is evident. (a, c) are x–y views; (b, d) are axial views obtained from optical sectioning collections. (Adapted from Diaspro, 2001b.) comes from a 700–1000 nm thick region about the focal point for objectives with NAs in the range of 1Á2–1Á4 (Brakenhoff et al. 1979; Wilson & Sheppard, 1984; Jonkman & Stelzer, 2001; Torok & Sheppard, 2001; Wilson, 2001). As a comparison, typical axial extension of the confocal volumes are of the order of 500–700 nm, although these values are reached at much lower collection efficiency than in the 2PE case. Finally, it is worth noting that, when considering in-depth imaging of thick samples, optical aberrations should be properly considered (de Grauw & Gerritsen, 2001; Diaspro et al. 2002b; Neil et al. 2002; Marsh et al. 2003).

3.4 Saturation effects in 2PE Besides photobleaching, an important factor is given by the saturation of the fluorescence emission. For excitation intensities, I, higher than a threshold value, Isat, the fluorescence 2PE in biological microscopy 109 emission increases less rapidly than%I for confocal 1PE, or%I 2 for 2PE. This is due to excitation rates that are larger than the fluorescence de-excitation rates (ground state depletion) or, in the case of pulsed 2PE, to excitation to higher excited states from the first excited state due to the high photon flux.

As for the ﬁrst possibility, we estimate that the number of photon pairs, na, that a ﬂuorescent molecule simultaneously absorbs during a single pulse is (Denk et al. 1990): 2 2 2 (2) d2ðÞÁlexc PaveÁ NA na / g 2 ,(7) tPfP 2hclexc where Pave is the time-averaged power of the beam, lexc is the excitation wavelength, NA is (2) the objective NA, tP is the pulse duration and fP is the pulse train repetition rate. The g factor is 1Á0, 0Á66 and 0Á59 for the Rectangular, Gaussian and Hyperbolic-secant-squared pulse shape

(Svelto, 1998). Ground state depletion occurs when nafPB1/tfl, i.e. when natflfPB1, where tfl is the fluorescence lifetime of the fluorescent molecule. Now, if we want at least one 2PE per pulse

(i.e. naB1) while keeping the intensity below the saturation threshold, the pulse repetition rate should be of the order of 1/tfl or less, which, for commonly used fluorescent molecules for which tflB10 ns means fPf100 MHz. The typical repetition rate of titanium:sapphire lasers used for 2PE (y80 MHz), just happened to be in the right range for cavity requirements (Girkin,

2003). Now, by taking the fluorescein fluorescent molecule as an example, for lexc=780 nm we % x3 2 obtain na 6Á7r10 Pave, where Pave is expressed in mW. We have assumed d2=38 GM at 780 nm [1 GM (Goppert–Mayer)=10x58 (m4Ás)] (Denk et al. 1990; Xu, 2001), NA=1Á4, x9 fP=100 MHz and tP=100 fs. Since for fluorescein tfl=4r10 s, saturation begins to occur at 20 mW (Koester et al. 1999; So et al. 2001). Besides the saturation effect, in 2PE higher-order transitions usually occur even before the saturation becomes evident. Several brilliant and effective comparisons between 1PE and 2PE on simple dyes have been previously reported (Mertz, 1998; Eggeling et al. 2005). It must be noted that these effects increase at reasonably low excitation powers. As an example, rhodamine, even at average powers (%4 mW of 2PE) does not show the fluorescence rate increasing quadratically with the excitation intensity (Mertz, 1998).

3.5 Fluorescence correlation spectroscopy Information on the internal dynamics of the sample can be obtained from the analysis of the fluctuation of the TPE fluorescence signal. Fluorescence spectroscopy combined with the unique spatial resolution features of TPE allows the localization of rare fluorescent components and the dynamics of chemical reactions in cellular compartments to be followed (Eigen & Rigler, 1994; Brock et al. 1998; Schwille et al. 1999a; Schwille, 2001; Harms et al. 2001; Chen, 2002; Chen et al. 2002). The analysis of the fluorescence fluctuations is usually performed by means of FCS. In this technique, the autocorrelation of the fluorescence signal is performed online by fast digital correlator boards. FCS is a rapidly developing field in its own right, and it is already recognized as an essential tool for the in vivo characterization of absolute concentrations, molecular interactions, and kinetic processes, such as diffusion and chemical reactions (Haustein & Schwille, 2004). In FCS we want to measure the light emitted from a few (%1–10) molecules with microsecond–second time resolution, much larger than the excited state lifetime (%1 ns). On this time-scale, which is typical of molecular diffusion to internal photodynamics and chemical kinetics, the fluorescence emission can be thought of as an instantaneous relaxation 110 A. Diaspro, G. Chirico and M. Collini process and the laser excitation is actually seen by the sample as a continuum wave due to the high repetition rates (%80 MHz). For this reason the time dependence of the excitation intensity is not usually taken into account. Moreover, since fluorescence emission is measured, the measured light intensity is the sum of the individual molecular contributions which are proportional to the local laser beam intensity (Thompson, 1991). In order to understand the origin of the fluorescence fluctuations due to molecular diffusion, let us assume that the laser profile in the focal plane (z=0), has a top-hat profile with radius w0. The fluorescence would then simply be proportional to the number N of molecules in the excitation volume and the fluctuations are related to the change of N, i.e. ndF2m/ nF2m=ndN2m/nN2m. For more realistic laser beam shapes, such as the GL shape (Born & Wolf, 1993) a complex mathematical treatment leads to a proportionality of the type ndF2m/nF2m=cndN2m/nN2m, where c is a geometric factor that depends on the shape of the collection efficiency function (Berland & Shen, 2003). Now, can we determine the number fluctuations in the observation volume? The mean number of molecules per excitation volume is nNm=CVexc, where C is the number concen- x15 tration (molecules/litres of solution) of the polymer in the sample. For the case Vexc=10 l x14 (=fl) and C=6r10 molecules/l, which corresponds to a concentration of 1 nM, we obtain % nNm 1. The probability P(N) of finding N molecules in Vexc is proportional (Eigen & Rigler, 1994) to the number of ways N molecules can be arranged in Vexc :

nN mN P(N )= exnN m: (8) N ! 2 2% According to this distribution, the number fluctuation is ndN m/nNm 1/nNm=1/(CVexc) and, therefore, the fluorescence fluctuations ndF2m/nFm2 scale as 1/C. The more diluted the polymer solution is, the higher the signal-to-noise ratio will be. Obviously we cannot increase the fluctuation amplitudes at will due to the effect of the background that reduces the measured fluctuations. However, a compromise must always be found between the average fluorescence signal, 2 2% x1 which scales as nNm, and the signal-to-noise ratio, S/N=ndF m/nFm (nNmVexc) (Koppel, 1974; Quian, 1990; Kask et al. 1997). Obviously S/N also depends on the total averaging 0Á5 time, Tm, and better S/N ratios can be reached by increasing Tm, since S=N Tm (Kask et al. 1997). Moreover, S/N is dependent on the molecular brightness, defined as e=d2(lexc)g(lem). This is especially true for the fast time range (%1 ms) of the autocorrelation function (ACF) (Koppel, 1974) that is related to internal photodynamics and chemical kinetics. For the diffusion dynamics the fluorescence fluctuations are slower (%100–300 ms), the dependence of S/N on the molecular brightness is less severe (Koppel, 1974), and lower excitation power and less bright dyes can be employed. In 2PE experiments, however, the saturation of the fluorescence output due to transitions to higher excited states (Mertz, 1998; Eggeling et al. 2005) may severely limit the applications to dim dyes.

3.5.1 Autocorrelation analysis

Now, when the average number of molecules is nNmf1 (Magde et al. 1972; Eigen & Rigler, 1994), the signal is at the background level for most of the observation time and occasionally we may detect a fluorescence burst corresponding to a molecule diffusing through the volume, such as the case reported in Fig. 7: since the duration of a fluorescence burst is related to 2PE in biological microscopy 111

100000 (a) C = 10 f M 10000

190 191 1000

100000 100 (b) C = 100 f M 10000

1000

100 100000 (c) Rate (Hz) C = 1 pM 10000

1000

100000 100 (d)

10000

1000 C = 10 pM

100 0 200 400 600 800 Time (s) Fig. 7. Fluorescence photon counts versus time collected through a two-photon excitation (TPE) from a solution of fluoresceine-labelled microspheres 20 nm in diameter at concentrations: C=10 fM (a); C=100 fM (b); C=1pM (c); and C=10 pM (d). Excitation wavelength=770 nm. The inset in panel (a) is a blow-up of the first peak in the trace of panel (a). the translational diffusion properties of the molecules, one can get estimates of the translational diffusion of the molecules similarly to photon correlation spectroscopy by means of the ACF:

ndF(s+t)dF(s)ms gF (t)= 2 : (9) nF(s)ms However, conversely to the case of photon correlation spectroscopy, much more information can be gained from higher-order correlation functions of the ﬂuorescence signal (Thompson, 1991) or the analysis of the distribution of photon counts (Chen et al. 1999; Kask et al. 1999).

The exact 2PE gF(t) cannot be analytically derived (Berland et al. 1995; Xu, 2001). However, it can be ﬁtted by a function of the type:

x1 x0Á5 gF (t)=gF, 0(1+t=txy) (1+t=tz ) =gF, 0GD(t, txy, tz ), (10)

2 2 where tffiffiffiffiffiffixy ffi w0 =8D and the axial diffusion time tz ffi z0 =8D is larger than field txy. Since for 2PE p w2 0 % % z0= 2p =lexc, for l=800 nm and NA=0Á9 we get w0 0Á54 mm and z0 0Á92 mm, while for 112 A. Diaspro, G. Chirico and M. Collini

NA=1Á4 we find w0/z0%1. It is of note that, in spite of the complexity of the exact relationship of the 2PE ACF derived by Berland et al. (1995), the time behaviour of the correlation functions in the TPE mode can still be analysed by means of a simple hyperbolic equation [e.g. Eq. (10)] for most of the microscopy-spectroscopy applications. As expected from the analysis performed of the number fluctuations, the zero lag-time correlation function, gF,0, is related to the concentration number of fluorophores nNm within the excitation volume: gF, 0=c=nN m, (11) where the contrast factor c=0Á076 for TPE. Since nFm=nNme, an estimate of the molecular % brightness can be obtained from the product: e nFmgF,0/c. In a case where the background contribution affects the measured count rate, nFmeasm=nFm+nFBm, the molecular brightness 2 2 must be corrected (Koppel, 1974; Thompson, 1991) as e=emeasnFm /(nFmeasm – nFBm) . This effect would lead to a decrease of the signal-to-noise ratio at extremely low sample concentrations. Besides translational diffusion, any molecular brightness fluctuation is reported by the ACF. In the case of unimolecular chemical reactions, or of internal photodynamics, in which a change of fluorescence yield is involved, the fluorescence signal fluctuates according to the process kinetics. Superimposed on these fluctuations we find those relative to the molecular diffusion through the excitation volume. The fluorescence ACF is then given by a superposition of an exponential relaxation and a diffusion term (Schwille, 2001):

(1xT +T exp [xt=tT ]) gF (t)=gF, 0 GD(t, txy, tz ): (12) 1xT

In this equation the function GD(t, txy, tz) represents the diffusion contribution to the ACF [Eq. (10)], tF is the characteristic time of the brightness fluctuation and T is the fraction of the molecules that are undergoing this fluctuation. We have assumed that (Widengren et al. 1994) the internal kinetics is completely decoupled from the diffusion process and that the relaxation time of the kinetics, tT, is much shorter than tx,y. Excited triplet-singlet transitions and molecular-binding kinetics are treated by means of Eq. (12) in order to get information on the rate constants of the process. In general for a uni-molecular reaction of the type B , D, where B is the bright species and D the dark species (quantum yield=0), tT and T are related to the kinetics constants kon and koff of the reaction as (Schwille, 2001): 9 x1 > tT=(kon+koff ) , = (13) kon > T = , ; kon+koff

3.5.2 Photon-counting histogram analysis Statistical requirements for the collection of the full-time decay of the ACF are quite severe.

On the other hand for most applications we are only interested in the measurement of gF(0) in order to obtain an estimate of the number of molecules per excitation volume. Such information can also be achieved by data analyses that are complementary to FCS (Chen et al. 2PE in biological microscopy 113

1999; Kask et al. 1999). For sampling times much smaller than the diffusion time tx,y, the photon-counting distribution, P(k), depends on the average number of molecules nNm in the excitation volume and the molecular brightness e, defined as the product of the excitation intensity, the sampling time, the molecular absorption cross-section and the fluorescence quantum yield. In the case of a single fluorescent species nNm and e can be measured from the first two moments, nkm and nk2m, of the photon-counting distribution (Chen et al. 1999; Chirico et al. 2000; Malengo et al. 2004): nkm2xnkm2 e= x1 c nkm (14) nN m=nkm=e

In the case of a multiple-species sample, one should apply a nonlinear fitting of the full photon-counting histogram (PCH) according to the numerical expressions given by Gratton and co-workers (Chen et al. 1999). The average nkm and nk2m, are simply given by: ! 9 XM > > nkm= ki M, => i=1 ! > (15) XM > 2 2 > nk m= ki M, ; i=1 where M is the total number of samplings and ki is the number of photons collected per sampling time at the ith sampling of the experiment. FCS can be performed also in a point-by-point fashion on cells thereby reconstructing an image in terms of molecular brightness, and local molecular concentration collected on well- resolved excitation volumes. This technique, called image correlation spectroscopy (ICS) and introduced by Petersen et al. (1993), conjugates the counting feature of FCS to high spatial resolution of microscopy. The basis of the application of ICS is a spatial autocorrelation analysis, at a fixed time, of fluorescence images on which single fluorescence centres are visible all over the field of view (Wiseman & Petersen, 1999). This allows measurement of the number N of fluorescent spots under observation according to the relation: ÀÁ ndF(x+j, y+g)dF(x, y)m c j2+g2 j g x, y gF ( , )= 2 = exp x 2 , (16) nF(x, y)mx, y N w0 where w0 is the beam waist of the laser on the focal plane and the symbol n_mx,y indicates an average over all the image pixels. Finally, recent image applications of PCH analysis have been presented (Malengo et al. 2004). If the fluorescence counts k(t, i, j) are acquired versus time (t=Ts,2Ts, _, NTs) over each pixel (x, y) with a sampling time Ts, the pixel content on the conventional fluorescence image is given by:

XNT 1 s I (i, j)= k(t, i, j), (17) M t=Ts 114 A. Diaspro, G. Chirico and M. Collini

(a) (b) 5·2 0 5·2 0 0·2 2·75 4·2 4·2 0·4 5·50 3·1 0·5 3·1 8·25 0·7 11·00 y ( µ m) y ( µ m) 2·1 0·9 2·1 13·75 1·1 16·50 1·0 1·0 1·2 19·25 1·0 2·1 3·1 4·2 5·2 1·4 5·24·23·12·11·0 22·00 x (µm) x (µm)

(c)

5·2 0 0·02 4·2 0·05 3·1 0·07 0·10 y ( µ m) 2·1 0·12 0·15 1·0 0·17 5·24·23·12·11·0 0·20 x (µm)

Fig. 8. PCH imaging of a nanocapsule stained with FITC (ﬂuorescein) (Malengo et al. 2004). The ﬁeld of view is 5Á2mmr5Á2 mm, acquisition rate 10 kHz, total acquisition time per pixel=30 s, x and y resolution=300 nm, excitation power at 100 mW. (a) Conventional image, I(i, j); (b) number image, nNm(i, i); (c) brightness image e(i, j).

where i=1, _ , m and j=1, _ , n spans the pixels of the image and M is the length of the acquisition buﬀer. On the other hand the second moment of the photon count distribution can be computed on each image pixel as:

XNT 1 s I (2)(i, j)= k2(t, i, j): (18) M t=Ts

By applying the algorithm given by Chen et al. (1999), the average numbers nNm, and average brightness e, can be computed pixel-by-pixel by the following set of equations: 9 (2) 2 I (i, j)xI (i, j) => e(i, j)c= x1, I (i, j) (19) ;> nN m(i, j)=I (i, j)=e(i, j):

As an example we report the images of fluorescent nanocapsules (Diaspro et al. 2002d; Diaspro, 2004b; Malengo et al. 2004) acquired with a spatial resolution of 300 nm. The PCH image [Eq. (19)] of one nanocapsule stained with fluorescein (see Fig. 8), indicates that the origin of fluorescence spatial changes in the sample are mainly due to differences in 2PE in biological microscopy 115

Table 2. Properties of some common ﬂuorescent molecules under two-photon excitation

Extrinsic ﬂuorophores l (nm) gd2 d2 Bis-MSB 691/700 6Á0¡1Á86Á3¡1Á8 Bodipy 920 17¡4Á9– Calcium Green 740–990 – y80 Calcoﬂuor 780/820 – – Cascade Blue 750–800 2Á1¡0Á6 y3 Coumarin 307 776, 700–800 19¡5Á5 y20 CY2 780/800 – – CY3 780 – – CY5 780/820 – – DAPI (free) 700/720 0Á16¡0Á05 y3Á5* Dansyl 700 1 – Dansyl hydrazine 700 0Á72¡0Á2– Dil 700 95¡28 – Filipin 720 – – FITC 740–820 – y25–38* Fluorescein (pHy11) 780 – 38¡9Á7 Fura-2 (free) 700 11 – Fura-2 (high Ca) 700 12 – Hoechst 780/820 – – Indo-1 (free) 700 4Á5¡1Á312¡4 Indo-1 (high Ca) 590/700 1Á2¡0Á42Á1¡0Á6 Lucifer Yellow 840–860 0Á95¡0Á3 y2 Nile Red 810 – – Oregon Green Bapta 1 800 – – Rhodamine B 840 – 210¡55 Rhodamine 123 780–860 – – Syto 13 810 – – Texas Red 780 – – Triple probe (DAPI, FITC, and Rhodamine) 720/740 – – TRITC (Rhodamine) 800–840 – –

brightness. In the example shown in Fig. 8, the brightness image [e(i, j); Fig. 8c] shows an almost constant value over the region expected for the shape of the nanocapsule as observed from the conventional image [I(i, j); Fig. 8a]. The number image nNm(i, j), instead shows (Fig. 8b) that at least in two distinct regions the number of molecules is larger than the average. The combination of ﬂuorescence ﬂuctuation spectroscopy and microscopy therefore allows us to obtain important direct information on the heterogeneity of the samples.

4. Two-photon-excited probes The probability of a nonlinear interaction between the excitation light beam and a fluorescent molecule depends on its two- or multi-photon cross-section. In the specific case of TPE, cross- sections are given in GM units (1 GM=10x50 cm4 sx1 photon). The successful application of 2PE to biological samples is partly due to the fact that commonly used fluorescent molecules could be efficiently excited by 2PE (Xu, 2001). 2PE cross-sections have been measured for a large number of ‘popular’ fluorescent molecules (Xu & Webb, 1996). Table 2 summarizes the properties of some common fluorescent molecules under TPE (So et al. 2000; Xu, 2001; 116 A. Diaspro, G. Chirico and M. Collini

(a) 1000 Bodipy xs

DAPI xs

100 DIL xs

FITC xs 10 Coumarin xs

Cascade Blue xs 1 Lucifer Yellow 790740690 990940890840 1040 1090 xs Rhodamine xs

0·1

0·01

0·001

(b) 1000 Ca-Crimson xs

Ca-Green xs 100 Ca-Orange wl

Fluo3 xs 10 Fura Ca xs

Indo+Ca xs 1 690 740 790 840 890 940 990 1040 Indo free xs

Fura free mean 0·1 xs

0·01

0·001 Fig. 9. Sample of measured cross-sections (GM) of common cellular ﬂuorescent molecules (a) and calcium markers (b) within the wavelength range of the most popular ultra-fast lasers, i.e. 690–1040 nm. (Data from http://microscopy.bio-rad.com/products/multiphoton/Radiance2100MP/mpspectra.htm).

Dickinson et al. 2003). Figure 9 shows a sample of measured cross-sections within the wavelength range of the most popular ultra-fast lasers (Girkin & Wokosin, 2001a). Moreover, newly engineered organic molecules, endowed with large two-photon absorption cross-sections have been recently developed (Albota et al. 1998; Abbotto et al. 2003; D’Alfonso et al. 2003). A practical ‘rule of thumb’, essentially valid for symmetrical fluorescent molecules, states that, as a function of the excitation wavelength, one may expect to have a TPE cross-section relative maximum at a wavelength twice that needed for one-photon excitation for a certain fluorescent molecule. However, this ‘rule of thumb’ is not fulfilled by more complex structures, for example most of the Green Fluorescence Protein (GFP) mutants (Blab et al. 2001; Fig. 10). 2PE in biological microscopy 117

380 400 420 440 460 480 500 380 400 420 440 460 480 500 520 540 10 50 eCFP 1·0 eGFP 2·0 8 40 )

0·8 ) 2 1·5 2

6 cm 30 0·6 cm –15 –15 (GM) (GM) 1·0 2PE 2PE 4 (10 20 (10 σ σ 0·4 OPE OPE σ 0·5 σ 2 0·2 10

0 0·0 0 0·0 750 800 850 900 950 1000 750 800 850 900 950 1000 1050 1000 Wavelength (nm) Wavelength (nm)

380 400 420 440 460 480 500 520 540 380 400 420 440 460 480 500 520 540 560 30 3·5 30 eYFP DsRed 3·0 0·8 ) )

2·5 2 2 20 20 0·6 cm 2·0 cm –15 (GM) –15 (GM) 2PE

2PE 1·5 0·4 (10 (10 σ σ 10 10 OPE 1·0 OPE σ σ 0·3 0·5 0 0·0 0 0·0 110010501000950900850800750 750 800 850 900 950 1000 1050 1100 1150 Wavelength (nm) Wavelength (nm)

Fig. 10. Absolute two-photon excitation (2PE) spectra times the quantum yield (action cross-section, sTPE, left axes) of eCFP, eGFP, eYFP, and DsRed in phosphate buﬀer (wavelength scale at bottom). The one photon cross-section (sOPE, right axes) are shown for comparison (solid lines, wavelength scale at top). (After Blab et al. 2001.)

In addition to extrinsic fluorescent molecules, 2PE fluorescence from NAD(P)H, flavo- proteins (Piston et al. 1995; So et al. 2000), tryptophan and tyrosine in proteins have been measured. Special mention is also warranted on the imaging of GFP, which is an important molecular marker for gene expression (Potter, 1996; Chalfie & Kain, 1998). GFP cross-sections are y6 GM (800 nm) and 7 GM (960 nm) in the case of wild and S65T type respectively. As a comparison one should consider that the cross-section for NADH, at the excitation maximum of 700 nm, is y0Á02 GM (So et al. 2000). This extension to autofluorescent molecules that can be effectively exploited without the need of UV excitation opens new relevant perspectives in 3D imaging of biological systems and also in connection to other types of nonlinear interactions, i.e. second- and third-harmonic generation (Herman & Tanke, 1998; Lakowicz, 1999; Zoumi et al. 2002; Zipfel et al. 2003). Table 3 summarizes, even if in a less than rigorous way due to the continuing work in progress on it, 2PE cross-section data from classes of intrinsically fluorescent molecules. By comparison we also report recent data about fluorescent semiconductor nanocrystals – quantum dots (Qds) (Hines & Guyot-Sionnest, 1996; Mattoussi et al. 2000; Peng & Peng, 2001) – that allow multi-colour imaging in demanding biological environments such as living tissue, i.e. living animals or organs and within cellular cytoplasm (Sokolov et al. 2003; Larson et al. 2003; Jaiswal & Simon, 2004). These fluorescent probes exhibit two-photon cross- sections of the order of thousands of GM and a wide range of emission wavelengths. They are indicated as good candidates for deep and multi-colour imaging (Chan et al. 2002; Parak et al. 2002; Akerman et al. 2002; Dubertret et al. 2002; Sokolov et al. 2003; Voura et al. 2004), despite some problems related to intermittent fluorescent behaviour and to a high propensity for 118 A. Diaspro, G. Chirico and M. Collini

Table 3. Two-photon excitation cross-section data from classes of intrinsically ﬂuorescent molecules

Intrinsic emitters l (nm) d2 GFP wt 800–850 y6 GFP S65T y960 y7 BFP 780/820 – CFP 780/840 – YFP 860/900 – EGFP 940–1000 y250 DsRed – Coral Red 960–990 y20–110 Citrine – Coral Yellow 950 y70 Phycoerythrin 1064 y300 Flavins y700–730 y0Á1–0Á8 NADH y690–730 y0Á02–0Á09 Retinol 700–830 y0Á07 Pyridoxine 690–710 y0Á008 Folic acid 700–770 y0Á007 Lipofuscin 700–850 – Collagen, elastin 700–740 – Qdots 700–1000 y2000–47000

aggregation. These facts promoted research activities in nano-sized fluorescent probes (Tokumasu & Dvorak, 2003; Gao et al. 2004; Jaiswal & Simon, 2004). As can be seen from 2PE cross-section tables and figures there is a very interesting and useful variety of ‘relative peaks’. The practical consequence is that unlike one-photon excitation when using 2PE one can find a wavelength that can be used for exciting several different molecules. The relevance of this fact is obvious, for example with respect to co-localization problems. One can try to find an optimal excitation wavelength for simultaneously priming fluorescence of different fluorochromes. This means that a real and effective multiple fluorescence co-localization of biological molecules, macromolecules, organelles, etc. can be obtained. Figure 11 shows examples of fluorescence molecular cross-sections demonstrating multiple fluorescence excitation in the case of 2PE (red line) with respect to the 1PE (green) case. Reduced photobleaching is an essential requirement for the fluorescence microscopy application of a fluorescent molecule (see also discussion in Section 3.2). While the 2PE scheme reduces overall photobleaching of the sample by limiting excitation to a small volume element, photobleaching within the excited area is not reduced (Brakenhoff et al. 1996). In fact, accelerated rates of photobleaching have been observed in bulk by using 2PE compared to conventional one-photon excitation (Patterson & Piston, 2000). Recent studies reported on the effect of TPE on the total amount of fluorescence that can be collected from a single immobilized molecule (Chirico et al. 2003b; Federici et al. unpublished data). For the four dyes considered in this study, namely: Indo-1, Rhodamine 6G, Fluorescein and Pyrene, the 2PE bleaching rate increases by a power of 3 of the excitation intensity. This brings the conclusion that a trade-off must be considered in order to limit bleaching, while gaining in emission by increasing the excitation intensity (Chirico et al. 2003a). However, the earlier fear of destroying the sensitive biological sample by direct absorption of the NIR light has also been shown to be exaggerated, although it always requires checking (Schonle & Hell, 1998; Hopt & Neher, 1999; Tirri et al. 2003). 2PE in biological microscopy 119

(a) (b) 1×103 1×102

1×101 1×102 1×10 1×101 1×10–1

1×10 1×10–2 600 700 800 900 1000 1100 600 700 800 900 1000 (c)(d) 1×102 1×101

1×10 1×101 1×10–1 1×10 1×10–2

–3 1×10–1 1×10 600 700 800 900 1000 1100 650 700 750 800850 900

Fig. 11. Rhodamine B (a), Coumarine (b), Fluoresceine (c) and Cascade Blue (d) one- (solid line) and two- photon (dotted line) excitation cross-sections (GM) (Xu, 2001). The wavelength scale has to be divided by 2 when considering the one-photon excitation case.

5. Design considerations for a 2PE fluorescence microscope 5.1 General aspects The original 2PE systems were mainly based around conventional confocal scanning systems and all of these demonstrated the principles of the technique and produced excellent results (Girkin, 2003). In principle, any laser scanning microscope can be modified to perform 2PE or multi-photon excitation microscopy by replacing the CW laser source with a pulse laser and optimizing some optical elements (Diaspro, 2001b; Centonze, 2002). Nowadays, ‘turn-key’ 2PE or multi-photon excitation microscopes are commercially accessible at a price ranging from 250 000 to 700 000 E/US$. The main parts for realizing a 2PE architecture are the following: a high peak-power laser delivering pulsed (fs or ps) IR or NIR photons at 100 MHz repetition rate and mW average power, a laser beam or sample scanning device, a high NA objective (>1), a high-throughput microscope pathway, and a high-sensitivity detection system (Denk et al. 1995; Konig et al. 1996c; Potter et al. 1996; So et al. 1996; Soeller & Cannell, 1996, 1999; Wokosin & White, 1997; Centonze & White, 1998; Wolleschensky et al. 1998; Diaspro et al. 1999b; Tan et al. 1999; Hopt & Neher, 1999; Fan et al. 1999; Wier et al. 2000; McCarthy, 1999; Mainen et al. 1999; Majewska et al. 2000; Nguyen et al. 2001; Diaspro, 2001b; Girkin & Wokosin, 2001a; Centonze, 2002; Iyer et al. 2002; Sanderson & Parker, 2003; Girkin, 2003; Muller et al. 2003; Malengo et al. 2004; Ridsdale et al. 2004). In a general scheme for a 2PE microscope, conventional excitation ability for confocal or wide-field fluorescence microscopy should be maintained. Within the general view, let us consider image formation modalities. Typically, 2PE or confocal microscopy images are built by raster scanning obtained by x–y mirrors of a 120 A. Diaspro, G. Chirico and M. Collini galvanometric-driven mechanical scanner (Ploem, 1987; Webb, 1996). This fact implies that the image acquisition rate is mainly determined by the mechanical properties of the scanner. Fast beam-scanning schemes can be realized that exploit the localized fluorescence by using multi- element lenses, thin beam splitters or, in general, a number of 2PE beams scanned through the sample simultaneously (Buist et al. 1998; Straub & Hell, 1998b; Fan et al. 1999; Tan et al. 1999; Straub et al. 2000; Hell & Anderson, 2001; Nielsen et al. 2001). In this case one should accurately consider the compromise between sensitivity, spatial resolution, temporal resolution and photobleaching (Girkin & Wokosin, 2001a; Girkin, 2003). However, in the most popular case of the utilization of scanning mirrors, enhanced silver coatings are prevalently used to optimize reflectivity of the IR excitation wavelengths. Then, the excitation light should reach the microscope objective passing through the minimum number of optical components and possibly along the shortest path. Typically, high NA objectives, with high IR transmission (70–80%), are used to maximize 2PE efficiency (Benham, 2002; Benham & Schwartz, 2002). While the x–y scanners provide lateral focal-point scanning, axial scanning can be achieved by means of different positioning devices, the most popular being: belt-driven systems using a DC motor and objective piezo nano-positioners (Diaspro, 2001b). Acquisition and visualization are typically computer controlled by dedicated software, that allows the control of different key parameters such as: light detector gain, spectral separation, acquisition channel, scanning speed, excitation laser beam, field of view, image size, z-axis positioning (Webb, 1996). Detection system is another critical issue mentioned here only in terms of general aspects (Wokosin et al. 1998; So et al. 2000; Girkin & Wokosin, 2001a). Photodetectors that can be used include photomultiplier tubes, avalanche photodiodes, and charge-coupled device (CCD) cameras (Denk et al. 1995; Murphy, 2001). Photomultiplier tubes are the most commonly used. This is due to their low cost, good sensitivity in the blue-green spectral region, high dynamic range, large size of the sensitive area, and single-photon counting mode availability (Hamamatsu Photonics, 1999). They usually provide a quantum efficiency around 20–40% in the blue-green spectral region that drops to <1% moving to the red region. This is a favourable condition under the 2PE regime since one wants to reject as much as possible those wavelengths above 680 nm, mainly used for excitation. Moreover, the large size of the sensitive area of photomultiplier tubes allows efficient collection of signal in the non-descanned mode (see Section 5Á2). On the other hand, avalanche photodiodes (APD) are excellent in terms of sensitivity exhibiting quantum efficiency close to 70–80% in the visible spectral range. Unfortunately, they are ex- pensive and the small active photosensitive area introduces some drawbacks in the detection scheme and requires special descanning optics (Farrer et al. 1999). Moreover, CCD cameras can also be used, especially in video rate multi-focal imaging (Buist et al. 1998; Straub & Hell, 1998b; Fuijta & Takamatsu, 2001; Girkin & Wokosin, 2001a). Another general aspect is related to the possibility given by the 2PE process of priming fluorescence in different molecules using a unique radiation wavelength (see also Section 4). This fact suggests that a very precise spectral separation is much to be desired (Farkas, 2001; Dickinson et al. 2001; Haraguchi et al. 2002; Zimmerman et al. 2003; Berg, 2004). Usually, the emitted fluorescence is collected by an objective and transferred to the detection system through a dichroic mirror along the emission path that realizes the first stage of spectral filtering. This device should also block reflected excitation. Then optical filters are inserted before emission can reach the detectors in order to select the emitted wavelength for a specific acquisition channel. In 2PE, due to the high peak excitation intensity, an additional barrier filter is needed to avoid mixing of the excitation and emission light at the detection system, which is positioned 2PE in biological microscopy 121 differently depending on the acquisition scheme being used. Recently, the introduction of new- generation scanning heads was accompanied by efficient spectral detection systems (Wright & Wright, 2002). Nikon invested in a separate filter box with the new model C1 (Ridsdale et al. 2004). This allows better spectral control than in the classical schemes completely integrated into the scanning head and the adaptation of monochromator or similar devices in a very simple and effective customized way. Zeiss developed on its LSM510 a spectral separation scheme based on multiple detectors. A highly efficient optical grating provides an innovative way of separating the fluorescence emissions in the incorporated Hamamatsu detectors, META (Dickinson et al. 2003). Here, the entire fluorescence signal is projected onto the 32 channels of the META detector acting as a spectral discriminator. Thus, a spectral signature can be acquired for each pixel of the scanned image and subsequently used for the digital spectral separation into component dyes (Dickinson et al. 2001). Unfortunately, some spectral gaps are in the red detection portion of the signal due to the characteristics of the detector itself. Also FluoView 1000 by Olympus has a spectral head made by mixing dichroics and grating-slit systems. Another comparatively new system is the Leica TCS SP2 AOBS. This is the natural evolution of the spectral confocal microscope SP1, designed to be a filter-free spectral confocal and multi-photon microscope. Spectral detection is performed by a prism spectrophotometer detector (Seyfried et al. 2003). This allows performance of continuous spectral detection without the need of changing optical filters. Leica Microsystems was the first company that introduced the new concept of a spectral scanning head (Calloway, 1999). In any case high- optical-density filters able to block the IR high-peak-power excitation radiation should be used (Diaspro, 2001b; Stanley, 2001). As a final general aspect, once the best quality image possible has been obtained then soph- isticated mathematical algorithms can be applied to enhance those fine details and features of interest to the biological researcher and to improve the quality of data to be used for 3D modelling (Weinstein & Castleman, 1971; Diaspro et al. 1990, 2000, 2004c; van der Voort & Strasters, 1995; Boccacci & Bertero, 2001; Vermolen et al. 2004). Recently, an image restoration web service has been established to obtain the best quality 3D dataset from wide-field, confocal or 2PE optically sectioned samples (Bonetto et al. 2004). Now, let us focus on four more specific aspects, namely: descanned and non-descanned imaging, lens objectives, laser sources, and an example of a practical realization.

5.2 Descanned and non-descanned 2PE imaging Two different approaches are currently utilized to perform 2PE, namely: descanned and non- descanned modes (Sandison & Webb, 1994; Cannel & Soellet, 1997). The former uses the very same optical pathway and mechanism employed in confocal laser scanning microscopy. The latter essentially optimizes the optical pathway by minimizing the number of optical elements encountered on the way from the sample to detectors, and increases the detection area. The non-descanned approach is possible in 2PE since the signal generated by the sample is exclusively originated from the focal volume (Centonze, 2002). The major implication of this behaviour is that is no longer necessary to re-image the diffraction-limited spot of emitted light to impose an imaging aperture able to cut off out-of-focus information. Figure 12 illustrates these two approaches. The non-descanned detection scheme is in tune with the need for collecting every excited photon possible (Pawley, 1995), with the rule that ‘The best optics are no optics!’ (Girkin & Wokosin, 2001a). When working with point-scanning laser excitation 122 A. Diaspro, G. Chirico and M. Collini systems short pixel dwell times (microseconds) are often used, which necessitate very high source intensities for sufficient signal-to-noise imaging. High laser intensities have a corre- spondingly high risk of photobleaching and saturation potential (Hopt & Neher, 1999). The best solution is one that improves the signal-to-noise ratio within the detection scheme. The descanned detection scheme allows increasing resolution at the cost of a drop in detection efficiency when pinhole diameter is reduced (Sheppard & Gu, 1990; Sandison & Webb, 1994; Gauderon et al. 1999; Torok & Sheppard, 2001). Conversely, the non-descanned mode requires that the confocal architecture is modified: the emitted radiation is collected using dichroic mirrors on the emission path or external detectors without passing through the galvanometric scanning mirrors or the scanning device being employed for point-like excitation (Piston et al. 1994; Williams et al. 1994). A photomultiplier tube can be placed immediately beneath the objective or above the condenser to collect all the fluorescence captured by the lens. The resulting shorter emission pathway, possibly with the use of demagnification lenses, ameliorates the collection of emitted photons from a greater solid angle (Centonze, 2002; Koester et al. 1999). This permits collection of more scattered photons emitted by fluorescent molecules, thereby increasing the signal-to-noise ratio especially when imaging very deeply in thick light- scattering samples (Centonze & White, 1998). Thus far, non-descanned imaging results in a significant increase in detection sensitivity (Wokosin & White, 1997; Centonze & White, 1998; Koester et al. 1999), although it makes the detector sensitive to ambient room light. All recent confocal microscopes allow adaptation of collection in both modes.

5.3 Lens objectives and pulse broadening For a fluorescence microscope, special criteria have to be satisfied (Benham, 2002; Benham & Schwartz, 2002) for the choice of the objective lens besides those common to any microscope (Keller, 1995). For 2PE, an adequate transmission in the IR regime has to be coupled with good collection efficiency towards the UV region. Moreover, the number of components, their shape and the type of glass should be optimized without affecting resolution properties in order to reduce pulse-width distortions due to group velocity dispersion (Wokosin et al. 1998; Wolleschensky et al. 1998, 2001; Netz et al. 2000). Although the collection efficiency of the time- averaged photon flux is dependent on the NA of the collecting lens, it was demonstrated that the total fluorescence generation is independent of the NA of the focusing lens when imaging thick and uniformly fluorescent samples (Xu, 2001). This is due to the fact that the increase of intensity, obtained by a sharper focusing (high NA), is counterbalanced by the shrinking of the excitation volume. Thus, the integral of fluorescence over the entire space remains constant. The very relevant practical consequence of this fact is that in 2PE measurements from thick samples, assuming no aberrations, the generated fluorescence is insensitive to the size of the focal spot. As a positive consequence, a moderate variation of the laser beam size would not affect the measurements. This depends on the fact that it is possible to collect all the generated fluorescence by using an appropriate (non-descanned) acquisition scheme. For point- scanning systems, the acceptance angle of the objective lens is related to the NA. Girkin & Wokosin (2001a) reported on the light collection performances of a variety of lenses. The 1Á4NA60r oil and 1Á2NA60r water lenses have the best collection potential, the 1Á3NAoil lenses can collect 85% compared to the best two lenses, while the 0Á75 NA air lenses and 1Á0NA water-dipping lens only collect 66% as much light. As usual, a compromise must be reached, as the higher NA lenses (recently 1Á65 by Olympus and 1Á45 by Nikon and Zeiss have become 2PE in biological microscopy 123

Excitation light Scanning system A

Detector DM Pinhole x–y Scanner z Non-descanned detector

Detector DM

Objective B Lens Focal Plane Fluorescence

Scattering specimen A B Multiphoton (descanned) Multiphoton (direct detection)

Fig. 12. Simpliﬁed optical schemes for de-scanned (A) and non-descanned (B) detection. Confocal pinhole can be used or fully opened. Images along the z-axis under (B) condition have a better signal-to-noise ratio than under (A) one. (Courtesy of Mark Cannel; adapted from Soeller & Cannell, 1999; courtesy of Ammasi Periasamy, image inset).

available) gain the light collection power at the loss of working distance. Typical working distances for oil immersion lenses with a NA of 1Á4 are y250 mm (which include the thickness of a cover slip that most commonly is 0Á17 mm). This gives a practical working distance of <100 mm, making them unsuitable for really deep imaging of intact tissue. As a general rule one selects a lens with as large a NA as possible giving the working distance required for the preparation under investigation. The 1Á4 NA lenses provide a good starting point for 2PE lens selection. In addition NA has a significant contribution in determining the signal-to- background and signal-to-noise ratios. These factors play a large role in determining whether a particular sample can be successfully imaged, and in particular on the maximum depth that can be visualized (Girkin & Wokosin, 2001a; Jonkman & Stelzer, 2001; de Grauw & Gerritsen, 2001). Table 4 shows a collection of transmission data for Nikon CFI60 series of objectives (Girkin & Wokosin, 2001a; Benham & Schwartz, 2002). Objective lenses and optical components play a role in pulse broadening and this influences the efficiency of the 2PE process. The short pulses typically used for 2PE or multi-photon imaging microscopy have bandwidths of the order of 15 nm (corresponding to%100 fs pulse time duration), and the passage through microscope optics produces a dispersion that results in an effective stretching of the pulse in time and reduction of the peak power (Muller et al. 1998; Netz et al. 2000; Girkin & Wokosin, 2001a). This is similar to an avalanche effect. Once the pulse has started to be stretched, the effect continues as it is due to the spectral bandwidth and not to the actual pulse length in the optical train (Girkin & Wokosin, 2001a). Now, considering an ideal chirp-free pulse at the entrance of an optical system having a pulse width tin, the pulse broadening B can be calculated as tout/tin where tout is the pulse width at 124 A. Diaspro, G. Chirico and M. Collini

Table 4. Collection of transmission data for Nikon CFI60 series of objectives

Working T %at T %at T %at Objective MAG/NA distance (mm) 350 nm 550 nm 900 nm CFI Plan 0Á13 <15 >71 51–70 Apochromat 100rOil IR 1Á40 CFI Plan Fluor 100rOil 1Á30 0Á20 51–70 >71 >71 CFI Fluor 60r1Á00 2Á00 51–70 >71 >71 Water dipping CFI Plan Fluor 60rOil 1Á25 0Á20 31–50 >71 51–70 CFI Fluor 40r0Á80 2Á00 51–70 >71 >71 Water dipping CFI Super (S) Fluor 40rOil 1Á30 0Á22 51–70 >71 >71 CFI Super (S) Fluor 20r0Á75 1Á00 51–70 >71 >71 CFI Fluor 10r0Á30 2Á00 51–70 >71 51–70 Water dipping CFI Super (S) Fluor 10r0Á50 1Á20 51–70 >71 >71

Table 5. Optical dispersion parameters for diﬀerent glass types

D at 800 nm (fs2 cmx1) Glass type

+251 CaF2 +300 quartz +389 FK-3 +445 BK7 +1030 SF2 +1600 SF10

the focal plane (Konig, 2000): qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 2 2 B=tout=tin= ðÞ1+7Á68(DL=tin) , (20)

Typical values for the optical dispersion parameter D for glass objectives at 800 nm are given in Table 5. Considering the optical path length factor L, a typical DL value of the whole microscope, including oil immersion high-NA objective is 5000 fs2 (Konig, 2000). Wolleschensky et al. (2001) reported a summary of the dispersion parameter for Zeiss microscope objective lenses measured at 800 nm. DL values (in fs2) are 1714, 1494, 2398, and 1531 (within an error of y10%) for 40r/0Á8 water IR Achroplan, 63r/0Á9 water IR Achroplan, 40r/1Á3 oil Plan Neoﬂuar, and 20r/0Á75 Plan Apochromat respectively. Pulse broadening factor for a 100 fs pulse was estimated to be between 1Á14 and 1Á23 (Wolleshensky et al. 2001). In order to minimize dispersion problems one should use pulses of around 150–200 fs (Konig, 2000). Operatively, a typical optical system, considering the lens objective eﬀect as predominant, will stretch a 200 fs pulse to 220 fs at y850 nm producing a peak power loss of 10%. Moving to picosecond pulses it should be borne in mind that 30% more average power is required for the same signal when compared with 100 fs pulses (Girkin & Wokosin, 2001a). 2PE in biological microscopy 125

5.4 Laser sources

Laser sources can be considered as a sort of key step for the development and dissemination of 2PE. Within the non-resonant 2PE framework, owing to the comparatively low cross-sections of fluorescent molecules, high photon flux densities are required, >1024 photons cmx2 sx1 (Konig, 2000). 2PE or multi-photon imaging typically requires y500 times more average power delivered at the sample than conventional confocal laser scanning microscopy in order to achieve a comparable number of excitation events within the focal volume (Girkin & Wokosin, 2001a). Using radiation in the spectral range of 600–1100 nm, excitation intensities in the MW-GW cmx2 are required. Colliding pulse dye lasers used in the early development of 2PE imaging (Denk et al. 1990; Valdemains et al. 1995) could not really be considered as practical systems for the long term (Girkin, 2003), due to the limited tunability, moderate output average power – 15 mW – and the considerable maintenance needed. However, a similar source with high-peak powers operating at around 600–630 nm, an unsual range for solid-state-based lasers (see below), has been recently developed and used for multi-photon imaging applications (McConnell et al. 2004). Solid-state-based lasers were introduced in 1992 with the development of femtosecond titanium:sapphire lasers (Curley et al. 1992; Fisher et al. 1997). Even if such systems are not perfect for the application they have become the laser of choice for most users (van de Vende & Gratton, 1995; Centonze, 2002; Girkin, 2003). This is especially due to the fact that all solid-state sources requiring standard mains electrical power supply and minimal cooling systems favoured the dissemination of 2PE and multi-photon imaging systems (Wokosin et al. 1997; Diaspro, 2004a). More recently a number of advances have been made that, when available commercially, may change the practical application of multi-photon microscopy (Girkin & Wokosin, 2001b; Girkin, 2003; Tirri et al. 2003). 2PE imaging has been achieved with significantly longer pulses than the most commonly used 100–200 fs case: picosecond (Hanninen et al. 1995) and CW (Hanninen et al. 1994) 2PE has been accomplished successfully. However, this would not be the preferred choice for most applications due to linear absorption and hence sample heating (Konig et al. 1995). Nowadays, most commercial systems operate around 100–200 fs and 80 MHz for historical reasons (see Table 6). The original synchronously pumped short-pulse sources were excited by large-frame ion lasers which happened to have a cavity length equivalent to an 80 MHz pulse repetition rate (Girkin, 2003), a fact particularly useful for fluorescence excitation (see Section 3Á4; Denk et al. 1995). Such a repetition rate, is high enough to allow rapid scanning – approximately 1 frame/s in a conventional system – but low enough that the fluorescence lifetime of the fluorophores does not cause excitation events to be wasted because the fluorescence has not decayed before the next excitation pulse arrives. Considering this, the ideal repetition rate would be around 80–200 MHz and an average power output around 200 mW. This means, considering the optical efficiency of the microscope, an average power at the sample of a few tens of mW. Table 6 also includes data about a new promising ytterbium-based source. This novel laser source, emitting high energy (20 nJ) femtosecond pulses, in a broad spectrum (250 nm), can be tuned from 950 to 1200 nm, without any laser adjustment, and delivers sub-300 fs pulses with a 10 nm spectral width (http://www.amplitude-systemes.com; Courjaud et al. 2001; Deguil et al. 2004). As recently reported by Hanninen’s group, low-cost lasers can be used to utilize the positive properties of TPE with certain compromises and well-designed experiments (Tirri et al. 2003). 126 .Dapo .CiioadM Collini M. and Chirico G. Diaspro, A.

Table 6. Laser sources for 2PE

Wavelength Repetition Laser material Company, model (nm) Pulse length rate (MHz) Power Comments Ti:Sapphire Coherent, Mira 700–980 <200 fs 76 0Á7W1Á3 W May require mirror change for full tuning Spectra Physics, 700–1000 <100 fs (or 80 0Á8W1Á4 W May require mirror change for full tuning Tsunami 2 ps as option) Coherent, Chameleon – 705–980 <140 fs 90 1Á7 W Hands free operation, computer controlled, XR compact design, single box Spectra Physics, Mai Tai 710–990 120 fs 80 1Á5 W Hands free operation, computer controlled, compact design, single box Time Bandwidth, Pallas 780–860 <100 fs 75 500 mW Time Bandwidth, Tiger 780–860 <100 fs 100 400 mW Femtosource 750–850 <12 fs 75 400 mW 600 mW Nd:YLF MicroLase/Coherent 1047 200 fs 120 500 mW Not presently available Scotland, BioLite Nd:Glass Time Bandwidth, 1058 <250 fs 100 >400 mW GLX200 Ytterbium Amplitude Systems 1030 <200 fs 50 1 W Demonstrated on dyes inc Texas Red and GFP; optional 950–1150 selectable Cr:LiSAF Highqlasers 850 100 fs 50 >1mW OPO Coherent and Spectra 350–1200 100 fs y200 mW Exact parameters depends crystal selected. Physics Requires titanium:sapphire as pump source

The pulse length given is the average pulse length through over the full tuning range of the system. Power is the average power at the peak of the tuning curve, typically 800 nm, or at the operating wavelength. Both Coherent Mira and Spectra Physics Tsunami systems can be pumped with a 5, 8 or 10 W green laser source. When two powers are given these are for 5 and 10 W pumped. Higher pump power may be needed for full tuning range. Between 925 and 960 nm titanium:sapphire lasers need to be purged to ensure that no water vapour is present within the laser cavity. Computer tunable systems are sealed to prevent this complication and are endowed of a pointing stability system that reduces the need of new alignment following tuning across a wide range of wavelengths. 2PE in biological microscopy 127

Fig. 13. Two-photon architectures at LAMBS (Laboratory for Advanced Microscopy, Bioimaging and Spectroscopy) incorporated in the new Research Centre of the University of Genoa for Correlative Microscopy (MicroScoBio) funded by IFOM (Italian Molecular Oncology Foundation). Under the auspices and grants of the National Institute for the Physics of Matter (INFM, Istituto Nazionale per la Fisica della Materia) this was the ﬁrst Italian 2PE architecture (Diaspro, 2001b). Leica TCS SP2 AOBS (front, centre) and PCM2000 Nikon (back, right) confocal laser scanning systems. They are operating sharing the very same Titanium Sapphire laser source. (Photograph taken at LAMBS.)

Once the laser source has been selected, one has to couple it with microscope optics. Wolleschensky and colleagues (2001) reported accurate data, and analysis of benefits and problems in using an optical fibre or direct coupling to launch the excitation within a confocal scanning head. Direct coupling allows the maximum flexibility, together with the minimum level of ‘extra’ problems at the excitation stage. In fact, even if the fibre coupling solution seems the best in terms of practicality and safety at a first glance, some practical problems can arise related to the fibre coupling solution itself. For example, due to high-order nonlinearities in the fibre, there is a limit regarding the maximum deliverable power, typically 60–100 mW. Moreover, because the fibre coupler needs some devices for maintaining pulse width and beam shape, it is not totally turn-key – demanding further control and alignment procedures especially when wavelength changes are required or laser output beam changes occur, due, for example to room temperature variations.

5.5 Example of a practical realization This section deals with an example of a practical realization of a 2PE microscope based on a commercial confocal laser scanning microscope (CLSM), Nikon PCM2000 (Nikon Instruments, Florence, Italy), as previously reported in detail (Diaspro et al. 1999b; Diaspro, 2001b). Figure 13 illustrates the 2PE laboratory at the University of Genoa showing the recent addition of a spectral CLSM, Leica SP2/AOBS (Leica Microsystems, Heidelberg GmbH, Germany). A core component of the 2PE microscope is the laser source. In this case, two mode-locked titanium:sapphire IR pulsed lasers are available, namely: a Tsunami 3960 (Spectra Physics Inc., Mountain View, CA, USA), pumped by a (5 W at 532 nm) solid-state laser Millennia V (Spectra 128 A. Diaspro, G. Chirico and M. Collini

Physics Inc.), and a Chameleon-XR (Coherent Inc., Santa Clara, CA, USA), a widely tunable, compact and hands-free laser diode, pumped by an incorporated high-power Verdi (Coherent Inc.) solid-state 532 nm laser (see also Table 6). This allows 2PE in the 680–980 nm range. Two excitation ranges are covered by the Tsunami, e.g. 680–830 and 730–900 nm, depending on the set of the mirrors actually mounted into the laser cavity, and a third range from 710 to 980 nm is achieved by Chameleon. This flexible configuration permits 2PE of a variety of fluorescent molecules normally excited by visible and UV radiation, including the GFP family, according to the cross-section behaviours given in Tables 2 and 3. Power and wavelength measurements are carried out using an RE201 model ultra-fast laser spectrum analyser (Ist-Rees, UK) and an AN2/10A-P model thermopile detector power meter (Ophir, Israel) that constitute the beam diagnostics module of the architecture. Recently we have added a home-made compact optical autocorrelator, which allows measurement of the width of laser pulses on the microscope objective plane (Cannone et al. 2003b). A special dichroic mirror set (CVI, USA), optimized for high-power ultra-short IR pulsed laser beams, is employed to bring the laser excitation directly into the confocal laser scanning head. Before entering into the PCM2000 or Leica SP2 scanning head, beam average power is brought to desired values using a neutral-density rotating wheel (Melles Griot, USA). We refer to the PCM2000 scanning head unless specified differently. An average power of 30 mW at the entrance of the scanning head produces an average power before the microscope objective of 9–15 mW, depending on the wavelength and on the alignment. The PCM2000 Nikon scanning head has a comparatively low number of optical components along the excitation pathway, and the resulting average power at the sample, i.e. at the focal plane of the objective, is estimated to be between 5 and 12 mW. This further loss of power is due to the transmission property of the lenses in the IR wavelength region (see Table 4). In terms of pulse width, considering a pulse duration in the range of 90–150 fs at the exit of the laser output window, after passing driving mirrors, neutral density filter and the scanning lens, a 1Á5–1Á8 times broadening was found when using a high NA objective (Diaspro, 2001b). This is in agreement with earlier reports (Hanninen & Hell, 1994; Soeller & Cannell, 1996). For the optical system described at this point, pulse widths in the range of 220–265 fs have been measured at the sample focal plane (Cannone et al. 2003b). However, in order to check the pulsing status of the laser source during the measurement sessions, one should continuously display the pulse condition. This can be easily done by monitoring the wavelength spectrum by means of an oscilloscope connected to the output of the spectrum analyser, as shown in Fig. 14. The pulsing condition of the laser can also be verified using a simple and inexpensive reflective grating. In this case the reflected image on a screen will be sharp for quasi-continuous emission and blurred for pulsed emission. This is due to the fact that the output of the pulsed laser beam is more spectrally broadened in the case of pulsed emission. Unfortunately, the Tsunami requires periodical re-alignment due to beam displacement following wavelength changes. This problem is completely overcome when using Chameleon since it is endowed with a beam-pointing system, named PowerTrack, allowing active alignment for long-term stability. Moreover, the utilization of Chameleon is currently preferred due to the fact that the laser output pulse width is stable around 120–140 fs. This fact keeps the broadening of pulse width limited. In fact, considering a 2PE microscope with acousto-optical modulator having a b value [group velocity dispersion; DL in Eq. (20)] of y13 500 fs2 at 800 nm, 60–80 fs pulses will broaden to 540 fs at the sample, while 120 fs pulses will broaden to 340 fs. Figure 15 shows calculated broadening of pulses due to dispersion in a 2PE microscope (www.coherentinc.com, 2002). More broadening will produce a considerable loss in 2PE efficiency according to Eq. (7). The other core component of the 2PE 2PE in biological microscopy 129

Beam diagnostic 2P laser

Tsunami Variable N.D. filter

1P laser Specimen TE 300 module

Z Millenia

PCM2000 2P optical pump

PCM2000 controller EZ 2000

Fig. 14. Overall scheme of the Nikon PCM2000-based two-photon microscope described in the text (Diaspro, 2001b). architecture is the scanning and imaging system. As anticipated, the scanning and acquisition system for microscopic imaging is based on a commercial single-pinhole scanning head Nikon PCM2000 (Nikon Instruments, Florence, Italy) mounted on the lateral port of an inverted microscope, Nikon Eclipse TE300. The Nikon PCM2000 has a simple, efficient and compact light path that makes it very appropriate for conversion to a 2PE microscope (Diaspro, 2001b). Figure 16 illustrates the optical pathways in the confocal laser scanning head. The optical resolution performances of the PCM2000 confocal laser scanning microscope when operating in conventional confocal mode, and using a 100r/1Á3 NA oil immersion objective, have been reported in detail elsewhere and are 180¡20 nm laterally and 510¡50 nm axially (Diaspro, 1999a). Better performances can be obtained by means of digital deconvolution methods (Diaspro et al. 1997, 2000). Under the TPE regime the scanning head operates in the ‘open pinhole’ condition; i.e. a wide-field descanned detection scheme is used (Diaspro, 2001b). Figure 17 gives an insight of the open scanning head. A dichroic mirror (1st) has been substituted in the original scanning head to allow excitation from 680 to 1050 nm (Chroma Inc., Brattleboro, VT, USA). The substituted dichroic mirror reflects very efficiently (>95%) from 680 to 1050 nm. The 50% cut-off is at y640 nm. This dichroic mirror transmits more than 90% radiation from 410 to 620 nm. The neutral density filter at the open-pinhole location of the original PCM2000 scanning head has been removed. Galvanometric mirrors are metal-coated (silver) on fused silica. The minimum pixel residence time is 1Á5 ms. A series of emission custom-made filters (Stanley, 2001), blocking IR radiation with different thresholds >650, >700, >720 nm to an optical 130 A. Diaspro, G. Chirico and M. Collini

(a) 700 = 17,500 fs 600

= 13.500 fs 500

400

300 ~ 60–80 fs typ.

200 Broadened pulse width (fs) Chameleon ~ 120 fs

100

0 0 100 200 300 400 Pulse width from laser (fs)

(b)

Fig. 15. (a) Calculated broadening of pulses due to dispersion in a 2PE typical microscope (see text, for a range of laser pulse widths). (b) The microscope, coupled with an ultra-fast laser spectrum analyser RE201 (Ist-Rees, UK), allows pulse control. (Courtesy of Mario Arace who purchased the oscilloscope in 1978.) density of 6–7 within 50 mW of beam power incident on the filters themselves. This allows the use of those fluorophores emitting in the red region of the spectrum and of growing interest. The optical filters more commonly utilized are the following: E650P, HQ 460/50, HQ 535/50, HQ 485/30 and HQ 405/30 (Chroma Inc.). More specifically, the E650P filter constitutes the base for the other HQ filters. This means that for some applications a kind of ‘sandwich’ made by optical filters can be easily realized thanks to the optically simple and accessible PCM2000 scanning head. 1PE mode is preserved by the switching of two optical pathways: the single- mode optical fibre used for 1PE and the 2PE direct coupling (Diaspro, 2001b). Switching from conventional to TPE allows retention of the focus and position on the sample as demonstrated 2PE in biological microscopy 131

PMT PCM2000

1 79 5 TE 300

x–y 3

268

Fig. 16. Details of the optical pathway of the confocal scanning head Nikon PCM2000. The excitation beam enters into the PCM2000 scanning head through an optical coupler (1) in order to reach the sample on the x–y–z stage (5). The beam passes through the pinhole holder (2) kept in open position, the galvanometric mirrors (3) and the scanning lens (4). Fluorescence generated from the sample (5) is delivered to the PMT through acquisition channels directed by two selectable mirrors (6, 8) via optical fibre (7, 9) coupling. One-photon and two-photon mode can be accomplished simply by switching from the single-mode optical fibre (one-photon), coupled to a module containing conventional laser sources (Ar–Ion, He–Ne green), to the two-photon optical coupler (TPOC) shown in Fig. 38, allowing Tsunami laser beam delivery (two- photon). Axial scanning for confocal and two-photon excitation (TPE) 3D imaging is actuated by means of two different positioning devices depending on the experimental circumstances and axial accuracy needed, namely: a belt-driven system using a DC motor (RFZ-A, Nikon, Japan) and a single objective piezo nano- positioner (PIFOC P-721-17, Physik Instrumente, Germany). The piezoelectric axial positioner allows an axial resolution of 10 nm within a motion range of 1000 nm in 100 nm steps and utilizes a linear variable differential transformer (LVDT) integrated feedback sensor (Diaspro, 2001b). in Figs 18 and 19. At the end of the collection pathway, a high throughput optical fibre delivers the emitted fluorescence from the scanning head to the PCM2000 control unit where photomultiplier tubes (R928, Hamamatsu, Japan) are physically plugged. Axial scanning for confocal and TPE 3D imaging is actuated by means of two different positioning devices depending on the experimental circumstances and axial accuracy needed, namely: a belt-driven system using a DC motor (RFZ-A, Nikon, Japan) and a single objective piezo nano-positioner (PIFOC P-721-17; Physik Instrumente, Germany). The piezoelectric axial positioner allows an axial resolution of 10 nm within a motion range of 1000 nm at 100 nm steps and utilizes a linear variable differential transformer (LVDT) integrated feedback sensor. Acquisition and visualization is completely computer controlled by dedicated open software (EZ2000; Coord, The Netherlands; http:// www.coord.nl). Image processing based on deconvolution algorithms is performed by means of a web-based package designed and implemented at the Laboratory for Advanced Microscopy, Bioimaging and Spectroscopy (LAMBS) (Diaspro et al. 2002b; Bonetto et al. 2004). Now, in order to evaluate the performances of the microscope some basic trials have to be performed, namely: evaluation of the fluorescence quadratic behaviour and of the PSF. As a first step, the quadratic relationship of the fluorescence intensity versus excitation power has to be demonstrated before speculating on the fluorescence signal. This can be simply accomplished by measuring fluorescence intensity from a standard or from the actual sample as a function 132 A. Diaspro, G. Chirico and M. Collini

Scanning head input

Scanning mirrors Emission filter

Scanning lens

Output channel D2 D1

Pinhole

Fig. 17. Nikon PCM 2000 confocal laser scanning head modified for two-photon excitation at LAMBS. Main modifications are: pinhole and neutral density filter removal in the large pinhole position; D1 dichroic optimized for IR transmission; free-space coupling input; additional barrier filter (Chroma Inc.) for avoiding collection of high-power peaks of the excitation beam (Diaspro, 2001b). of the excitation power. Special attention has to be given to the acquisition conditions. They should be kept constant during the acquisition session or calibrated for the intensity range. Figure 20 shows the 2PE trend obtained from a solution of fluoresceine (Diaspro, 2001b). The evaluation of the PSF can be performed using fluorescent microspheres of dimensions below optical resolution, i.e. 5200 nm or thin fluorescent layers. In the first case one determines the 3D PSF (Diaspro et al. 1999b). In the second case, one measures the axial behaviour of the optical system (Schrader et al. 1998). PSF measurements reported here are referred to a planachromatic Nikon 100r,1Á4 NA immersion oil objectives with enhanced transmission in the IR region. Lateral and axial resolutions were evaluated as 210¡40 and 700¡50 nm respectively (Diaspro, 2001b). The 3D imaging capabilities of the 2PE microscope is also shown in Fig. 21. A DNA sample marked with DAPI was imaged using the intrinsic optical sectioning ability of the TPE of fluorescent molecules. The 3D assembly of the sample is clearly evident even in the case of the thick sample being imaged, i.e. a sea urchin egg. Optical sections exhibit different structural details at high signal-to-noise ratio without any image processing. Figure 22 reports autofluorescence optical slices of another thick biological sample, e.g. the cistis of Colpoda. This very same architecture is also used for single-molecule detection and spectroscopic measurements (Diaspro et al. 2001; Chirico et al. 2001a; Cannone et al. 2003a). Recently at LAMBS a new 2PE microscope based on a Leica SP2 AOBS confocal laser scanning microscope has been established. Laser coupling is realized utilizing an extra laser port available at the scanning head. Since this confocal laser scanning microscope is endowed with a high-performance spectral detection unit, it is ideal for spectral analysis of autofluorescence signals and for other nonlinear interactions like SHG. Figure 23 shows an image of a protozoan exhibiting 2PE in biological microscopy 133

(a)

20 µm

(b)

20 µm

Fig. 18. Optical sectioning of ﬂuorescent microspheres demonstrated in confocal and two-photon excitation (TPE) mode after switching from (a) one- to (b) two-photon mode using the TPOC developed at LAMBS. It is worth noting that after switching sample positioning is retained.

(a)(b)

Fig. 19. Same biological cell to demonstrate switching from (a) one- to (b) two-photon excitation mode. In two-photon excitation mode the internal structure of the nucleus is clearly visible, i.e. chromatin DNA marked by DAPI. This ﬁgure clearly shows the control of positioning after mode switching. 134 A. Diaspro, G. Chirico and M. Collini

3·0

2·5

2·0

1·5

1·0 Flourescence intensity (AU) 0·5

0 0 5 10 15 20 25 30 35 40 45 50 55 P average (mW)

Fig. 20. Quadratic behaviour check for imaging under the two-photon excitation regime through the plot of ﬂuorescence intensity versus excitation average power (Diaspro, 2001b). autoﬂuorescence and a second-harmonic signal on the backscattering pathway when excited at 720 nm (Campagnola et al. 1999; Diaspro et al. 2002c).

6. Applications This section, for obvious reasons, is far from being exhaustive. It reports some of the most relevant aspects, in the authors’ opinion, in relation to TPE and related methods. Interested readers can ﬁnd other applications and stimuli in some books and special issues on this subject (Pawley, 1995; Hell, 1996; Lakowicz, 1999; Diaspro, 1998, 1999a, b, 2001a, 2004a; Periasamy, 2001; Matsumoto, 2002; Masters, 2002; Ferguson, 2003; Periasamy & Diaspro, 2003; Feijo & Moreno, 2004).

6.1 Biological applications of 2PE 2PE is growing tremendously in terms of applications in many areas of biology, physics, medicine, and engineering. Its capability of deep imaging makes it extremely valuable for in vivo imaging. The related technology is still developing rapidly and its range of utilization is broadening. The ﬁrst 2PE applications to biological cells were presented by Denk and co-workers (1990) within the neurosciences, and most of the applications presented up to now are related to calcium mapping in cells, the intact brain and brain slices (Denk et al. 1994; Oheim et al. 2001; Helmchen & Denk, 2002; Tsai et al. 2003; Theer et al. 2003). Recently Taki et al. (2004) have presented developments in ratiometric dyes for the detection of zinc ions by means of 2PE that are also promising for biological applications.

6.1.1 Brain images Signalling mechanisms at individual synapses can be monitored in brain slices. The ability of 2PE to provide high-resolution images within scattering turbid media has a key role for these 2PE in biological microscopy 135

0-12·2 µm 3-11·3 µm 6-10·4 µm 9-9·4 µm 12-8·6 µm

15-7·7 µm 18-6·8 µm 21-5·9 µm 24-5·0 µm 27-4·1 µm

30-3·2 µm 33-2·3 µm 36-1·3 µm 39-0·4 µm 42-0·4 µm

45-1·3 µm 48-2·3 µm 51-3·1 µm 54-4·0 µm 57-5·0 µm

60-5·8 µm 63-6·8 µm 66-7·7 µm 69-8·5 µm 72-9·5 µm

Fig. 21. Three-dimensional optical slices from a sea urchin egg marked by DAPI, two-photon excitation at 720 nm. The heterochromatin distribution within the female pronucleus is visible. The whole egg has a diameter of 80 mm while the nucleus is 10 mm (as reference this is the maximum visible diameter). In conventional wide-ﬁeld microscopy one could see only a bright spot of confusion from the nucleus. (Sample by Carla Falugi, Department of Biology, University of Genoa.)

studies (Beaurepaire et al. 2001). Oertner and colleagues (2002) found that the amount of glutamate released per action potential changes during short-term facilitation, suggesting a multi- vescicular release of glutamate. Moreover, Sabatini et al. (2002) investigated calcium buﬀering capacity in spines of pyramidal neurons using 2PE. Examples of these applications are given in Fig. 24, showing a calcium-related image of rat granule cerebrellar cells loaded with Indo-1 acetoxymethyl (AM). The cellular network has been optically sectioned at a moderate average power on the focal plane, 2 mW, at 720 nm excitation wavelength and by focusing with a 100r/1Á3 NA objective. The complex and intricate organization in Purkinje cells are shown in Fig. 25. 2PE calcium imaging of isolated cells can be used to monitor the activity of distinct neurons in brain tissue in vivo. Recently, Stosiek and colleagues (2003) introduced a versatile approach for loading membrane-permeant ﬂuorescent indicator dyes in large populations of cells. They 136 A. Diaspro, G. Chirico and M. Collini

Fig. 22. Optical sections from Colpoda’s cistis. From these images was possible to detect the different layers of membrane composition by means of the autofluorescence signal, primed at 780 nm, as well as small nuclei membranes visible as small dots in round-shaped structures. (Sample by Paola Ramoino, DIPTERIS, University of Genoa.) established a pressure ejection-based local dye-delivery protocol that can be used for a large spectrum of membrane-permeant indicator dyes, including Calcium Green-1 AM-ester, Fura-2 AM, Fluo-4 AM, and Indo-1 AM. This dye-loading protocol was successfully applied in mouse brain tissue in vivo and in vitro at any developmental stage from newborn to adult. Thus, these results demonstrated the suitability of 2PE imaging for real-time analyses of intact neuronal circuits with the resolution of individual cells. Another outstanding application is given by a study of temporal and spatial dynamics of Ca2+ signalling in mouse cortical neurons (Stutzmann et al. 2003). Dynamic processes in the brain require long-term imaging that a 2PE approach can uniquely provide while limiting the bleaching of out-of-focus planes in the sample. It is possible to study structural reorganization in the brain on a slow time-scale compared to neural activity. This can be done by utilizing cranial windows similar to the approach used in cancer research (Jain et al. 2002) where the skull is removed over a small area and permanently replaced with a cover glass. TPE imaging is also possible through the thinned skull of living animals (Christie et al. 2001; Yoder & Kleinfeld, 2002). The overall approach is minimally invasive and allows stable imaging over a period of weeks. Christie and colleagues (2001) provided the first demonstration of growth and stability of senile plaques in a mouse model of Alzheimer’s disease using in vivo multi-photon microscopy. Figure 26 shows a top-down projection of senile plaques in the brain. In a related recent study Nitsch et al. (2004) have shown, for the first time, that within the complex cellular network of living brain tissue, specific T cells contact neurons in which they induce calcium 2PE in biological microscopy 137

200 Nx 100

200

400

600

800 934 900 600 800 200 400

Fig. 23. Three-dimensional view of a second-harmonic generation and autofluorescence from Paramecium primaurelia multimodal image obtained by means of two-photon excitation at 810 nm. As a first check, also used for two-photon excitation autofluorescence, the laser was taken out of mode locking and the signals vanished. This fact indicates that the signals’ origin was due to nonlinear processes, which were also verified by a quadratic dependence on the laser power. Moreover, the laser was scanned between 750 and 830 nm with the 405 nm emission filter kept fixed. No signal was detected at 405 nm within a range of y5nmat y810 nm. Finally, the potential second-harmonic generation image appeared bleach resistant. Diaspro’s group is indebted to Colin Sheppard, Tony Wilson, Bruce Tromberg and Guy Cox for critical and useful discussions about the still unclear origin of such a signal on the back-scattering pathway (Diaspro et al. 2002c). (Sample prepared by Paola Ramoino, DIPTERIS, University of Genoa.) oscillations resulting in a lethal increase in neuronal calcium levels. This in vivo study, which reveals important details for the understanding of multiple sclerosis, has employed TPE ion-sensitive dyes. Wang and colleagues (2003) produced a very impressive work combining the 2PE method with the mapping of the activity of a large number of olfactory neurones in the fly brain by means of a genetically targeted endogenous calcium probe used in conjunction with a diverse sensory input to the olfactory receptors. Very recently Vest et al. (2004) have tried to validate, also by means of 2PE, the hypothesis that calcium aids the formation of domains of ordered lipids within erythrocyte membranes by interacting directly with the inner leaflet of the cell membrane. Changes in membrane order were assessed with steady-state fluorescence spectroscopy and 2PE with an environment-sensitive probe, laurdan. 138 A. Diaspro, G. Chirico and M. Collini

Fig. 24. Granule rat cerebrallar cell loaded with Indo-1 AM, a calcium-binding dye. This UV-excitable ﬂuorescent molecule has been excited at 720 nm at a moderate average power at the focal plane of 2 mW. (Courtesy of Alessandro Esposito, LAMBS.)

Fig. 25. Purkinje cell labelled with Oregon Green. Calcium ion concentration is mapped by means of a colour scale from blue (low concentration level) to red (maximum concentration level). (Courtesy of Professor Cesare Usai, Institute of Biophysics, National Research Council, Genoa, Italy.) 2PE in biological microscopy 139

Fig. 26. Top-down projection of senile plaques in the brain of a living transgenic mouse (Tg2576). This image is from an x–y–z volume of 500r615r200 mm3 (Christie et al. 2001). (Image by B. J. Bacskai, [email protected]; downloaded from Biorad site: http://microscopy.bio-rad.com/ gallery7.htm).

6.1.2 Applications on the kidney 2PE has been used to capture high-quality images and study the function of otherwise inaccessible cell types and complex cell structures of the juxtaglomerular apparatus (JGA) in living preparations of the kidney (Peti-Peterdi et al. 2002). This structure has multiple cell types that exhibit a complex array of functions, which regulate the process of filtrate formation and renal haemodynamics. For the first time, it was possible to report on high- resolution 3D morphology of isolated, perfused kidney glomeruli, tubules, and JGA. Calcium imaging of the glomerulus and JGA (Fig. 27), demonstrated the utility of 2PE in capturing the complexity of events and effects that are exerted by the specific hypertensive autacoid angiotensin II. 2PE in vivo has been used to study the renal excretion of sulfonefluorescein (SF) in normal and cystic rat kidneys (Tanner et al. 2004). Figure 28a shows a low power view of the kidney surface of a normal rat infused with SF. SF intensities in proximal tubules are not uniform, but are higher in late proximal tubules than in early proximal tubules. Figure 28b shows a represen- tative high-power view from a non-pathological kidney. Proximal tubule cell SF fluorescence intensity clearly exceeds that of the plasma and the tubule lumen. This brilliant work by Tanner and colleagues (2003) clearly demonstrates the ability of TPE microscopy to study quantitatively the renal excretion of fluorescent probes in vivo.

6.1.3 Mammalian embryos Squirrel and co-workers (1999) studied mammalian embryos over 24 h without compromising viability. Two-photon microscopy permitted long-term ﬂuorescence observations of the dynamics of 3D cyto-architecture in highly photosensitive specimens such as mammalian 140 A. Diaspro, G. Chirico and M. Collini

AA EA MD cTAL

Fig. 27. Visualization of various glomerular and juxtaglomerular apparatus (JGA) structures in situ. Glomerulus is perfused through the aﬀerent arteriole (AA) with attached cortical thick ascending limb (cTAL) and macula densa (MD). The image shows the Ca2+ map of the double-perfused AA and cTAL containing the MD. Tissue was loaded with Indo-1. G, glomerulus; EA, eﬀerent ateriole (bar=10 mm).

(a)(b)

Fig. 28. (a) Low power (20r objective) view of the surface of the kidney of an anaesthetized rat kidney during constant intravenous infusion of a solution of sulfone fluorescein (SF). SF is an organic anion secreted by proximal tubules, and it accumulates in proximal tubule cells, especially in late proximal segments. Distal tubules (arrowheads) do not secrete SF (scale=90 mm). (b) Higher power (60r objective) view of normal kidney. Proximal tubule cell SF fluorescence intensity averages that of peritubullar capillary blood plasma (fine arrows) and is also higher than in the proximal tubule lumen. The distal tubule cells do not take up SF, and their nuclei stain brightly (blue) with the Hoechst dye 33342. SF is concentrated in the distal tubule lumen due to glomerula filtration of SF, secretion by upstream proximal tubule segments, and water reabsorption along the nephron (scale=30 mm). (Image courtesy of George Tanner, Indiana University School of Medicine.) 2PE in biological microscopy 141 embryos (White et al. 2001). Recently, Isogai et al. (2003) have used time-lapse multi-photon microscopy to image living Tg(fli1:EGFP)y1 zebrafish embryos and to examine how a patterned, functional network of angiogenic blood vessels is generated in the early vertebrate trunk. Since protein expression patterns are a primary determinant of tissue function, a method to access macroscopic distances at subcellular levels of detail is of great interest. Jacobs and colleagues (2003) have shown how to combine two-photon microscopy with a novel image montage method (fast-beam blanking coupled with mathematical alignment tools) in order to extend the limited field of view of laser scanning microscopes. These authors have shown the effectiveness of the combined approach to the analysis of the distribution of connexin-46, visualized across equatorial sections of the rat mammalian lens. Figure 29 shows an overview taken from the equatorial lens radius where gross changes in cell morphology with increasing depth into the lens are evident (Jacobs et al. 2003). 2PE has also allowed visualization of the lateral organization of cellular membranes. Gratton and colleagues (Gaus et al. 2003) have directly visualized the membrane lipid structure of living macrophages, providing evidence that the membrane coverage by lipid rafts and their fluidity are principally governed by cholesterol content, thereby providing strong support for the lipid raft hypothesis. A flourishing of work (Arnulphi et al. 2005; Benninger et al. 2005; Sanchez & Gratton, 2005; Wang et al. 2005; Zoumi et al. 2005) in this direction makes it difficult to track all recent developments achieved by 2PE in this field. A recent comprehensive review on the methods for detecting microdomains in intact cell membranes has also appeared (Lagerholm et al. 2005).

6.1.4 Applications to immuno-response New preparations, fluorescent probes and imaging techniques are providing the means to observe the behaviour of cells in the tissue environment of lymphoid organs. When combined with two-photon laser microscopy, intravital imaging of surgically exposed lymph nodes provides a unique view of lymphocyte migration and antigen presentation as it occurs within the living animal. Cahalan et al. (2002, 2003), have reached relevant results that indicate that lymphocytes migrate randomly within lymphoid organs, and that lymphocyte contact with antigen- presenting cells may be a stochastic process rather than guided by chemokine gradients. These results have been confirmed and extended in a recent 2PE study of the interaction of the dendritic cells with T cells within intact lymph nodes in the absence of relevant antigen (Miller et al. 2004). In general the main advantage of TPE microscopy in the study of the immunological response lies in the possibility of performing in vivo observations as documented in recent experiments on the tracking of pathogenic myelin basic protein-specific CD4(+) effector T cells in early CNS lesions of experimental autoimmune encephalomyelitis (Kawakami et al. 2005).

6.1.5 Myocytes Time fluctuations of the concentration of the reduced form of the nicotine adenine dinucleotide (NADH) have been studied by time-series of 2PE images of resting myocytes. These data (Blinova et al. 2004) suggest for the first time that the cellular NADH fluorescence is dominated by the sampling noise of the instruments and not significantly modified by the biological processes. More conclusive studies on myocytes by 2PE have investigated the spatial distribution of intracellular sodium concentration in rat ventricular myocytes, showing that sodium diffusion 142 A. Diaspro, G. Chirico and M. Collini

0·5 mm (a)

0·1 mm (b)

0·02 mm (c)

Fig. 29. Demonstration of the range of spatial scales encompassed by the composite image data. The sample is rat mammalian lens. (a) Overview taken from the equatorial lens radius, showing gross changes in cell morphology with increasing depth into the lens. (a) Five-fold magnification of the boxed region in (a) showing local cell morphology. (a) Five-fold magnification of the boxed region in (a) showing subcellular morphological detail. Images have been also taken (not shown) at distances of 500 mm and 700 mm into the lens respectively. Note that at this resolution the change in the expression pattern of labelled gap junctions (red) can be clearly identified. The arrow (c) indicates a small gap junction plaque on the narrow side of hexagonal fibre cells. The extent of the displayed data in the horizontal direction is indicated above each image. All images were extracted from the same original data. (Adapted from Jacobs et al. 2003.) in cardiac myocytes is slow with respect to trans-sarcolemmal sodium transport rates, although the mechanisms responsible are unclear (Despa et al. 2004). Moreover, Lindegger & Niggli (2005) compared UV and diffraction-limited two-photon photolysis of caged Ca2+ in the investigation of Ca2+ release channels (ryanodine receptors, RyRs). These authors found opposite results when using UV or TPE photolysis inferring that changes of RyR open probabilities after b-adrenergic stimulation, enhancing local regenerativity and reliability of Ca2+ signalling.

6.1.6 Retina Imanishi et al. (2004) have recently established, by means of 2PE imaging, the presence of previously uncharacterized structures, very distinct from other cellular organelles in the retina. Besides this use of 2PE microscopy that has allowed in vivo experiments with unique insights in the function of these structures, Das et al. (2003) used TPE microscopy to reconstruct 2PE in biological microscopy 143

Fig. 30. Three-dimensional view of the mature sperm head of the octopus Eledone cirrhosa, loaded with DAPI, from 12 optical sections (Diaspro et al. 1997). (Courtesy of Silvia Scaglione; image processing and visualization by Fabio Mazzone, Francesco Di Fato and Silvia Scaglione at LAMBS and BioLab, University of Genoa, Italy.) cell divisions in living zebrafish embryonic retinas. Even more recently, Niell & Smith (2005) showed how to provide controlled visual stimuli to larval zebrafish, while performing two- photon imaging of tectal neurons loaded with a fluorescent calcium indicator, allowing the determination of visual response properties in intact fish.

6.1.7 DNA imaging DNA imaging is possible by using UV excitable dyes whose fluorescence can also be primed by 2PE. Figure 30 shows images of the helical structure of sperm heads of the octopus Eledone cirrhosa obtained by TPE of UV-excitable fluorescent molecules. A 3D representation of the helical structure shown in Fig. 30, is realized by assembling 2D images from a stack of 64, 512r512r8 bits, taken 200 nm apart along the optical axis. The mature helical sperm head has a pitch of 0Á6–0Á75 mm, an outer radius of 0Á25–0Á3 mm, and is 43 mm long. From single 2D images it is not possible to get information about the spatial organization of the sperm head: the handiness, as in the confocal case, can be determined only using the completely assembled dataset (Diaspro et al. 1997; Difato et al. 2004). Of greater interest is the capability of TPE microscopy to track DNA in living cells. By the combination of two-photon and confocal microscopy, three main apoptosis parameters (the change of nuclear morphology, collapse of mitochondrial membrane potential, and increase of intracellular calcium) were recorded simultaneously for single As2O3-induced Molt-4 cells (Wang et al. 2004). In a recent review Errington et al. (2005) have discussed how to perform DNA imaging or tracking in living cells (for anti-cancer research purposes) by means of two-photon laser scanning microscopy. Tracking drug delivery in subcellular compartments, with the mapping of sites of critical 144 A. Diaspro, G. Chirico and M. Collini drug–DNA interactions was particularly addressed (Errington et al. 2005). Recently, dual-colour two-photon fluorescence cross-correlation spectroscopy was used to detect single-stranded gamma tubulin DNA (Berland, 2004). Even if this work was carried out in solution, it constitutes the starting point for measurements and imaging in more complex environments such as within living cells.

6.1.8 FISH applications Konig’s group has applied 2PE to multi-colour fluorescence in situ hybridization (FISH) (Levsky & Singer, 2003). They performed 3D FISH to detect specific sequences within single DNA molecules in cells and tissues. They simultaneously excited the visible fluorescence of a wide range of FISH fluorochromes, such as FITC, DAC, Cy3, Cy5, Cy5.5, Rhodamine, Spectrum Aqua, Spectrum Green, Spectrum Orange, Jenfluor, and Texas Red as well as of DNA/ chromosome stains, for example Hoechst 33342, DAPI, SYBR green, propidium iodide, ethidium homodimer, and Giemsa. In addition to the advantage of using only one excitation wavelength for a variety of fluorochromes, multi-photon excitation provided the possibility of 3D fluorescence imaging. The technology is of great impact in human genetics for the diagnosis of numerical chromosome aberrations and micro-deletions. In particular, Konig et al. (2000a) have obtained the intra-nuclear localization of FISH-labelled chromosome territories in inter- phase nuclei of amniotic fluid cells by multi-colour 3D images. In the same work Konig and co-workers obtained optical sectioning of Hoechst 33342- labelled DNA within living culture cells and within tissue of living tumour-bearing mice (Konig et al. 2000a) by using the high-light penetration depth at 770 nm. Figure 31 shows imaging of centrometric multiple fluorescent probes in human biopsies (Konig et al. 2000b).

6.2 2PE imaging of single molecules 2PE has typically 0Á2 mm radial resolution. As such it does not provide the resolution to image single chromophores and fluorescent or labelled biomolecules embedded in entrapping matrices. However, when imaging sparse samples, one gets isolated fluorescent spots that have the size of the PSF. We can ascribe those spots to single-molecule emission by performing a number of statistical checks on the amount of photons emitted, on their polarization (Ha et al. 1996; Bopp et al. 1998) and on the emission dynamics (Chirico et al. 2001a). Single-molecule optical spectroscopy by optical methods has recently acquired major interest for its unique ability to identify and characterize the properties of single molecular entities (Magde et al. 1972; Xie & Lu, 1999; Schwille, 2001). Here, we focus on single-molecule visualization (far-field) using TPE-primed fluorescence emission (Mertz et al. 1995; Sonneleitner et al. 1999; Diaspro et al. 2001). Following the pioneering work by Sanchez et al. (1997) on two-photon imaging of single rhodamine B glass immobilized molecules, spatially resolved applications of ultra-sensitive 2PE fluorescence have shown promising results (Sonneleitner et al. 2000; Chirico et al. 2001a). Two basic issues of any type of single-molecule imaging are related to diminish the background signal, either residual scattering or fluorescence, and to discriminate between the signals arising from single molecules and those that correspond to small molecular aggregates. The latter issue can only be tackled by following the time evolution of the fluorescence emission of molecular aggregates, which may be degraded by the prolonged exposure to the exciting radiation as shown in Fig. 32. Moreover, one can discriminate single molecules from aggregates considering that 2PE in biological microscopy 145

2·0 µm 2·5 µm 3·0 µm 3·5 µm

4·0 µm 4·5 µm 5·0 µm 5·5 µm

Fig. 31. Imaging of centromeric fluorescent probes in human biopsies by 3D two-photon multicolour FISH (Konig et al. 2000a). The localization of Spectrum Green (green colour) labelled C-4 probes, Spectrum Orange (red colour) labelled C-X probes and Spectrum Aqua (blue colour) labelled C-Y probes are depicted in a 10 mm thick kidney cryosection. The nuclear area was imaged after counterstaining with ethidium bromide (adapted from Konig et al. 2000b). on an image the distributions of the pixel content show discrete peaks at levels that are all multiples of that corresponding to the dimmest spot revealed in the substrates (Fig. 33) (Cannone et al. 2003a; Chirico et al. 2003a). TPE may also lead to an increase of the signal- to-noise ratio for single-molecule detection, due to the tiny volumes (%0Á1 fl) obtained without the need of spatially filtering the emission signal as in confocal microscopy. A systematic study of single-molecule detection by 2PE has been reported on dyes (Pyrene, Rhodamine 6G, Fluoresceine and Indo-1) of increasing complexity. They are embedded in silica gels (Mozzarelli & Bettati, 2001) and illuminated by 770 nm radiation of a 100 fs, 80 MHz pulsed titanium:sapphire (Chirico et al. 2003b) through a scanning head. The fluorescence output, collected with a 225 ms resolution, scales as the square of the exciting power (see lower inset of Fig. 34) and it is apparently constant until a sudden drop in the background level (Fig. 34). Due to a remarkable temperature dependence of this bleaching behaviour, the phenomenon has been interpreted as a temperature-induced structural change of the dyes due to the IR absorption that is not re-radiated into fluorescence. Moreover, the bleaching rate computed on the single molecules scales as a power law of the excitation power with an exponent >2 (in the range 2Á1–2Á6) depending on the complexity of the chemical structure (see upper inset in Fig. 34). Similar results were also found in solution by Patterson & Piston (2000). Since the fluorescence output is always scaling with P2, the total number of photons that can be collected by 2PE fluorescence spectroscopy is decreasing with the excitation power as Px0Á5. This result indicates that the best observation conditions are obtained with very sensitive detectors and low excitation intensities. A second example that we report is the detection of the TPE fluorescence of a single GFP mutant, called E2GFP (Cinelli et al. 2001) trapped in wet silica gels (Chirico et al. 2004). The typical image of GFP loaded gels, collected at 785 nm, is shown in the inset of Fig. 35. The fluorescence kinetics are followed for%110 s and show a bleaching of the E2GFP molecule 146 A. Diaspro, G. Chirico and M. Collini

1·0 5 4 3 0·5 2 Intensity Intensity 1

0·0 0 0123456 0123456 Time Time

Fig. 32. Single and multiple ﬂuorescent molecular aggregate trend. Comparison between single molecule (left) and aggregate (right) intensity decay (Chirico et al. 2003a). (Courtesy of Fabio Cannone, LABS and INFM Milano, Bicocca.)

40 30 20 10·5 20

Intensity (AU) 10 0

Numbers 10 18·9 28·8 39·7 45·2 0 0 605040302010 Integrated fluorescence per spot (AU)

Fig. 33. Distribution of the intensity of the spots of an image of a glass side cleaned and prepared by spin coating a C=310 nm Rhodamine 6G solution. The image was acquired with residence time 3 ms, and average excitation power 7 mW. Top (inset): ﬂuorescence of the peaks in the distribution, in order of increasing intensity (Cannone et al. 2003a). 2PE in biological microscopy 147

20 1 0·1

Bleaching rate (Hz) 81216 Power (mW) 0

20 Fluorescence (AU)

20 20 0 100 403020

Fluorescence Power (mW) 0 020406080 100 Time (s)

Fig. 34. Typical fluorescence signal versus time for single molecules of (a) fluorescein (.), (b) Rhodamine 6G ($), (c) Indo-1 (%), and (d) Pyrene (m)at6Á7 mW of excitation power. Panel (a) (inset): typical distribution of the bleaching time for fluorescein. Panel (d) (inset): fluorescence signal (arbitrary units) per single molecule versus the excitation power. The average has been computed on at least 80 molecules. The 2 symbols are as in the main panel. The data are fitted to a law of the type AexcrP . Panel (a) (inset): average bleaching rate, CB, of Rhodamine 6G ($), fluorescein (.), Indo-1 (%) and Pyrene (m), versus the excitation power. The solid lines are fits to a power law, BrPC, with C varying from 2Á1to2Á6. after%85 s. After 10 s of bleaching, the sample is scanned 40 times at 720 nm (open squares in Fig. 35) finding that the E2GFP has recovered fluorescence (last filled square in Fig. 35). Imaging of individual molecules in a phospholipid membrane has also been obtained by TPE (Sonnleitner et al. 1999). Two-photon laser scanning microscopy at 700 nm was also used for detecting single Cy3-labelled lipids on the plasma membrane of living human coronary artery smooth muscle cells (Sonnleitner et al. 2000). Fluorescence images of cells, taken with a mean power of 6 mW and a dwell time of 50 ms per pixel, showed peaks from single Cy3-labelled lipids diffusing on the cell membrane. This work clearly demonstrates that single-molecule detection with 2PE is also possible on cells in vivo. This has opened further perspectives to utilize the advantages of 2PE, e.g. the reduced background, increased lateral and axial resolution and the possibility of synchronous multi-colour experiments, for single-molecule imaging in living biological cells (Harms et al. 2001, Heinze et al. 2004). Finally we cite a recent TPE investigation of the unfolding of a GFP mutant (GFPmut2) performed at the single-molecule level (Cannone et al. 2005). In this case the TPE emission has been discriminated in two acquisition channels that correspond to the emission of the neutral and the anionic form of the GFP chromophore. The complex interplay of these two 148 A. Diaspro, G. Chirico and M. Collini

10 Fluorescence (AU)

050100 Time (s)

Fig. 35. Fluorescence kinetics of a single E2GFP molecule embedded in a silica gel. The illumination is at 785 nm, average power=4 mW for the first 95 s. Then the image is scanned at 720 nm, average power 4 mW, for 10 s (open squares). The image is then scanned once more at 785 nm, average power 4 mW (last filled square). Inset: typical fluorescence image of a 15r15 mm2 field of E2GFP silica gels. conformational/protonation states in the unfolding and refolding kinetics has been investigated in detail, showing that subtle memory effects are taking place in the unfolding of GFP. Even more recently a high time resolution (10 ms) investigation of the TPE-primed emission of the same mutant has shown oscillations in the GFP emission a few tens of milliseconds before the unfolding event (Baldini et al. 2005). The two emission levels correspond to the two protonation states and the characteristic frequencies of the oscillatory behaviour lie in the 400–1000 Hz range. More notably the oscillations can be driven by both electric and acoustic fields (Baldini et al. 2005).

6.3 FCS applications The possibility of coupling FCS to scanning microscopy is extremely attractive as a sensitive method of probing local diffusion in cells and in vivo, and to distinguish Brownian diffusion from active transport phenomena (Schwille, 2001), which are common in cells. The 2PE version of FCS takes further advantage of the low background of the IR excitation in the cell and from the possibility of simultaneously exciting widely different chromophores. This peculiarity of TPE makes it ideal for applications in fluorescence cross-correlation experiments as documented by the effort of Schwille’s group (Heinze et al. 2000; Haustein & Schwille, 2004; Kim et al. 2005; Jahnz & Schwille, 2005). It must be noted that FCS is in itself a highly sensitive technique not free of artefacts and particular care must be taken in the choice of the fluorescent probe, which could display different photophysics when excited by 1PE or 2PE, or could interact with im- mobile cellular structures (Schwille et al. 1999a, b). The actual extent of the advantages of 2PE in FCS, for example in limiting the sample photodamage in the cellular environments, have been investigated by Gratton and co-workers among the first groups (Berland et al. 1995, 1996). In particular, Berland et al. (1995) applied these techniques to characterize the intracellular environment by measuring the diffusion coefficients of fluorescent beads, 7 and 15 nm in diameter, injected into the cytoplasm of 2PE in biological microscopy 149

Buffer 0·8 Cytosol Membrane IgE receptor 0·6 D=3×10–10 cm2/s

0·4 Autocorrelation 0·2

D=3×10–6 cm2/s 0·0 1×10–3 0·01 0·1 1 10 100 1000 10000 Time (ms)

Fig. 36. Various autocorrelation curves demonstrating the enormous difference in motility between buffer solution and cytosol. (Courtesy of Petra Schwille and Elke Haustein, MPI Gottingen, Germany.) mouse fibroblast cells. Figure 36, taken from Schwille (2001), clearly shows the different motility of fluorophores in different cellular compartments. The decay times of the ACFs vary by several orders of magnitude, with diffusion coefficients that span a range 3r10x6 cmx2 sx1

Gratton’s laboratory has also recently presented a detailed study on the possibility of applying PCH analysis to the study of the motility of proteins in cellular compartments (Chen et al. 2002). These authors have characterized the molecular properties of autofluorescence and transiently expressed enhanced green fluorescence protein (EGFP) in the nucleus and in the cytoplasm of HeLa cells both by FCS and PCH analysis. Because the concept of molecular brightness is crucial for PCH analysis, they have determined the statistical accuracy of its measurement under in vivo conditions, finding a molecular brightness of EGFP that is a factor of 10 higher than the brightness of the autofluorescence. The molecular brightness of EGFP measured in the nucleus, the cytoplasm, and in vitro are identical and one of the main conclusions of this study is that molecular brightness is a very stable and predictable quantity for cellular measurements. Further recent applications of 2PE FCS to the study of molecular diffusion in cellular and solution environments have appeared. Ruan et al. (2002) fused cytosolic adenylate kinase (AK1, a ubiquitous enzyme) and its isoform (AK1b) with EGFP and expressed the chimera proteins in HeLa cells. By using TPE scanning fluorescence imaging, Ruan et al. were able to directly visualize the localization of AK1-EGFP and AK1b-EGFP in live cells and to detect different diffusion properties of the two isoforms depending on their location within the cytoplasm and on the plasma membrane (Ruan et al. 2002). Besides the possibility of investigating molecular motion, FCS is also capable of detecting the internal photodynamics of the fluorophores and assess how it is affected by the different environments. The Webb group has thoroughly investigated the photodynamic properties of mutants of GFP in solution (Haupts et al. 1998; Heikal et al. 2001) and also in cells (Schwille et al. 1999a, b). The internal photodynamics of GFP was investigated at different pH, with one- and two-photon excitation, finding two fast blinking processes. Only the fastest one was sensible to the pH and was ascribed to the protonation of the chromophoric moiety of GFP, while the second was attributed to a cis-trans conformational change as also confirmed by theoretical investigations (Voityuk et al. 1998). Further characterizations of the heterogeneity of the fluorescence emission of proteins by means of two-photon correlation spectroscopy have appeared for mutants of GFP, Coral Red protein (DsRed) (Heikal et al. 2000) and O-acetylserine sulfhydrylase (Chirico et al. 2001b). The FCS method has also been used recently to measure the very slow diffusion of receptor clusters in cultured cells (Srivastava & Petersen, 1998). Additionally, a two-colour image cross- correlation spectroscopy (ICCS) variant has been used for co-localization studies at the plasma membrane of influenza virus (Brown et al. 1999). A pioneering work on TPE ICS in cells appeared in 2000, where the possibility of TPE co-localizing and simultaneously exciting two different fluorophores has been exploited to perform image cross-correlation (Wiseman et al. 2000). Finally, a recent application of 2PE related to molecular diffusion consists of an interesting blend of imaging and un-caging properties of the phenomenon. Reported for the first time, is the experimental evidence of 2PE photolysis of a caged proton compound, 2-nitrobenzaldehyde (o-NBA), monitored using a new sensor system that utilizes fluorescent-labelled nanocapsules, i.e. a fluorescent nanostructured shell of micrometric size and nanometric thickness (Diaspro et al. 2003). The photo-labile compound undergoes one-photon absorption in the UV range (200–380 nm), and the mechanism that leads to proton release is based on the well-known 2-nitrobenzyl photochemistry, which has been used for many photoactivable-caged compounds. Because the use of UV excitation can cause biological damage, the multi-photon un-caging 2PE in biological microscopy 151

(a)(b)

Fig. 37. Two-photon excitation images of FITC-doped polyelectrolyte nanocapsules [(PAH/PSS)3, 3rd and 5th layer FITC-labelled]. Fluorescein emission was excited at a wavelength of 720 nm, in the presence of 2-nitrobenzaldehyde, before (a) and after (b) 2-nitrobenzaldehyde photolysis that was induced by high- energy scanning with multi-photon excitation at 720 nm. The decrease of ﬂuorescence intensity monitors the uncaging eﬀect (Diaspro et al. 2003). process is preferable. Using a femtosecond titanium:sapphire laser operating at 720 nm, with a pulse width of 200 fs at the sample, delivered through the 2PE microscope (described in Section 5), and a 1-min exposure time with high power (45–50 mW), an appreciable photolysis of 2-nitrobenzaldehyde was obtained (see Fig. 37). One of the relevant aspects in this work is the combined utilization of 2PE both for imaging and for an active role like that of the highly localized un-caging agent.

6.4 Signals from nonlinear interactions SHG is a second-order nonlinear optical process that is confined to regions that lacks a centre of symmetry. Interested readers can see the brilliant recent review by Campagnola & Loew (2003) about this important nonlinear technique. The origin of SHG is the dissipation of power in the sample due to a quadratic response of the microscopic currents to the electric field. It is a coherent process reminiscent of the single-photon Rayleigh scattering and, therefore, intrinsically different from 2PE fluorescence where a quantum absorption occurs, rapidly followed by emission of radiation. Since SHG is a nonlinear optical process, the microscopy application of this technique retains the 3D capabilities of 2PE. Several additional aspects make SHG microscopy very powerful. Since the excitation uses NIR wavelengths, this method is well suited for studying intact tissue as with 2PE. Moreover the SHG signals have well-defined polarizations, and SHG polarization anisotropy can be used to determine the absolute orientation and degree of organization of proteins in tissues, and to enhance the image contrast. Since SHG does not arise from absorption, in-plane photodamage is greatly reduced. In addition, 2PE fluorescence images can be collected in a separate data channel simultaneously with SHG. Despite these attractive properties, SHG has only recently been used for biological imaging applications (Campagnola et al. 1999; Moreaux et al. 2000; Zoumi et al. 2002; Diaspro et al. 2002c; Zipfel et al. 2003). A powerful advance is that obtained in coupling 2PE and SHG imaging on the very same detection optical path, involving different contrast mechanisms, to get 152 A. Diaspro, G. Chirico and M. Collini

Fig. 38. Colour-coded image of immunostained collagen fibres in the RAFT. The image is an overlay of two images obtained from the same sample site by using an SBG39 (322–654 nm) wide-pass emission filter and a 520/40 nm bandpass emission filter for 800 nm excitation, and P=60 mW. Collagen fibers are shown in cyan, and the spotted staining pattern of the antibody is shown in yellow (bar, 3 mm). (Adapted from Zoumi et al. 2002.)

complementary information regarding biological system structure and functioning. 2PE fluorescence is generally measured in epi-illuminating geometry but the forward propagating nature of SHG seemed to restrict SHG microscopy to the transmission detection mode. Zipfel and colleagues (2003a) achieved multi-colour nonlinear microscopy of living tissue using 2PE and 3PE intrinsic fluorescence combined with SHG by supermolecular structures producing images with the resolution and detail of standard histology without the use of exogenous stains. Imaging of intrinsic indicators within tissue, such as nicotinamide adenine dinucleotide, retinol, indoleamines, and collagen provided crucial physiological information. They demonstrated applications involving a range of intrinsic molecules and molecular assemblies that enable direct visualization of tissue morphology, cell metabolism, and disease states such as Alzheimer’s disease and cancer. In vivo SHG signals of intrinsic components were generated by scanning small regions of the specimen. Since collagen has been demonstrated to be a very effective ‘upconverter’ of light by SHG there is a growing number of applications in biomedical imaging. Different types of collagen can be distinguished at low perturbation and without specific sample preparations (Roth & Freund, 1979; Cox et al. 2002; Zoumi et al. 2002) Figure 38 illustrates a colour-coded image, which displays SHG signals in cyan originated from type I collagen and antibody fluorescence in yellow revealing that the fibres in the raft (membrane microdomains enriched in cholesterol and glycosphingolipids) correspond to collagen (Zoumi et al. 2002). The fact that SHG is collected in the forward direction, due to its physical inherent properties, hampered potential experiments especially in thick or in optical configurations where it is not possible to place forward detectors. Recently reflected SHG signals were collected from the Tromberg and Diaspro groups (Zoumi et al. 2002; Diaspro et al. 2002c) opening new application perspectives. Mohler and colleagues (2003) have recently shown how structural protein arrays 2PE in biological microscopy 153 consisting largely of collagen, myosin, and tubulin, and their associated proteins can be imaged in three dimensions with high contrast and resolution by laser-scanning SHG microscopy. In particular, structural protein arrays that are highly ordered and birefringent produce large SHG signals without the need for any exogenous labels. The authors, combining SHG with TPE GFP imaging were able to infer the molecular origin of the SHG contrast in Caenorhabditis elegans sarcomeres. From the Laboratoire de Neurophysiologie et Nouvelles Microscopies (CNRS, Paris, France), the combination of 2PE and SHG allowed a new outstanding possibility of fast-targeted control of the local inter-leaflet distribution of markers in biological membranes. In fact, Pons et al. (2002) demonstrated an interesting mechanism of localized photoinduced flip- flop of stilbazolium markers in model lipid bilayer membranes. The flip-flop mechanism and dynamics were determined by combined 2PE fluorescence and SHG microscopy. Upon illumination of labelled membranes with a femtosecond laser beam, two-photon absorption induced photoisomerization, with a significant increase in the cis marker population, whose flip-flop rate was determined to be at least 1000 times faster than that for trans markers. Other interesting applications of high-order optical generation have appeared recently. Third- harmonic generation can also be exploited to form images without the need of fluorescent markers (Mueller et al. 1998; Squier et al. 1998). Moreover surface-enhanced second-harmonic generated (S-SHG) signals have been detected from granular gold structures exhibiting local plasmon resonance (Anceau et al. 2003). Here, a TPE microscopy technique was used to perform high spatial resolution S-SHG imaging. Polarization measurements of local S-SHG responses revealed field anisotropy in the enhancement regions and, furthermore, proved the incoherent and strongly depolarized nature of the emission, which is attributed to ultra-fast fluctuations of the enhancement location.

7. Conclusions Considering its impact on basic and applied research, including new diagnostic methods, the development of 2PE and multi-photon excitation imaging may be viewed as a great revolution for a new era in optical microscopy. Since 2PE broke into the scientific arena, it has grown in an unpredictably fast way, and has generated and still produces a tremendous number of related techniques. New experiments can be designed and performed and comparing one- and two- photon experiments can perform a critical reading of past results. It is true that a few impediments exist to the understanding and full exploitation of 2PE. One of the major limitations in this direction is the difficulty of predicting or measuring two-photon absorption spectra of the fluorescent molecules used. A further extremely practical disadvantage of 2PE resides in the cost of appropriate laser sources. The expectation is that their use will probably increase as cheaper and more reliable laser sources are developed. Furthermore, one should consider that in-plane optical resolution for 2PE is lower than in the conventional excitation case even if the overall signal-to-background ratio is larger (Jonkman & Stelzer, 2001). Photobleaching and photodamage can be greater than in 1PE at least in the focal plane (Drummond et al. 2002) indicating that the technique is better suited for thick samples than thin ones within this context. A precise control of the influence of pulse duration on imaging efficiency and potential photodamage would require an accurate control of pulse width in the focal plane that is not easy to accomplish on-line in an intensive utilization of the 2PE microscope (Koester et al. 1999). Finally, due to high peak powers, 3PE or plasma generation can affect imaging of biological samples (Konig et al. 1999b; Koester et al. 1999), and in pigmented samples, 154 A. Diaspro, G. Chirico and M. Collini single-photon absorbers such as haemoglobin, melanin or chlorophyll can cause damage through heating (Tauer, 2002). Despite these potential drawbacks 2PE and related techniques can be considered as a mainstay of the modern biological research milieu and a bright perspective in optical microscopy. In fact, 2PE brings an intrinsic 3D resolution, the absence of background fluorescence, and the attractive possibility of exciting UV-excitable fluorescent molecules at IR wavelengths increasing sample penetration. In 2PE, the fluorescence emission occurs at a wavelength substantially shorter than the excitation wavelength, therefore reducing Rayleigh and Raman scattering contribution and increasing the signal-to-noise ratio. This fact makes single-molecule experiments, including spectroscopic ones, easier than in the conventional excitation case. On the nanotechnological side, 2PE architectures can be easily turned into active devices combining their 3D imaging ability with the possibility of performing destructive localized actions. Moreover, a further benefit in establishing a 2PE optical system is the flexibility in choosing the measurement modality favoured by the simplification of the optical design. As a matter of fact, a 2PE microscope offers a number and variety of measurement options without changing any optics or hardware. This means that one can really get multi-modal and multi-functional information within the same experiment from the very same sample. Finally, it should be remembered that photobleaching and natural fluorescence are strictly confined in the focal volume of event, and multiple excitations of fluorescent molecules can be more easily and efficiently accomplished than by using conventional excitation. For these reasons, 2PE is rapidly becoming an essential imaging system for thick samples and live cells, even within their own organs or tissues.

8. Acknowledgements

The authors are grateful to Alessandra Gliozzi, Giancarlo Baldini, Salvatore Cannistaro, Cristiana Ricci, Paolo Sapuppo and Enrico Gratton for believing in the 2PE project. A. D. is personally indebted to several scientists who provided helpful comments for the realization of the 2PE set-up, among them Enrico Gratton, David Piston, Tony Wilson, Colin Sheppard, Karsten Konig, Joseph Lakowicz, Weiming Yu, Steve Potter, Mary Dickinson, Martin Hoppe, Rolf Borlinghaus, Irmtraud Steinmez, Cesare Usai, Hans Gerritsen, Jeffrey Larson, Stan Schwartz, Peter So, Min Gu, Ammasi Perisamy, Fred Brakenhoff, Barry Masters, Michael Stanley, Osamu Nakamura, Stefan Hell, Christian Soeller, Marc Cannel. Special thanks are due to John Girkin for providing data on laser sources and important comments. All the users of the MPLSM (mplsm- [email protected]) and Confocal ([email protected]) mail- ing list are acknowledged. The authors are indebted to their co-workers at LAMBS and LABS, namely (in random order): Mario Faretta, Paolo Bianchini, Silke Krol, Fabio Cannone, Silvia Scaglione, Raffaella Magrassi, Laura D’Alfonso, Francesca Bertani, Mirko Corosu, Federico Federici, Sabrina Beretta, Alessandro Esposito, Cesare Usai, Paola Ramoino, Francesco Difato, Andrea Gerbi, Valentina Caorsi, Davide Mazza, Giuseppe Vicidomini, Mattia Pesce, Ilaria Testa, Marc Schneider, Munish Chanana, Francesca Cella, Federica Morotti, Emiliano Ronzitti. All the authors are indebted to their respective families. Special thanks are due to Brad Amos, Davide Neuhaus and Andrea Cranton. A. D. dedicates this work to the memory of Osamu Nakamura, and all the authors to Gianfranco Menestrina. The TPE project is performed under the auspices and grants of the National Institute for the Physics of Matter, Italy. Main sources of grants are the Fondazione San Paolo Torino, RTN Nanocapsule EU, IFOM and MIUR-FIRB grants. 2PE in biological microscopy 155

9. References

ABBOTTO, A., BEVERINA, L., CHIRICO, G., FACCHETTI, A., BASTIAENS,P.I.&HELL, S. W. (eds.) (2004). Recent ad- FERRUTI, P., GILBERTI,M.&PAGANI, G. A. (2003). vances in light microscopy. Journal of Structural Biology Crosslinked poly(amido-amine)s as superior matrices 147, 1–89. for chemical incorporation of highly efficient BEAUREPAIRE, E., OHEIM,M.&MERTZ, J. (2001). Ultra- organic nonlinear optical dyes. Macromolecular Rapid deep two-photon fluorescence excitation in turbid Communication 24, 397–402. media. Optics Communications 188, 25–29. AKERMAN, M. E., CHAN, W. C., LAAKONEN, P., BHATIA, BELTRAME, F., BIANCO, B., CASTELLARO,G.&DIASPRO,A. S. N. & RUOSLAHTI, E. (2002). Nanocrystal targeting in (1985). Fluorescence, absorption, phase-contrast, holo- vivo. Proceedings of the National Academy of Sciences USA 99, graphic and acoustical cytometries of living cells. 12617–12621. In Interactions Between Electromagnetic Fields and Cells (eds. A. Chiabrera, C. Nicolini & H. P. Schwan), AGARD, D. A. (1984). Optical sectioning microscopy: pp. 483–498. NATO ASI Series, vol. 97. New York and cellular architecture in three dimensions. Annual Review London: Plenum Press Publishing. of Biophysics 13, 191–219. BENEDETTI, P. (1998). From the histophotometer to the ALBOTA, M., BELJONNE, D., BREDAS, J. L., EHRLICH, J. E., confocal microscope: the evolution of analytical FU, J. Y., HEIKAL, A. A., HESS, S. E., KOGEJ, T., LEVIN, microscopy. European Journal of Histochemistry 42, 11–17. M. D., MARDER, S. R., MCCORD-MAUGHON, D., PERRY, BENHAM, G. S. (2002). Practical aspects of objective lens J. W., ROCKEL, H., RUMI, M., SUBRAMANIAM, G., WEBB, selection for confocal and multiphoton digital imaging W. W., WU,X.L.&XU, C. (1998). Design of organic techniques. In Cell Biological Applications of Confocal molecules with large two-photon absorption cross Microscopy, 2nd edn (ed. B. Matsumoto), pp. 245–299. sections. Science 281, 16153–16156. Methods in Cell Biology series, vol. 70. San Diego, AMOS, B. (2000). Lessons from the history of light mi- London: Academic Press. croscopy. Nature Cell Biology 2, E151–E152. BENHAM,G.S.&SCHWARTZ, S. (2002). Suitable micro- AMOS, W. B. (1998). Multiphoton imaging. In Current scope objectives for multiphoton digital imaging. In Protocols in Cytometry (eds. Z. Darzynkiewicz et al.), Multiphoton Microscopy in the Biomedical Sciences II (eds. A. pp. 2.9.1–2.9.11. New York: John Wiley & Sons. Periasamy & P. T. C. So). Proceedings of SPIE 4620, AMOS,W.B.&WHITE, J. G. (2003). How the confocal 36–47. laser scanning microscope entered biological research. BENNINGER, R. K. P., ONFELT, B., NEIL, M. A. A., DAVIS, Biology of the Cell 95, 335–342. D. M. & FRENCH, P. M. W. (2005). Fluorescence ima- ANCEAU, C., BRASSELET, S., ZYSS,J.&GADENNE, P. (2003). ging of two-photon linear dichroism: cholesterol Local second-harmonic generation enhancement on depletion disrupts molecular orientation in cell mem- gold nanostructures probed by two-photon microscopy. branes. Biophysical Journal 88, 609–622. Optics Letters 28, 713–715. BERG, R. H. (2004). Evaluation of spectral imaging for ANDREWS, D. L. (1985). A simple statistical treatment of plant cell analysis. Journal of Microscopy. 214, 174–181. multiphoton absorption. American Journal of Physics 53, BERLAND, K. (2004). Detection of specific DNA seuqences 1001–1002. using dual-color two-photon fluorescence correlation spectroscopy. Journal of Biotechnology 108, 127–136. ARNULPHI, C., SANCHEZ, S. A., TRICERRI, M. A., BERLAND,K.&SHEN, G. (2003). Excitation saturation GRATTON,E.&JONAS, A. (2005). Interaction of in two-photon fluorescence correlation spectroscopy. human apolipoprotein A-I with model membranes Applied Optics 42, 5566–5576. exhibiting lipid domains. Biophysical Journal 89, 285–295. BERLAND, K. M., SO, P. T. C., CHEN, Y., MANTULIN,W.W. ARAGON,S.R.&PECORA, R. (1975). Fluorescence corre- &GRATTON, E. (1996). Scanning two-photon fluctu- lation spectroscopy and Brownian rotational diffusion. ation correlation spectroscopy: particle counting Biopolymers 14, 119–137. measurements for detection of molecular aggregation. ARNDT-JOVIN, D. J., NICOUD, R. M., KAUFMANN,J.&JOVIN, Biophysical Journal 71, 410–420. T. M. (1985). Fluorescence digital-imaging microscopy BERLAND, K. M., SO,P.T.C.&GRATTON, E. (1995). in cell biology. Science 230, 13330–13335. Two-photon fluorescence correlation spectroscopy: AXE, J. D. (1964). Two-photon processes in complex method and application to the intracellular environ- atoms. Physics Review 136, 42–45. ment. Biophysical Journal 68, 694–701. BALDINI, G., CANNONE,F.&CHIRICO, G. (2005). Pre- BERNS, M. W. (1976). A possible two-photon effect in vitro unfolding resonant oscillations of single green fluor- using a focused laser beam. Biophysical Journal 16, escent protein molecules. Science 309, 1096–1100. 973–977. BARAD, Y., EIZENBER, H., HOROWITZ,M.&SILBERBERG,Y. BEWERSDORF,J.&HELL, S. W. (1998). Picosecond pulsed (1997). Nonlinear scanning laser microscopy by third two-photon imaging with repetition rates 200 MHz and harmonic generation. Applied Physics Letters 70, 922–924. 400 MHz. Journal of Microscopy 191, 28–38. 156 A. Diaspro, G. Chirico and M. Collini

BHAWALKAR, J. D., KUMAR, N. D., ZHAO,C.F.&PRASAD, neuroblastoma nuclei shown by confocal scanning laser P. N. (1997). Two-photon photodynamic therapy. microscopy. Nature 317, 748–749. Journal of Clinical Laser Medicine and Surgery 15, 201–204. BROCK, R., HINK,M.A.&JOVIN, T. M. (1998). BIANCO,B.&DIASPRO, A. (1989). Analysis of the three Fluorescence correlation microscopy of cells in the dimensional cell imaging obtained with optical micro- presence of autofluorescence. Biophysical Journal 75, scopy techniques based on defocusing. Cell Biophysics 2547–2557. 15, 189–200. BROWN, C. M., ROTH, M. G., HENIS,Y.I.&PETERSEN, BIRGE, R. R. (1986). Two-photon spectroscopy of protein- N. O. (1999). An internalization-component influenza bound fluorophores. Accounts of Chemical Research 19, Hemagglutinin mutant causes the redistribution of AP- 138–146. 2 to existing coated pits and is colocalized with AP-2 BIRKS, J. B. (1970). Photophysics of Aromatic Molecules. in clathrin free clusters. Biochemistry 38, 15166–15173. London: Wiley Interscience. BUIST, A. H., MULLER, M., SQUIER,J.&BRAKENHOFF,G.J. BLAB, G. A., LOMMERSE, P. H. M., COGNET, L., HARMS, (1998). Real time two-photon absorption microscopy G. S. & SCHMIDT, T. (2001). Two-photon excitation using multi point excitation. Journal of Microscopy 192, action cross-sections of the autofluorescent proteins. 217–226. Chemical Physics Letters 350, 71–77. CAHALAN, M. D., PARKER, I., WEI,S.H.&MILLER,M.J. (2002). Two-photon tissue imaging: seeing the immune BLINOVA, K., COMBS, C., KELLMAN,P.&BALABAN,R.S. (2004). Fluctuation analysis of mitochondrial NADH system in a fresh light. Nat Rev Immunol. 2, 872–880. fluorescence signals in confocal and two-photon CAHALAN, M. D., PARKER, I., WEI,S.H.&MILLER,M.J. microscopy images of living cardiac myocytes. Journal (2003). Real-time imaging of lymphocytes in vivo. Current Opinion in Immunology of Microscopy (Oxford) 213, 70–75. 15, 372–377. CALLIS, P. R. (1997). Two-photon-induced fluorescence. BOCCACCI,P.&BERTERO, M. (2001). Image restoration Annual Review of Physical Chemistry 48, 271–297. methods: basics and algorithms. In Confocal and Two- CALLOWAY, C. B. (1999). A confocal microscope with photon Microscopy: Foundations, Applications and Advances spectrophotometric detection. Microscopy and Micro- (ed. A. Diaspro), pp. 253–270. New York: Wiley-Liss, analysis 5 (Suppl. 2 Proceedings), 460–461. Inc. CAMPAGNOLA, P., MEI-DE WEI,LEWIS,A.&LOEW,L. BONETTO, P., BOCCACCI, P., SCARITO, M., DAVOLIO, M., (1999). High-resolution nonlinear optical imaging of EPIFANI, M., VICIDOMINI, G., TACCHETTI, C., RAMOINO, live cells by second harmonic generation. Biophysical P., USAI,C.&DIASPRO, A. (2004). Three-dimensional Journal 77, 3341–3351. microscopy migrates to the web with ‘powerup your CAMPAGNOLA,P.J.&LOEW, L. M. (2003). Second- microscope’. Microscopy Research and Technique 64, 196– harmonic imaging microscopy for visualizing bio- 203. molecular arrays in cells, tissues and organisms. Nature BONNET, G., KRICHEVSKY,O.&LIBCHABER, A. (1998). Biotechnology 21, 1356–1360. Kinetics of conformational fluctuations in DNA CAMPAGNOLA, P. J., MILLARD, A. C., TERASAKI, M., HOPPE, hairpin-loops. Proceedings of the National Academy of Sciences P. E., MALONE,C.J.&MOHLER, W. A. (2002). Three- USA 95, 8602–8606. dimensional high-resolution second-harmonic gener- BOOTH,M.J.&HELL, S. W. (1998). Continuous wave ation imaging of endogenous structural proteins in excitation two-photon fluorescence microscopy biological tissues. Biophysical Journal 81, 493–508. exemplified with the 647-nm ArKr laser line. Journal of CANNONE, F., BOLOGNA, S., CAMPANINI, B., DIASPRO, A., Microscopy 190, 298–304. BETTATI, S., MOZZARELLI,A.&CHIRICO, G. (2005). BOREJDO, J. (1979). Motion of myosin fragments during Tracking unfolding and refolding of single GFPmut2 actin-activated ATPase: fluorescence correlation spec- molecules. Biophysical Journal 89, 2033–2045. troscopy study. Biopolymers 18, 2807–2820. CANNONE, F., CHIRICO, G., BALDINI,G.&DIASPRO,A. BORN,M.&WOLF, E. (1993). Principles of Optics, 6th edn. (2003b). Measurement of the laser pulse width on the Oxford: Pergamon Press. microscope objective plane by modulated autocorr- BRAKENHOFF, G. J., BLOM,P.&BARENDS, P. (1979). elation method. Journal of Microscopy 210, 149–157. Confocal scanning light microscopy with high aperture CANNONE, F., CHIRICO,G.&DIASPRO, A. (2003a). Two- immersion lenses. Journal of Microscopy 117, 219–232. photon interactions at single fluorescent molecule level. BRAKENHOFF, G. J., MULLER,M.&GHAUHARALI,R.I. Journal of Biomedical Optics 8, 391–395. (1996). Analysis of efficiency of two-photon versus CARLSSON, K., WALLEŃ,P.&BRODIN, L. (1989). Three single-photon absorption of fluorescence generation dimensional imaging of neurons by confocal micro- in biological objects. Journal of Microscopy 183, 140–144. scopy. Journal of Microscopy 155, 15–26. BRAKENHOFF, G. J., VAN DER VOORT, H. T. M., VAN CENTONZE, V. E. (2002). Introduction to multiphoton SPRONSEN, E. A., LINNEMANS,W.A.M.&NANNINGA, excitation imaging for the biological sciences. In Cell N. (1985). Three-dimensional chromatin distribution in Biological Applications of Confocal microscopy, 2nd edn 2PE in biological microscopy 157

(ed. B. Matsumoto), pp. 129–148. Methods in Cell controllable elements in bioelectronics. Applied Physics Biology series, vol. 70. San Diego, London: Academic Letters 79, 3353–3355. Press. COURJAUD, A., DEGUIL,N.&SALIN, F. (2001). High-power CENTONZE,V.E.&WHITE, J. G. (1998). Multiphoton diode-pumped Yb:KGW ultrafast laser. Advanced Solid excitation provides optical sections from deeper within State Lasers 68, 161–163. scattering specimens than confocal imaging. Biophysical COX, G., KABLE, E., JONES, A., FRASER, I., MANCONI,F.& Journal 75, 2015–2024. GORRELL, M. D. (2003). 3-Dimensional imaging of CHALFIE,M.&KAIN, S. (1998). Green Fluorescent Protein. collagen using second harmonic generation. Journal of New York: Wiley Liss. Structural Biology 141, 53–62. CHAN, W. C., MAXWELL, D. J., GAO, X., BAILEY, R. E., COX, G., MANCONI,F.&KABLE, E. (2002). Second har- HAN,M.&NIE, S. (2002). Luminescent quantum dots monic imaging of collagen in mammalian tissue. In for multiplexed biological detection and imaging. Multiphoton Microscopy in the Biomedical Sciences, II (eds. A. Current Opinion in Biotechnology 13, 40–46. Periasamy & P. T. C. So). Proceedings of SPIE 4620, CHEN, Y., MUELLER, J. D., RUAN,Q.&GRATTON,E. 148–156. (2002). Molecular brightness characterization of egfp COX,G.&SHEPPARD, C. J. R. (2004). Practical limits of in vivo by fluorescence fluctuation spectroscopy. resolution in confocal and non-linear microscopy. Biophysical Journal 82, 133–144. Microscopy Research and Technique 63, 18–22. CURLEY, P. F., FERGUSON, A. I., WHITE,J.G.&AMOS, CHEN, Y., MULLER, J. D., SO,P.T.C.&GRATTON,E. (1999). The photon counting histogram in fluorescence W. B. (1992). Application of a femtosecond mode- fluctuation spectroscopy. Biophysical Journal 77, 553– locked Ti-sapphire laser to the field of laser-scanning Optical and Quantum Electronics 567. microscopy. 24, 851–859. D’ALFONSO, L., CHIRICO, G., COLLINI, M., BALDINI, G., CHIRICO, G., BETTATI, S., MOZZARELLI, A., CHEN, Y., DIASPRO, A., RAMOINO, P., ABBOTTO, A., BEVERINA,L.& MU¨ LLER,J.D.&GRATTON, E. (2001b). Molecular PAGANI, G. A. (2003). New two-photon excitation heterogeneity of O-acetylserine sulfhydrylase by two- chromophores for cellular imaging. Proceedings of SPIE photon excited fluorescence fluctuation spectroscopy. 5139, 27–39. Biophysical Journal 80, 1973–1985. DAS, T., PAYER, B., CAYOUETTE,M.&HARRIS,W.A. CHIRICO, G., CANNONE, F., BALDINI,G.&DIASPRO,A. (2003). In vivo time-lapse imaging of cell divisions (2003b). A two-photon thermal bleaching of single during neurogenesis in the developing zebrafish retina. fluorescent molecules. Biophysical Journal 84, 588–598. Neuron 37, 597–609. CHIRICO, G., CANNONE, F., BERETTA,S.&DIASPRO,A. DEGUIL, N., MOTTAY, E., SALIN, F., LEGROS,P.& (2001a). Single molecule studies by means of the two- CHOQUET, D. (2004). Novel diode-pumped infrared photon fluorescence distribution. Microscopy Research and tunable laser system for multi-photon microscopy. Technique 55, 359–364. Microscopy Research and Technique 63, 23–26. CHIRICO, G., CANNONE,F.&DIASPRO, A. (2003a). Single DENK, W. (1996). Two-photon excitation in functional molecule photodynamics by means of one- and two- biological imaging. Journal of Biomedical Optics 1, photon approach. Journal of Physics (D). Applied Physics 296–304. 36, 1682–1688. DENK, W., DELANEY, K. R., GELPERIN, A., KLEINFELD, D., CHIRICO, G., CANNONE, F., DIASPRO, A., BOLOGNA, S., STROWBRIDGE, D. W., TANK,D.W.&YUSTE, R. (1994). PELLEGRINI, V., NIFOSI,R.&BELTRAM, F. (2004). Multi- Anatomical and functional imaging of neurons using photon switching dynamics of single green fluorescent 2-photon laser scanning microscopy. Journal of proteins. Physical Review (E ). Statistical, Nonlinear and Soft Neuroscience Methods 54, 151–162. Matter Physics 70 ( 3 pt 1), 030901. DENK, W., PISTON,D.&WEBB, W. W. (1995). Two- CHIRICO, G., OLIVINI,F.&BERETTA, S. (2000). photon molecular excitation in laser scanning mi- Fluorescence excitation volume in two-photon micro- croscopy. In Handbook of Confocal Microscopy (ed. J. B. scopy by autocorrelation spectroscopy and photon Pawley), pp. 445–457. New York: Plenum Press. counting histogram. Applied Spectroscopy 54, 1084–1090. DENK, W., STRICKLER,J.H.&WEBB, W. W. (1990). Two- CHRISTIE, R. H., BACKSAI, B. J., ZIPFEL, W. R., WILLIAMS, photon laser scanning fluorescence microscopy. Science R. M., KAJDASZ, S. T., WEBB,W.W.&HYMAN,B.T. 248, 73–76. (2001). Growth arrest of individual senile plaques in DENK,W.&SVOBODA, K. (1997). Photon upmanship: a model of Alzheimer’s disease observed by in vivo why multiphoton imaging is more than a gimmick. multiphoton microscopy. Journal of Neuroscience 21, Neuron 18, 351–357. 858–864. DESPA, S., KOCKSKAMPER, J., BLATTER,L.A.&BERS,D.M. CINELLI, R. A. G., PELLEGRINI, V., FERRARI, A., FARACI, P., (2004). Na/K pump-induced [Na](i) gradients in rat NIFOSI, R., TYAGI, M., GIACCA,M.&BELTRAM,F. ventricular myocytes measured with two-photon (2001). Green fluorescent proteins as optically microscopy. Biophysical Journal 87, 1360–1368. 158 A. Diaspro, G. Chirico and M. Collini

DIASPRO, A. (1998). Two-photon fluorescence excitation. excitation fluorescence microscopy. In Functional A new potential perspective in flow cytometry. Minerva Imaging (ed. D. Farkas). Proceedings of SPIE 4622, 47–53. Biotecnologica 11, 87–92. DIASPRO, A., KROL, S., CAVALLERI, O., SILVANO,D.& DIASPRO, A. (guest editor). (1999a). Two-photon micro- GLIOZZI, A. (2002d). Microscopical characterization of scopy. Microscopy Research and Technique 47, 163–212. nanocapsules templated on ionic crystals and biological DIASPRO, A. (guest editor). (1999b). Two-photon exci- cells toward biomedical applications. IEEE Transactions tation microscopy. IEEE Engineering in Medicine and on Nanobioscience 1, 110–115. Biology 18, 16–99. DIASPRO,A.&ROBELLO, M. (2000). Two-photon exci- DIASPRO, A. (ed.) (2001a). Confocal and Two-photon tation of fluorescence for three-dimensional optical Microscopy: Foundations, Applications, and Advances. New imaging of biological structures. Journal of Photochemistry York: Wiley-Liss. and Photobiology (B) 55, 1–8. DIASPRO, A. (2001b). Building a two-photon microscope DIASPRO, A., SARTORE,M.&NICOLINI, C. (1990). Three- using a laser scanning confocal architecture. In Methods dimensional representation of biostructures imaged in Cellular Imaging (ed. A. Periasamy), pp. 162–179. New with an optical microscope: I. Digital optical sectioning. York: Oxford University Press. Image and Vision Computing 8, 130–141. DIASPRO, A. (2004a). Rapid dissemination of two-photon DIASPRO, A., SILVANO, D., KROL, S., CAVALLERI,O.& excitation microscopy prompts new applications. GLIOZZI, A. (2002a). Single living cell encapsulation Microscopy Research and Technique 63, 1–2. in nano-organized polyelectrolyte shells. Langmuir 18, DIASPRO, A. (2004b). Nanocapsules: a European 5047–5050. Community interdisciplinary network in the nanobio- DIASPRO, A., VICIDOMINI, G., BOCCACCI, P., BONETTO, P., sciences. IEEE Transactions on Nanobioscience 3, 1–2. BERTERO, M., SCARITO, M., DAVOLIO,M.&EPIFANI,M. DIASPRO, A. (2004c). Introduction: image processing in (2002b). ‘Power-up your microscope’ (http:// optical microscopy. Microscopy Research and Technique 64, www.lambs.it). 87–88. DICKINSON, M. E., BEARMAN, G., TILLE, S., LANSFORD,R. DIASPRO, A., ANNUNZIATA, S., RAIMONDO,M.&ROBELLO, &FRASER, S. E. (2001). Multi-spectral imaging and M. (1999a). Three-dimensional optical behaviour of linear unmixing adds a whole new dimension to laser a confocal microscope with single illumination and scanning fluorescence microscopy. BioTechniques 31, detection pinhole through imaging of subresolution 1272–1279. beads. Microscopy Research and Technique 45, 130–131. DICKINSON, M. E., SIMBURGER, E., ZIMMERMANN, B., DIASPRO, A., ANNUNZIATA,S.&ROBELLO, M. (2000). WATERS,C.W.&FRASER, S. E. (2003). Multiphoton Single-pinhole confocal imaging of sub-resolution excitation spectra in biological samples. Journal of sparse objects using experimental point spread function Biomedical Optics 8, 329–338. and image restoration. Microscopy Research and Technique DICKSON, L. D. (1970). Characteristics of a propagating 51, 464–468. gaussian beam. Applied Optics 9, 1854–1861. DIASPRO, A., BELTRAME, F., FATO, M., PALMERI,A.& DIFATO, F., MAZZONE, F., SCAGLIONE, S., FATO, M., RAMOINO, P. (1997). Studies on the structure of sperm BELTRAME, F., KUBINOVA, L., JANACECK, J., RAMOINO,P. heads of Eledone cirrhosa by means of CLSM linked &DIASPRO, A. (2004). Improvement in volume esti- to bioimage-oriented devices. Microscopy Research and mation from confocal sections after image deconvolu- Technique 36, 159–164. tion. Microscopy Research and Technique 64, 151–155. DIASPRO, A., CHIRICO, G., FEDERICI, F., CANNONE,F.& DITTRICH,P.S.&SCHWILLE, P. (2002). Spatial two- BERETTA, S. (2001). Two-photon microscopy and photon fluorescence cross-correlation spectroscopy spectroscopy based on a compact confocal scanning for controlling molecular transport in microfluidic head. Journal of Biomedical Optics 6, 300–310. structures. Analytical Chemistry 74, 4472–4479. DIASPRO, A., COROSU,M.&RAMOINO, P. (1999b). DRUMMOND, D. R., CARTER,N.&CROSS, R. A. (2002). Adapting a compact confocal microscope system to a Multiphoton versus confocal high resolution z- two-photon excitation fluorescence imaging architec- sectioning of enhanced green fluorescent microtubules: ture. Microscopy Research and Technique 47, 196–205. increased multiphoton photobleaching within the focal DIASPRO, A., FEDERICI, F., VIAPPIANI, C., KROL, S., plane can be compensated using a Pockels cell and dual PISCIOTTA, M., CHIRICO, G., CANNONE,F.&GLIOZZI,A. widefield detectors. Journal of Microscopy (Oxford ) 206, (2003). Two-photon photolysis of 2-nitrobenzaldehyde 73–76. monitored by fluorescent-labeled nanocapsules. Journal DUBERTRET, B., SKOURIDES, P., NORRIS, D. J., NOIREAUX, V., of Physical Chemistry (B) 107, 11008–11012. BRIVANLOU,A.H.&LIBCHABER, A. (2002). In vivo DIASPRO, A., FRONTE, P., RAIMONDO, M., FATO, M., DE imaging of quantum dots encapsulated in phospholipid LEO, G., BELTRAME, F., CANNONE, F., CHIRICO,G.& micelles. Science 298, 1759–1762. RAMOINO, P. (2002c). Functional imaging of living EGGELING, C., VOLKMER,A.&SEIDEL, C. A. M. (2005). paramecium by means of confocal and two-photon Molecular photobleaching kinetics of rhodamine 6G by 2PE in biological microscopy 159

one- and two-photon induced confocal fluorescence GAO, X., CUI, Y., LEVENSON, R. M., CHUNG,L.W.K.& microscopy. ChemPhysChem 6, 791–804. NIE, S. (2004). In vivo cancer targeting and imaging EHRENBERG,M.&RIGLER, R. (1974). Rotational Brownian with semiconductor quantum dots. Nature Biotechnology motion and fluorescence intensity fluctuations. Chemical 22, 969–976. Physics 4, 390–401. GAUDERON, R., LUKINS,R.B.&SHEPPARD, C. J. R. (1999). EIGEN,M.&RIGLER, R. (1994). Sorting single molecules: Effects of a confocal pinhole in two-photon micro- application to diagnostics and evolutionary biotech- scopy. Microscopy Research and Technique 47, 210–214. nology. Proceedings of the National Academy of Sciences USA GAUS, K., GRATTON, E., KABLE, E. P. W., JONES, A. S., 91, 5740–5747. GELISSEN, I., KRITHARIDES,L.&JESSUP, W. (2003). ERRINGTON, R. J., AMEER-BEG, S. M., VOJNOVIC, B., Visualizing lipid structure and raft domains in living PATTERSON, L. H., ZLOH,M.&SMITH, P. J. (2005). cells with two-photon microscopy. Proceedings of the Advanced microscopy solutions for monitoring the National Academy of Sciences USA 100, 15554–15559. kinetics and dynamics of drug-DNA targeting in living GIRKIN, J. M. (2003). Optical physics enables advances in cells. Advances Drug Delivery Reviews 57, 153–167. multiphoton imaging. Journal of Physics (D): Applied ESPOSITO, A., FEDERICI, F., USAI, C., CANNONE, F., Physics 36, R250–R258. HIRICO OLLINI IASPRO C , G., C ,M.&D , A. (2004). Notes GIRKIN,J.M.&WOKOSIN, D. (2001a). Practical multi- on theory and experimental conditions behind two- photon microscopy. In Confocal and Two-photon photon excitation microscopy. Microscopy Research and Microscopy: Foundations, Applications and Advances (ed. A. Technique 63, 12–17. Diaspro), pp. 207–236. New York: Wiley-Liss, Inc. FAHEY, P. F., BARAK, L. S., ELSON, E. L., KOPPEL, D. E., GIRKIN,J.M.&WOKOSIN, D. (2001b). Novel compact WOLF,D.E.&WEBB, W. W. (1977). Lateral diffusion sources for multiphoton microscopy. Progress in Bio- in planar lipid bilayers. Science 195, 305–306. medical Optics and Imaging 2, 186–191. FAN, G. Y., FUJISAKI, H., MIYAWAKI, A., TSAY, R.-K. & GO¨ PPERT-MAYER, M. (1931). U¨ ber Elementarakte mit zwei TSIEN, R. Y. (1999). Video-rate scanning two-photon Quantenspru¨ngen. Annals of Physics 9, 273–295. excitation fluorescence microscopy and ratio imaging GOSCH, M., BLOM, H., HOLM, J., HEINO,T.&RIGLER,R. with cameleons. Biophysical Journal 76, 2412–2420. (2000). Hydrodynamic flow profiling in microchannel FARKAS, D. L. (2001). Spectral microscopy for quantitative structures by single molecule fluorescence correlation cell and tissue imaging. In Methods in Cellular Imaging spectroscopy. Analytical Chemistry 72, 3260–3265. (ed. A. Periasamy), pp. 345–363. New York: Oxford GOSNELL,T.R.&TAYLOR, A. J. (eds.) (1991). Selected Papers University Press. on Ultrafast Laser Technology. SPIE Milestone series. FEIJO,J.A.&MORENO, N. (2004). Imaging plant cells by Bellingham, USA: SPIE Press. two-photon excitation. Protoplasma 223, 1–32. GRATTON,E.&VAN DE VEN, M. J. (1995). Laser sources FERGUSON, A. (2003). Special issue on biophotonics. for confocal microscopy. In Handbook of Confocal Journal of Physics (D ): Applied Physics 36,1. Microscopy (ed. J. B. Pawley), pp. 69–97. New York: FEYNMAN, R. P. (1985). QED: The Strange Theory of Light and Matter. Princeton: Princeton University Press. Plenum Press. GRATTON, E., BARRY, N. P., BERETTA,S.&CELLI,A. FISHER, W. G., WATCHER, E. A., ARMAS,M.&SEATON,C. (1997). Titanium:sapphire laser as an excitation source (2001). Multiphoton fluorescence microscopy. Methods in two-photon spectroscopy. Applied Spectroscopy 51, 25, 103–110. 218–226. GRYCZYNSKI, I., MALAK,H.&LAKOWICZ, J. R. (1996). FRANKEN, P. A., HILL, A. E., PETERS,C.W.&WEINREICH, Multiphoton excitation of the DNA stains DAPI and G. (1961). Generation of optical harmonics. Physical Hoechst. Bioimaging 4, 138–148. Review Letters 7, 118–119. GU,M.&SHEPPARD, C. J. R. (1995). Comparison of three- FRENCH, T., SO, P. T. C., WEAVER, D. J., COELHO-SAMPAIO, dimensional imaging properties between two-photon T. & GRATTON, E. (1997). Two-photon fluorescence and single-photon fluorescence microscopy. Journal lifetime imaging microscopy of macrophage-mediated of Microscopy 177, 128–137. antigen processing. Journal of Microscopy 185, 339–353. GULOT, E., GEORGES, P., BRUN, A., FONTAINE-AUPART, FRIEDRICH, D. M. (1982). Two-photon molecular spectro- M. P., BELLON-FONTAINE,M.N.&BRIANDET, R. (2002). scopy. Journal of Chemical Education 59, 472–483. Heterogeneity of diffusion inside microbial biofilms FRIEDRICH,D.M.&MCCLAIN, W. M. (1980). Two-photon determined by fluorescence correlation spectroscopy molecular electronic spectroscopy. Annual Review of under two-photon excitation. Photochemistry and Physical Chemistry 31, 559–577. Photobiology 75, 570–578. GANNAWAY,J.N.&SHEPPARD, C. J. R. (1978). Second HANNINEN,P.E.&HELL, S. W. (1994). Femtosecond harmonic imaging in the scanning optical microscope. pulse broadening in the focal region of a two-photon Optical and Quantum Electronics 10, 435–439. fluorescence microscope. Bioimaging 2, 117–121. 160 A. Diaspro, G. Chirico and M. Collini

HANNINEN, P. E., SCHRADER, M., SOINI,E.&HELL,S. (1998). Two-photon near- and far-field fluorescence (1995). Two-photon excitation fluorescence micro- microscopy with continuous-wave excitation. Optics scopy using a semiconductor laser. Bioimaging 3, 70–75. Letters 23, 1238–1240. HANNINEN, P. E., SOINI,E.&HELL, S. W. (1994). HELLWARTH,R.&CHISTENSEN, P. (1974). Nonlinear Continous wave excitation two-photon fluorescence optical microscopic examination of structures in poly- microscopy. Journal of Microscopy 176, 222–225. crystalline ZnSe. Optics Commications 12, 318–322. HARAGUCHI, T., SHIMI, T., KOUJIN, T., HASHIGUCHI,N.& HELMCHEN,F.&DENK, W. (2002). New developments in HIRAOKA, Y. (2002). Spectral imaging fluorescence multiphoton microscopy. Current Opinion in Neurobiology microscopy. Genes Cells 7, 881–887. 12, 593–601. HARMS, G. S., COGNET, L., LOMMERSE, P. H. M., BLAB, HINES,M.A.&GUYOT-SIONNEST, P. (1996). Synthesis and G. A. & SCHMIDT, T. (2001). Autofluorescent proteins characterization of strongly luminescing ZnS-Capped in single-molecule research: applications to live cell CdSe nanocrystals. Journal of Physical Chemistry 100, imaging microscopy. Biophysical Journal 80, 2396–2408. 468–471. HAUGHLAND, P. R. (2002). Handbook of Fluorescent Probes and HOPT,A.&NEHER, E. (1999). Highly non-linear photo- Research Chemicals. Eugene, OR: Edn Molecular Probes. damage in two-photon fluorescence microscopy. HAUPTS, U., MAITI, S., SCHWILLE,P.&WEBB, W. W. (1998). Biophysical Journal 80, 2029–2036. Dynamics of fluorescence fluctuations in green fluor- ICENOGLE,R.D.&ELSON, E. L. (1983). Fluorescence escent protein observed by fluorescence correlation correlation spectroscopy and photobleaching recovery spectroscopy. Proceedings of the National Academy of Sciences of multiple binding reactions. II. FPR and FCS USA 95, 13573–13578. measurements at low and high DNA concentrations. HAUSTEIN,E.&SCHWILLE, P. (2004). Single-molecule Biopolymers 22, 1949–1966. spectroscopic methods. Current Opinion in Structural IMANISHI, Y., BATTEN, M. L., PISTON, D. W., BAEHR,W.& Biology 14, 531–540. PALCZEWSKI, K. (2004). Noninvasive two-photon HEIKAL, A. A., HESS, S. T., BAIRD, G. S., TSIEN,R.Y.& imaging reveals retinyl ester storage structures in the WEBB, W. W. (2000). Molecular spectroscopy and eye. Journal of Cell Biology 164, 373–383. dynamics of intrinsically fluorescent proteins: coral red ISOGAI, S., LAWSON, N. D., TORREALDAY, S., HORIGUCHI,M. (dsRed) and yellow (Citrine). Proceedings of the National &WEINSTEIN, B. (2003). Angiogenic network formation Academy of Sciences USA 97, 11996–12001. in the developing vertebrate trunk. Development 130, HEIKAL, A. A., HESS,S.T.&WEBB, W. W. (2001). 5281–5290. Multiphoton molecular spectroscopy and excited-state JACOBS, M. D., DONALDSON, P. J., CANNELL,M.B.& dynamics of enhanced green fluorescent protein SOELLER, C. (2003). Resolving morphology and anti- (EGFP): acid-base specificity. Chemical Physics 274, body labeling over large distances in tissue sections. 37–55. Microscopy Research and Technique 62, 83–91. HEINZE, K. G., KOLTERMANN,A.&SCHWILLE, P. (2000). JAHNZ,M.&SCHWILLE, P. (2005). An ultrasensitive site- Simultaneous two-photon excitation of distinct labels specific DNA recombination assay based on dual-color for dual-color fluorescence cross-correlation analysis. fluorescence cross-correlation spectroscopy. Nucleic Proceedings of the National Academy of Sciences USA 97, Acid Research 33, e60. 10377–10382 [Erratum in: Proceedings of the National JAISWAL,J.K.&SIMON, S. (2004). Potentials and pitfalls Academy of Sciences USA 98, 2001, 777]. of fluorescent quantum dots for biological imaging. HEINZE, K. G., JAHNZ,M.&SCHWILLE, P. (2004). Triple- Trends in Cell Biology 14, 497–504. color coincidence analysis: one step further in following JONKMAN,J.&STELZER, E. (2001). Resolution and contrast higher order molecular complex formation. Biophysical in confocal and two-photon microscopy. In Confocal Journal 86, 506–516. and Two-photon Microscopy: Foundations, Applications and HELL, S. W. (1996). Nonlinear optical microscopy. Bio- Advances (ed. A. Diaspro), pp. 101–126. New York: imaging 4, 121–123. Wiley-Liss, Inc. HELL, S. W. (2003). Toward fluorescence nanoscopy. KAISER,W.&GARRET, C. G. B. (1961). Two-photon 2+ Nature Biotechnology 21, 1347–1355. excitation in CaF2 :Eu . Physical Review Letters 7, HELL,S.W.&ANDERSON, V. (2001). Space-multiplexed 229–231. multifocal nonlinear microscopy. Journal of Microscopy KASK, P., GUNTHER,R.&AXHAUSEN, P. (1997). Statistics 202, 457–463. accuracy in fluorescence fluctuation experiments. HELL, S. W., BAHLMANN, K., SCHRADER, M., SOINI, A., European Biophysics Journal 25, 163–169. MALAK, H., GRYCZYNSKI,I.&LAKOWICZ, J. R. (1996). KASK, P., PALO, K., ULLMANN,D.&GALL, K. (1999). Three-photon excitation in fluorescence microscopy. Fluorescence-intensity distribution analysis and its Journal of Biomedical Optics 1, 71–74. application in biomolecular detection technology. HELL, S. W., BOOTH, M., WILMS, S., SCHNETTER, C. M., Proceedings of the National Academy of Sciences USA 96, KIRSCH, A. K., ARNDT-JOVIN,D.J.&JOVIN,T.M. 13756–13761. 2PE in biological microscopy 161

KASTEN, F. H. (1989). The origin of modern fluorescence KONIG, K., SO, P. T. C., MANTULIN, W. W., TROMBERG, microscopy, chapter 1. In Cell Structure and Function by B. J. & GRATTON, E. (1996a). Two-photon excited Microspectrofluorometry. San Diego: Academic Press. lifetime imaging of autofluorescence in cells during KAWAKAMI, N., NAGERL, U. V., ODOARDI, F., BONHOEFFER, UVA and NIR photostress. Journal of Microscopy 183, T., WEKERLE,H.&FLUGEL, A. (2005). Live imaging of 197–204. effector cell trafficking and autoantigen recognition KONIG, K., RIEMANN, I., FISCHER,P.&HALBHUBER,K.J. within the unfolding autoimmune encephalomyelitis (1999b). Intracellular nanosurgery with near infrared lesion. Journal of Experimental Medicine 201, 1805–1814. femtosecond laser pulses. Cell and Molecular Biology 45, KELLER, H. E. (1995). Objective lenses for confocal 195–201. microscopy. In Handbook of Biological Confocal Microscopy, KOPPEL, D. E. (1974). Statistical accuracy in fluorescence 2nd edn. (ed. J. B. Pawley), pp. 111–126. New York and correlation spectroscopy. Physics Review (A) 10, 1938– London: Plenum Press. 1945. KIM, S. A., HEINZE, K. G., BACIA, K., WAXHAM,M.N. KOPPEL, D. E., MORGAN, F., COWAN,A.E.& &SCHWILLE, P. (2005). Two-photon cross-correlation CARSON, J. H. (1994). Scanning concentration corre- analysis of intracellular reactions with variable stoichi- lation spectroscopy using the confocal laser micro- ometry. Biophysical Journal 88, 4319–4336. scope. Biophysical Journal 66, 502–507. KIRKPATRICK, S. M., NAIK,R.R.&STONE, M. O. (2001). LAGERHOLM, B. C., WEINREB,G.E.&JACOBSON,K. Nonlinear saturation and determination of the two- (2005). Detecting microdomains in intact cell mem- photon absorption cross section of green fluorescent branes. Annual Review of Physical Chemistry 56, 309–336. protein. Journal of Physical Chemistry (B) 105, 2867–2873. LARSON, D. R., ZIPFEL, W. R., WILLIAMS, R. M., CLARK, KO¨ HLER, R. H., CAO, J., ZIPFEL, W. R., WEBB,W.W.& S. W., BRUCHEZ, M. P., WISE,F.W.&WEBB,W.W. HANSON, M. (1997). Exchange of protein molecules (2003). Water-soluble quantum dots for multiphoton through connections between higher plant plastids. fluorescence imaging in vivo. Science 300, 1434–1436. Science 276, 2039–2042. LAKOWICZ, J. R. (1999). Principles of Fluorescence Microscopy. KO¨ HLER, R. H., SCHWILLE, P., WEBB,W.W.&HANSON,M. New York: Plenum Press. (2000). Active transport of GFP through plastid tubules Leica Microsystems Heidelberg GmbH (1999). Two- quantified by fluorescence correlation spectroscopy. photon-microscopy comparison of femtosecond and Journal of Cell Science 113, 392–393. picosecond technology. Version: RK240699 (www. KOESTER, H. J., BAUR, D., UHL,R.&HELL, S. W. (1999). sct.ub.es:802/PDF/Leica/Application_Letter_14_ Ca2+ fluorescence imaging with pico- and femtosecond two_photon.pdf ). Confocal Application Letters 14, 1–7. two-photon excitation: signal and photodamage. LEVSKY,J.M.&SINGER, R. H. (2003). Fluorescence in situ Biophysical Journal 77, 2226–2236. hybridization: past, present and future. Journal of Cell KONIG, K. (2000). Multiphoton microscopy in life Science 116, 2833–2838. 2+ sciences. Journal of Microscopy 200, 83–104. LINDEGGER,N.&NIGGLI, E. (2005). Paradoxical SR Ca KONIG, K., BECKER, T. W., FISCHER, P., RIEMANN,I.& release in guinea-pig cardiac myocytes after beta- HALBHUBER, K. J. (1999a). Pulse-length dependence adrenergic stimulation revealed by two-photon photo- of cellular response to intense near-infrared laser pulses lysis of caged Ca2+. Journal of Physics (London) 565, in multiphoton microscopes. Optics Letters 24, 113–115. 801–813. KONIG, K., GOHL, A., LIEHR, T., LONCAREVIC,I.F.& LOUDON, R. (1983). The Quantum Theory of Light. London: RIEMANN, I. (2000b). Two-photon multicolor FISH: a Oxford University Press. versatile technique to detect specific sequences with in LOUISELL, W. H. (1973). Quantum Statistical Properties of single DNA molecules in cells and tissues. Single Molecule Radiation. New York: Wiley. 1, 41–51. MAGDE, D., ELSON,E.&WEBB, W. W. (1972). Thermo- KONIG, K., LIANG, H., BERNS,M.W.&TROMBERG,B.J. dynamic fluctuations in a reacting system: measurement (1995). Cell damage by near-IR microbeams. Nature by fluorescence correlation spectroscopy. Physical Review 377, 20–21. Letters 29, 705–708. KONIG, K., RIEMANN, I., FISCHER,P.&HALBHUBER,K.J. MAGDE, D., WEBB,W.W.&ELSON, E. L. (1978). (2000a). Multiplex FISH and three-dimensional DNA Fluorescence correlation spectroscopy. III. Uniform imaging with near infrared femtosecond laser pulses. translation and laminar flow. Biopolymers 17, 361–376. Histochemistry and Cell Biology 11, 4337–4345. MAITI, S., SHEAR, J. B., WILLIAMS, R. M., ZIPFEL,W.R.& KONIG, K., SIMON,U.&HALBHUBER, K. J. (1996b). 3D WEBB, W. W. (1997). Measuring serotonin distribution resolved two-photon fluorescence microscopy of in live cells with three-photon excitation. Science 275, living cells using a modified confocal laser scanning 530–532. microscope. Cell and Molecular Biology (Noisy-le-grand) 42, MALENGO, G., MILANI, R., CANNONE, F., KROL, S., 1181–1194. DIASPRO,A.&CHIRICO, G. (2004). High sensitivity 162 A. Diaspro, G. Chirico and M. Collini

optical microscope for single molecule spectroscopy MOZZARELLI,A.&BETTATI, S. (2001). Functional proper- studies. Review of Scientific Instruments 75, 2746–2751. ties of immobilized proteins. In Advanced Functional MARSH, P. N., BURNS,D.&GIRKIN, J. M. (2003). Practical Molecules and Polymers, vol. 4 (ed. H. S. Nalwa), pp. implementation of adaptive optics in multiphoton 55–97. Tokyo: Gordon and Breach Science Publishers. microscopy. Optics Express 11, 1123–1130. NAKAMURA, O. (1993). Three-dimensional imaging MASTERS, B. R. (1996). Selected Papers on Confocal Microscopy. characteristics of laser scan fluorescence microscopy: SPIE Milestone series. Bellingham, WA: SPIE Press. two-photon excitation vs. single-photon excitation. MASTERS, B. R. (2002). Selected Papers on Multiphoton Optik 93, 39–42. Excitation Microscopy. SPIE Milestone series. Bellingham, NAKAMURA, O. (1999). Fundamentals of two-photon WA: SPIE Press. microscopy. Microscopy Research and Technique 47, 165– MASTERS,B.R.&SO, P. T. (2004). Antecedents of two- 171. photon excitation laser scanning microscopy. Microscopy NEIL, M. A. A., JUSKAITIS, R., BOOTH, M. J., WILSON, T., Research and Technique 63, 3–11. TANAKA,T.&KAWATA, S. (2002). Active aberration MATSUMOTO, B. (ed.) (2002). Cell Biological Applications of correction for the writing of three-dimensional optical Confocal Microscopy, 2nd edn. Methods in Cell Biology memory devices. Applied Optics 41, 1374–1379. series, vol. 70. San Diego, London: Academic Press. NETZ, R., FEURER, T., WOLLESCHENSKY,R.&SAUERBREY, MATTOUSSI, H., MAURO, J. M., GOLDMAN, E. R., R. (2000). Measurement of the pulse front distortion ANDERSON, G. P., SUNDAR, V. C., MIKULEC,F.V.& in high numerical aperture lenses. Applied Physics B70, BAWENDI, M. G. (2000). Self-assembly of CdSe-ZnS 833–837. quantum dot bioconjugates using an engineered re- NGUYEN, Q.-T., CALLAMARAS, N., HSIEH,C.&PARKER,I. combinant protein. Journal of the American Chemical Society (2001). Contrusction of a two-photon microscope for 122, 12142–12150. video-rate Ca2+ imaging. Cell Calcium 30, 383–393. MCCARTHY, D. C. (1999). A start on parts for multiphoton NIELL,C.M.&SMITH, S. J. (2005). Functional imaging microscopy. Photonics Spectra 33, 80–88. reveals rapid development of visual response properties MCCONNELL, G., GIRKIN, J. M., FERGUSON, A. I., SMITH,G. in the zebrafish tectum. Neuron 45, 941–951. &GURNEY, A. (in press). All-solid-state sub-picosecond NIELSEN, T., FRICKE, M., HELLWEG,D.&ANDRESEN,P. optical parametric oscillator laser system of two-photon (2001). High efficiency beam splitter for multifocal fluorescence imaging. Optics Letters. multiphoton microscopy. Journal of Microscopy 201, MERTZ, J. (1998). Molecular photodynamics involved 368–376. in multi-photon excitation fluorescence microscopy. NITSCH, R., POHL, E. E., SMORODCHENKO, A., INFANTE- European Physics Journal (D) 3, 53–66. DUARTE, C., AKTAS,O.&ZIPP, F. (2004). Direct impact MERTZ, J., XU,C.&WEBB, W. W. (1995). Single molecule of T cells on neurons revealed by two-photon micro- detection by two-photon excited fluorescence. Optics scopy in living brain tissue. Journal of Neuroscience 24, Letters 20, 2532–2534. 2458–2464. MILLER, M. J., HEJAZI, A. S., WEI, S. H., CAHALAN,M.D. OERTNER, T. G., SABATINI, B. L., NIMCHINSKY,E.A.& &PARKER, I. (2004). T cell repertoire scanning is pro- SVOBODA, K. (2002). Facilitation at single synapses moted by dynamic dendritic cell behavior and random probed with optical quantal analysis. Nature Neuroscience T cell motility in the lymph node. Proceedings of the 5, 657–664. National Academy of Sciences USA 101, 998–1003. OHEIM, M., BEAUREPAIRE, E., CHAIGNEAU, E., MERTZ,J.& MINSKY, M. (1988). Memoir of inventing the confocal CHARPAK, S. (2001). Two-photon microscopy in scanning microscope. Scanning 10, 128–138. brain tissue: parameters influencing the imaging depth. MOHLER, W., MILLARD,A.C.&CAMPAGNOLA, P. J. (2003). Journal of Neuroscience Methods 111, 29–37 [Erratum: 112, Second harmonic generation imaging of endogenous 205]. structural proteins. Methods 29, 97–109. PALMER,A.G.D.&THOMPSON, N. L. (1989). High-order MOREAUX, L., SANDRE,O.&MERTZ, J. (2000). Membrane fluorescence fluctuation analysis of model protein imaging by second harmonic generation microscopy. clusters. Proceedings of the National Academy of Sciences USA Journal of the Optical Society of America (B) 17, 1685–1694. 86, 6148–6152. MULLER, M., SCHMIDT, J., MIRONOV,S.L.&RICHTER, PARAK, W. J., BOUDREAU, R., LE GROS, M., GERION, D., D. W. (2003). Construction and performance of a ZANCHET, D., MICHEEL, C. M., WILLIAMS, S. C., custom-built two-photon laser scanning system. Journal ALIVISATOS,A.P.&LORABELL, C. (2002). Cell motility of Physics (D): Applied Physics 36, 17147–17157. and metastatic potential studies based on quantum dot MULLER, M., SQUIER, J., WILSON,K.R.&BRAKENHOFF, imaging of phagokinetic tracks. Advanced Materials 14, G. J. (1998). 3D microscopy of trasparent objects 882–885. using third-harmonic generation. Journal of Microscopy PASTIRK, I., DELA CRUZ, J. M., WALOWICZ, K. A., LOZOVOY, 191, 266–274. V. V. & DANTUS, M. (2003). Selective two-photon 2PE in biological microscopy 163

microscopy with shaped femtosecond pulses. Optics RIDSDALE, A., MICU,I.&STYS, P. (2004). Conversion of Express 11, 1695–1701. the Nikon C1 confocal laser-scanning head for multi- PATTERSON,G.H.&PISTON, D. W. (2000). Photo- photon excitation on an upright microscope. Applied bleaching in two-photon excitation microscopy. Bio- Optics 43, 1669–1675. physical Journal 78, 2159–2162. ROBINSON, J. P. (2001). Current Protocols in Cytometry. New PAWLEY, J. B. (ed.) (1995). Handbook of Biological Confocal York: John Wiley & Sons. Microscopy. New York: Plenum Press. ROTH,S.&FREUND, I. (1979). Second harmonic PENG,Z.A.&PENG, X. (2001). Formation of high-quality generation in collagen. Journal of Chemical Physics 70, CdTe, CdSe, and CdS nanocrystals using CdO as 1637–1643. precursor. Journal of the American Chemical Society 123, RUAN, Q., CHEN, Y., GRATTON, E., GLASER,M.& 183–184. MANTULIN, W. W. (2002). Cellular characterization of PENNISI, E. (1997). Biochemistry: photons add up to adenylate kinase and its isoform: two-photon excitation better microscopy. Science 275, 480–481. fluorescence imaging and fluorescence correlation PERIASAMY, A. (ed.) (2001). Methods in Cellular Imaging. New spectroscopy. Biophysical Journal 83, 3177–3187. York: Oxford University Press. SABATINI, B. L., OERTNER,T.G.&SVOBODA, K. (2002). PERIASAMY,A.&DIASPRO, A. (2003). Multiphoton The life-cycle of Ca++ ions in dendritic spines. Neuron microscopy. Journal of Biomedical Optics 8, 327–328. 33, 439–452. PETER,M.&AMEER-BEG, S. M. (2004). Imaging molecular SAKO, Y., SEKIHATA, A., YANAGISAWA, Y., YAMAMOTO, M., interactions by multiphoton FLIM. Biology of the Cell 96, SHIMADA, Y., OZAKI,K.&KUSUMI, A. (1997). 231–236. Comparison of two-photon excitation laser scanning PETERSEN, N. O., HODDELIUS, P. L., WISEMAN, P. W., microscopy with UV-confocal laser scanning micro- SEGER,O.&MAGNUSSON, K. E. (1993). Quantitation of scopy in three-dimensional calcium imaging using the membrane receptor distributions by image correlation fluorescence indicator Indo-1. Journal of Microscopy 185, spectroscopy: concept and application. Biophysical 9–20. Journal 65, 1135–1146. SANCHEZ, E. J., NOVOTNY, L., HOLTOM,G.R.&XIE,X.S. PETI-PETERDI, J., MORISHIMA, S., BELL,P.D.&OKADA,Y. (1997). Room temperature fluorescence imaging and (2002). Two-photon excitation fluorescence imaging of spectroscopy of single molecules by two-photon the living juxtaglomerular apparatus. American Journal of excitation. Journal of Physical Chemistry (A) 101, Physiology – Renal Physiology 283, F197–F201. 7019–7023. PISTON, D. W. (1999). Imaging living cells and tissues by SANCHEZ,S.A.&GRATTON, E. (2005). Lipid-protein two-photon excitation microscopy. Trends in Cell Biology interactions revealed by two-photon microscopy and 9, 66–69. fluorescence correlation spectroscopy. Accounts of PISTON, D. W., KIRBY, M. S., CHENG, H., LEDERER,W.J. Chemical Research 38, 469–477. &WEBB, W. W. (1994). Two-photon excitation fluor- SANDERSON,M.J.&PARKER, I. (2003). Video-rate confocal escence imaging of three-dimensional calcium ion microscopy. Methods in Enzymology 360, 447–480. activity. Applied Optics 33, 662–669. SANDISON,D.R.&WEBB, W. W. (1994). Background re- PLOEM, J. S. (1987). Laser scanning fluorescence micro- jection and signal-to-noise optimization in the confocal scopy. Applied Optics 26, 3226–3231. and alternative fluorescence microscopes. Applied Optics PONS, T., MOREAUX,L.&MERTZ, J. (2002). Photoinduced 33, 603–615. flip-flop of amphiphilic molecules in lipid bilayer SCHNEIDER, M., BAROZZI, S., TESTA, I., FARETTA,M.& membranes. Physical Review Letters 89, 288104. DIASPRO, A. (2005). Two-photon activation and POST, J. N., LIDKE, K. A., RIEGER,B.&ARNDT-JOVIN,D.J. excitation properties of PA-GFP in the 720–920-nm (2005). One- and two-photon photoactivation of a region. Biophysical Journal 89, 1346–1352. paGFP-fusion protein in live Drosophila embryos. SCHONLE,A.&HELL, S. W. (1998). Heating by absorption FEBS Letters 579, 325–330. in the focus of an objective lens. Optics Letters 23, POTTER, S. M. (1996). Vital imaging: two-photons are 325–327. better than one. Current Biology 6, 1596–1598. SCHRADER, M., HOFMANN,U.G.&HELL, S. W. (1998). QUIAN, H. (1990). On the statistics of fluorescence corre- Ultrathin fluorescent layers for monitoring the axial lation spectroscopy. Biophysical Chemistry 38, 49–57. resolution in confocal and two-photon fluorescence QIAN,H.&ELSON, E. L. (1990). Distribution of molecular microscopy. Journal of Microscopy 191, 135–140. aggregation by analysis of fluctuation moments. SCHWILLE, P. (2001). Fluorescence correlation spectro- Proceedings of the National Academy of Sciences USA 87, scopy and its potential for intracellular applications. Cell 5479–5483. Biochemistry and Biophysics 34, 383–408. RENTZEPIS, P. M., MITSCHELE,C.J.&SAXMAN,A.C. SCHWILLE, P., HAUPTS, U., MAITI,S.&WEBB,W.W. (1970). Measurement of ultrashort laser pulses by three- (1999a). Molecular dynamics in living cells observed photon fluorescence. Applied Physics Letters 17, 122–124. by fluorescence correlation spectroscopy with one- and 164 A. Diaspro, G. Chirico and M. Collini

two-photon excitation. Biophysical Journal 77, 2251– systems for in vivo molecular imaging of cancer. 2265. Technology in Cancer Research & Treatment 2, 489–503. SCHWILLE,P.&HEINZE, K. G. (2001). Two-photon SONNLEITNER, M., SCHU¨ TZ, G., KADA,G.&SCHINDLER,H. fluorescence cross-correlation spectroscopy. Chem- (2000). Imaging single lipid molecules in living cells PhysChem 2, 269–272. using two photon excitation. Single Molecules 1, 182–183. SCHWILLE, P., KORLACH,J.&WEBB, W. (1999b). SONNLEITNER, M., SCHUTZ,G.J.&SCHMIDT, T. (1999). Fluorescence correlation spectroscopy with single Imaging individual molecules by two-photon excitation. molecule sensitivity on cell and model membranes. Chemical Physics Letters 300, 221–226. Cytometry 36, 176–182. SPENCE, D. E., KEAN,P.N.&SIBBETT, W. (1991). 60-fsec SEYFRIED, V., BIRK, H., STORZ,R.&ULRICH, H. (2003). pulse generation from a self-mode-locked Ti:sapphire Advances in multispectral confocal imaging. Proceedings laser. Optics Letters 16, 42–45. of SPIE 5139, 147–157. SQUIER, J. A., MULLER, M., BRAKENHOFF,G.J.&WILSON, SHEPPARD,C.J.&GU, M. (1990). Image formation in two- K. R. (1998). Third harmonic generation microscopy. photon fluorescence microscopy. Optik 86, 104–106. Optical Express 3, 315–324. SHEPPARD,C.J.&KOMPFNER, R. (1978). Resonant scan- SQUIRREL, J. M., WOKOSIN, D. L., WHITE,J.G.&BARISTER, ning optical microscope. Applied Optics 17, 2879–2885. B. D. (1999). Long-term two-photon fluorescence imaging of mammalian embryos without compromising SHEPPARD,C.J.&WILSON, T. (1980). Image formation in confocal scanning microscopes. Optik 55, 331–342. viability. Nature Biotechnology 17, 763–767. SRIVASTAVA,M.&PETERSEN, N. O. (1998). Diffusion of SHEPPARD,C.J.R.&CHOUDHURY, A. (1977). Image formation in the scanning microscope. Optica Acta 24, transferrin receptor clusters. Biophysical Chemistry 75, 1051–1073. 201–211. STANLEY, M. (2001). Improvements in optical filter SHIH, Y. H., STREKALOV, D. V., PITTMAN,T.D.&RUBIN, design. In Multiphoton Microscopy in the Biomedical Sciences M. H. (1998). Why two-photon but not two photons? (eds. A. Periasamy & P. T. C. So). Proceedings of SPIE Fortschritte der Physik 46, 627–641. 4642, 52–61. SHOTTON, D. M. (1989). Confocal scanning optical micro- STARR,T.E.&THOMPSON, N. L. (2001). Total internal scopy and its applications for biological specimens. reflection with fluorescence correlation spectroscopy: Journal of Cell Science 94, 175–206. combined surface reaction and solution diffusion. SHOTTON, D. M. (1993). Electronic Light Microscopy. Biophysical Journal 80, 1575–1584. Techniques in Modern Biomedical Microscopy, vol. 1. New STELZER, E. H. K., HELL, S., LINDEK, S., PICK, R., STORZ, York: Wiley-Liss. C., STRICKER, R., RITTER,G.&SALMON, N. (1994). Non- SINGH,S.&BRADLEY, L. T. (1964). Three-photon linear absorption extends confocal fluorescence micro- absorption in naphtalene crystals by laser excitation. scopy into the ultra-violet regime and confines the Physical Review Letters 12, 162–164. illumination volume. Optical Communications 104, SO, P. T. C., BERLAND, K. M., FRENCH, T., DONG,C.Y. 223–228. &GRATTON, E. (1996). Two photon fluorescence STOSIEK, C., GARASHUK, O., HOLTHOFF,K.&KONNERTH, microscopy: time resolved and intensity imaging. In A. (2003). In vivo two-photon calcium imaging of Fluorescence Imaging Spectroscopy and Microscopy (eds. X. F. neuronal networks. Proceedings of the National Academy of Wang & B. Herman), pp. 351–373. Chemical Analysis Sciences USA 100, 7319–7324. Series, vol. 137. New York: J. Wiley & Sons. STRAUB,M.&HELL, S. W. (1998a). Fluorescence lifetime SO, P. T. C., DONG, C. Y., MASTERS,B.R.&BERLAND, three-dimensional microscopy with picosecond pre- K. M. (2000). Two-photon excitation fluorescence cision using a multifocal multiphoton microscope. microscopy. Annual Review of Biomedical Engineering 2, Applied Physics Letters 73, 1769–1771. 399–429. STRAUB,M.&HELL, S. W. (1998b). Multifocal multi- SOELLER,C.&CANNELL, M. B. (1996). Construction of a photon microscopy: a fast and efficient tool for 3D two-photon microscope and optimisation of illumi- fluorescence imaging. Bioimaging 6, 177–184. nation pulse duration. Pflugers Archiv: European Journal STRAUB, M., LODEMANN, P., HOLROYD, P., JAHN,R.& of Physiology 432, 555–561. HELL, S. W. (2000). Live cell imaging by multifocal SOELLER,C.&CANNELL, M. B. (1999). Two-photon multiphoton microscopy. European Journal of Cell Biology microscopy: imaging in scattering samples and three- 79, 726–734. dimensionally resolved flashed photolysis. Microscopy STUTZMANN, G. E., LA FERLA,F.M.&PARKER, I. (2003). Research and Technique 47, 182–195. Ca2+ signalling in mouse cortical neurons studied SOKOLOV, K., AARON, J., HSU, B., NIDA, D., GILLENWATER, by two-photon imaging and photoreleased inositol A., FOLLEN, M., MACAULAY, C., ADLER-STORTHZ, K., triphosphate. Journal of Neuroscience 23, 758–765. KORGEL, B., DESCOUR, M., PASQUALINI, R., ARAP, W., SVELTO, O. (1998). Principles of Lasers, 4th edn. New York: LAM,W.&RICHARDS-KORTUM, R. (2003). Optical Plenum Press. 2PE in biological microscopy 165

SYTSMA, J., VROOM, J. M., DE GRAUW,C.J.&GERRITSEN, VAN DER VOORT,H.T.M.&BRAKENHOFF, G. J. (1988). H. C. (1998). Time-gated fluorescence lifetime imaging Determination of the 3-dimensional optical properties and microvolume spectroscopy using two-photon of a confocal scanning laser microscope. Optik 78, excitation. Journal of Microscopy 191, 39–51. 48–53. TAKI, M., WOLFORD, J. L. & O’HALLORAN, T. V. (2004). VAN DER VOORT,H.T.M.&STRASTERS, K. C. (1995). Emission ratiometric imaging of intracellular zinc: Restoration of confocal images for quantitative image design of a benzoxazole fluorescent sensor and its analysis. Journal of Microscopy 178, 165–181. application in two-photon microscopy. Journal of the VERMOLEN, B. J., GARINI,Y.&YOUNG, I. T. (2004). 3D American Chemical Society 126, 712–713. restoration with multiple images acquired by a modified TAN, Y. P., LLANO, I., HOPT, A., WURRIEHAUSEN,F.& conventional microscope. Microscopy Research and NEHER, E. (1999). Fast scanning and efficient photo- Technique 64, 113–125. detection in a simple two-photon microscope. Journal of VEST, R. S., GONZALES, L. J., PERMANN, S. A., SPENCER, E., Neuroscience Methods 92, 123–135. HANSEN, L. D., JUDD,A.M.&BELL, J. D. (2004). TANNER, G. A., SANDOVAL,R.M.&DUNN, K. W. (2004). Divalent cations increase lipid order in erythrocytes and Two-photon in vivo microscopy of sulfonefluorescein susceptibility to secretory phospholipase A2. Biophysical secretion in normal and cystic rat kidneys. American Journal 86, 2251–2260. Journal of Physiology – Renal Physiology 286, F152–F160. VOITYUK, A. A., MICHEL-BEYERLE,M.E.&RO¨ SCH,N. (1998). Quantum chemical modeling of structure and TAUER, U. (2002). Advantages and risks of multiphoton microscopy in physiology. Experimental Physiology 87, absorption spectra of the chromophore in green fluor- 709–714. escent proteins. Chemical Physics 231, 13–25. VOURA, E. B., JAISWAL, J. K., MATTOUSSI,H.&SIMON, THEER, P., HASAN,M.T.&DENK, W. (2003). Two-photon S. M. (2004). Tracking metastatic tumor cell extra- imaging to a depth of 1000 microm in living brains by vasation with quantum dot nanocrystals and fluor- use of a Ti:Al2O3 regenerative amplifier. Optics Letters escence emission-scanning microscopy. Nature Medicine 28, 1022–1024. 10, 993–998. THOMPSON, N. (1991). Fluorescence correlation spectro- WANG, H. F., FU, Y., ZICKMUND, P., SHI,R.Y.&CHENG, scopy. In Topics in Fluorescence Spectroscopy, vol. I, J. X. (2005). Coherent anti-stokes Raman scattering Techniques (ed. J. R. Lakowitz). New York: Plenum imaging of axonal myelin in live spinal tissues. Press. Biophysical Journal 89, 581–591. THOMPSON,N.L.&AXELROD, D. (1983). Immunoglobulin WANG, J. W., WONG, A. M., FLORES, J., VOSSHALL,L.B.& surface-binding kinetics studied by total internal re- AXEL, R. (2003). Two-photon calcium imaging reveals flection with fluorescence correlation spectroscopy. an odor-evoked map of activity in the fly brain. Cell 112, Biophysical Journal 43, 103–114. 271–282. TIRRI, M., VAARNO, J., SOINI,J.T.&HANNINEN, P. (2003). WANG,X.F.&HERMAN, B. (1996). Fluorescence Imaging Low cost lasers challenge ultrafast systems in two- Spectroscopy and Microscopy. New York: Wiley-Liss. photon excitation applications. Opto-electronics Review 11, WANG, Y., WANG, X. F., WANG,C.&MA, H. (2004). 39–44. Simultaneously multi-parameter determination of TOKUMASU,F.&DVORAK, J. (2003). Development and hematonosis cell apoptosis by two-photon and con- application of quantum dots for immunocytochemistry focal laser scanning microscopy. Journal of Clinical of human erythrocytes. Journal of Microscopy 211, Laboratory Analysis 18, 271–275. 256–261. WEBB, R. H. (1996). Confocal optical microscopy. Reports TOROK,P.&SHEPPARD, C. J. R. (2001). The role of on Progress in Physics 59, 427–471. pinhole size in high aperture two and three-photon WEINSTEIN,M.&CASTLEMAN, K. R. (1971). Recon- microscopy. In Confocal and Two-photon Microscopy: structing 3-D specimens from 2-D section images. Foundations, Applications and Advances (ed. A. Diaspro), Proceedings of SPIE 26, 131–138. pp. 127–152. New York: Wiley-Liss, Inc. WHITE,J.G.&AMOS, W. B. (1987). Confocal microscopy TSAI, P. S., FRIEDMAN, B., IFARRAGUERRI, A. I., THOMPSON, comes of age. Nature 328, 183–184. B. D., LEV-RAM, V., SCHAFFER, C. B., XIONG, Q., TSIEN, WHITE, J. G., AMOS,W.B.&FORDHAM, M. (1987). An R. Y., SQUIER,J.A.&KLEINFELD, D. (2003). All-optical evaluation of confocal versus conventional imaging of histology using ultrashort laser pulses. Neuron 39, biological structures by fluorescence light microscopy. 27–41. Journal of Cell Biology 105, 41–48. TSIEN, R. Y. (1998). The green fluorescent protein. Annual WHITE, J. G., SQUIRRELL,J.M.&ELICEIRI, K. W. (2001). Review of Biochemistry 67, 509–544. Applying multiphoton imaging to the study of mem- TSIEN, R. Y. (1999). Monitoring cell calcium. In Calcium as brane dynamics in living cells. Traffic 2, 775–780. a Cellular Regulator (eds. E. Carafoli & C. Klee), WHITE,N.S.&ERRINGTON, R. J. (2000). Improved laser pp. 28–54. New York: Oxford University Press. scanning fluorescence microscopy by multiphoton 166 A. Diaspro, G. Chirico and M. Collini

excitation. Advances in Imaging and Electron Physics 113, WOLLESCHENSKY, R., DICKINSON,M.&FRASER,S.E. 249–277. (2001). Group-velocity dispersion and fiber delivery WIDENGREN,J.&RIGLER, R. (1996). Mechanisms of in multifocal laser scanning microscopy. In Confocal photobleaching investigated by fluorescence correlation and Two-photon Microscopy: Foundations, Applications and spectroscopy. Bioimaging 4, 149–157. Advances (ed. A. Diaspro), pp. 178–190. New York: WIDENGREN, J., RIGLER,R.&METS, U. (1994). Triplet- Wiley-Liss, Inc. state monitoring by fluorescence correlation spectro- WOLLESCHENSKY, R., FEURER, T., SAUERBREY,R.&SIMON, scopy. Journal of Fluorescence 4, 255–258. U. (1998). Characterization and optimization of a laser WIER, W. G., BALKE, C. W., MICHAEL,J.A.&MAUBAN, scanning microscope in the femtosecond regime. J. R. (2000). A custom confocal and two-photon digital Applied Physics B67, 87–94. laser scanning microscope. American Journal of Physiology WRIGHT,S.J.&WRIGHT, J. D. (2002). Introduction to 278, H2150–H2156. confocal microscopy. In Cell Biological Applications of WILLIAMS, R. M., PISTON,D.W.&WEBB, W. W. (1994). Confocal Microscopy, 2nd edn. (ed. B. Matsumoto), Two-photon molecular excitation provides intrinsic pp. 1–86. Methods in Cell Biology series, vol. 70. San 3-dimensional resolution for laser-based microscopy Diego, London: Academic Press. and microphotochemistry. FASEB Journal 8, 804–813. XIE,X.S.&LU, H. P. (1999). Single molecule enzymology. Journal of Biological Chemistry 274, 15967–15970. WILSON, T. (1990). Confocal Microscopy. London: Academic Press. XU, C. (2001). Cross-sections of fluorescence molecules in multiphoton microscopy. In Confocal and Two-photon WILSON, T. (2001). Confocal microscopy: basic principles and architectures. In Confocal and Two-photon Microscopy: Microscopy: Foundations, Applications and Advances (ed. A. Foundations, Applications and Advances (ed. A. Diaspro), Diaspro), pp. 75–99. New York: Wiley-Liss, Inc. XU,C.&WEBB, W. W. (1996). Measurement of two- pp. 19–38. New York: Wiley-Liss, Inc. photon excitation cross-sections of molecular fluor- WILSON,T.&SHEPPARD, C. J. R. (1984). Theory and Practice ophores with data from 690 nm to 1050 nm. Journal of Scanning Optical Microscopy. London: Academic Press. of the Optical Society of America B 13, 481–491. WISE, F. (1999). Lasers for two-photon microscopy. In YODER,E.J.&KLEINFELD, D. (2002). Cortical imaging Imaging: A Laboratory Manual (eds. R. Yuste, F. Lanni & through the intact mouse skull using two-photon A. Konnerth), pp. 18.1–18.9. Cold Spring Harbor: Cold excitation laser scanning microscopy. Microscopy Research Spring Harbor Press. and Technique 56, 304–305. WISEMAN,P.W.&PETERSEN, N. O. (1999). Image corre- ZHANG, J., CAMPBELL, R. E., TING,A.&TSIEN,R.Y. lation spectroscopy. II. Optimization for ultrasensitive (2002). Creating new fluorescent probes for cell detection of preexisting platelet-derived growth factor- biology. Nature Review Molecular Biology 3, 906–918. beta receptor oligomers on intact cells. Biophysical Journal ZIMMERMAN, T., RIETDORF,J.&PEPPERKOK, R. (2003). 76, 963–977. Spectral imaging and its applications in live cell WISEMAN, P. W., SQUIER, J. A., ELLISMAN,M.H.& microscopy. FEBS Letters 546, 87–92. WILSON, K. R. (2000). Two-photon image correlation ZIPFEL, W. R., WILLIAMS, R. M., CHRISTIE, R., YU NIKITIN, spectroscopy and image cross-correlation spectroscopy. A., HYMAN,B.T.&WEBB, W. W. (2003a). Live tissue Journal of Microscopy 200, 14–25. intrinsic emission microscopy using multiphoton- WOKOSIN, D. L., CENTONZE, V. E., CRITTENDEN,S.& excited native fluorescence and second harmonic gen- WHITE, J. (1996). Three-photon excitation fluorescence eration. Proceedings of the National Academy of Sciences USA imaging of biological specimens using an all-solid-state 100, 7075–7080. laser. BioImaging 4, 208–214. ZIPFEL, W. R., WILLIAMS,R.M.&WEBB, W. W. (2003b). WOKOSIN, D. L., CENTONZE, V. E., WHITE, J. G., Nonlinear magic: multiphoton microscopy in the bi- ARMSTRONG, D., ROBERTSON,G.&FERGUSON,A.I. osciences. Nature Biotechnology 21, 1369–1377. (1997). All-solid-state ultra-fast lasers facilitate multi- ZOUMI, A., DATTA, S., LIAW, L. H. L., WU, C. J., photon excitation fluorescence imaging. IEEE Journal MANTHRIPRAGADA, G., OSBORNE,T.F.&LAMORTE, of Special Topics in Quantum Electronics 2, 1051–1065. V. J. (2005). Spatial distribution and function of sterol WOKOSIN, D. L., DYMOTT,M.&WHITE, J. G. (1998). Pulse regulatory element-binding protein 1a and 2 homo- width considerations for multiphoton excitation laser and heterodimers by in vivo two-photon imaging and scanning fluorescence imaging. Proceedings of SPIE spectroscopy fluorescence resonance energy transfer. 3260A, 115–122. Molecular and Cellular Biology 25, 2946–2956. WOKOSIN,D.L.&WHITE, J. G. (1997). Optimization of ZOUMI, A., YEH,A.&TROMBERG, B. J. (2002). Imaging the design of a multiple-photon excitation laser scan- cells and extracellular matrix in vivo by using second- ning fluorescence imaging system. In Three-dimensional harmonic generation and two-photon excited fluor- Microscopy: Image Acquisition and Processing IV. Proceedings escence. Proceedings of the National Academy of Sciences USA of SPIE 2984, 25–29. 99, 11014–11019.