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Dual-Color 4Pi-Confocal Microscopy with 3D-Resolution in the 100 Nm Range

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Dual-Color 4Pi-Confocal Microscopy with 3D-Resolution in the 100 Nm Range Ultramicroscopy 90 (2002) 207–213 Dual-color 4Pi-confocal microscopy with 3D-resolution in the 100 nm range Hiroshi Kano, Stefan Jakobs, Matthias Nagorni, Stefan W. Hell* High Resolution Optical Microscopy Group, Max-Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37070 Gottin. gen, Germany Received 21 November 2000; received in revised form 2 July 2001 Abstract We report the development of simultaneous two-color channel recordingin 4Pi-confocal microscopy. A marked increase of spatial resolution over confocal microscopy becomes manifested in 4Pi-confocal three-dimensional (3D) data stacks of dual-labeled objects. The fundamentally improved resolution is verified both with densely labeled fluorescence beads as well as with membrane labeled fixed Escherichia coli. The synergistic combination of dual-color 4Pi-confocal recording with image restoration results in dual-color imaging with a 3D resolution in the 100 nm range. r 2002 Elsevier Science B.V. All rights reserved. 1. Introduction the typically 3.5 times sharper main maximum [1]. The side-lobes are removed mathematically by a By providinga fundamental improvement of linear filter, in which case a typical axial resolution axial sectioning, 4Pi-confocal microscopy [1] is a of 130–140 nm is achieved at a two-photon promisingdevelopment in three-dimensional (3D) excitation wavelength of 700–800 nm [3]. Alterna- far-field fluorescence microscopy. The 4Pi-confo- tively, the effective 3D-point spread function cal microscope owes its superior sectioningcap- (PSF) of the 4Pi-confocal microscope is used ability to the coherent addition of the spherical altogether to restore the 4Pi-confocal image data wavefronts produced by two opposingobjective with a non-linear restoration algorithm [4]. Image lenses of high numerical aperture. In its simplest restoration not only removes the side-lobes, but version, also referred to as 4Pi-confocal micro- also leads to a further improvement of the spatial scopy of type A [2], the two objective lenses are resolution by about 50%, so that a spatial illuminated by coherent beams that are brought to resolution of the order of 100 nm is achieved in constructive interference in the common focal all directions [4]. Similar resolution has also been point. The use of two-photon excitation is reported by a related method, I5M, that is based particularly advantageous since it leads to a on a wide-field approach, featuringthe advantage reduction of the two side-lobes that accompany of parallel data acquisition [5]. However, this method is seriously challenged by a higher and *Correspondingauthor. Tel.: +49-551-201-1366; fax: +49- more complex side-lobe structure. 551-201-1085. This superior spatial resolution of 4Pi-confocal E-mail address: [email protected] (S.W. Hell). microscopy has been demonstrated in a series of 0304-3991/02/$ - see front matter r 2002 Elsevier Science B.V. All rights reserved. PII: S 0304-3991(01)00132-2 208 H. Kano et al. / Ultramicroscopy 90 (2002) 207–213 applications, most notably in the imaging of the excitation was performed with a mode-locked actin [4] and the microtubule network [6] of fixed, Titanium Sapphire laser (Coherent, Inc.) operating glycerol mounted mouse fibroblast cells. More at the wavelength of 800 nm, whose beam was recently, 4Pi-confocal microscopy has been shown expanded and divided in two parts by a beam to be viable for imaging watery specimens, so that splitter. Each part illuminated the sample through for the first time the imaging of live specimens at one of the two oil objective lenses of 1.4 numerical this resolution was possible [7]. Dual-color detec- aperture (Leica 100X, Planapo), whereby the tion is an important extension to high resolution optical path lengths were matched with a precision imaging and particularly to 4Pi-confocal micro- of a few microns. The foci of the two lenses were scopy because the tacklingof many biological superimposed in space usinga piezo-stageoperat- problems requires precise knowledge about the ingin a closed loop mode. The emitted fluores- spatial distribution of differently labeled proteins, cence was collected by the objective lens on the organelles etc. Strictly speaking, co-localization of left-hand side and passed a dichroic mirror and the objects with different spectral properties, such as tube lens. To define the two color channels for color or fluorescence lifetime, has only little do detection, the fluorescence was separated by a long with the increase of resolution beyond classical wave pass dichroic mirror with an edge at 540 nm. barriers. But for a series of applications such as Each stream of fluorescence light was converged at fluorescence activated in situ hybridization (FISH) the core of an optical fiber and guided toward a or protein colocalization studies it is a viable single photon counting avalanche photo diode option to improve the spatial distribution infor- (Perkin–Elmer, Inc.). The detection system in mation [8]. conjunction with the finite openingof the fiber Successful dual channel detection faces technical provide confocality to the microscope with an challenges both in confocal fluorescence micro- openingcorresponded to 89% of the back scopy and even more so in its 4Pi-confocal projected Airy disk. counterpart, because the effect of longitudinal chromatic aberrations of the two detection chan- 2.2. Image processing nels must be eliminated [8]. Recently, we have shown that sub-resolution distance measurements Two kinds of data processingwere used for with a precision of 1 nm can be accomplished in a removingthe side-lobes in 4Pi-confocal micro- dual-color detection 4Pi-confocal axial image scan scopy. First, the 4Pi-confocal raw data were point- [9]. Here, we report for the first time 4Pi-confocal deconvolved, that is the lobes were removed by a imaging of extended dual-colored fluorescence linear filter applied in each axial line [3]. In order specimens, such as densely packed sub-resolution to establish the point-deconvolution by a linear fluorescence beads, as well as doubly labeled filter, only the relative location and height of the E. coli. We demonstrate a strikingincrease in two sidelobes have to be known. This 1D resolution over dual-color confocal fluorescence deconvolution is fast enough to be carried out microscopy. duringimageacquisition. We note that the effect of the point-deconvolution is restricted to the removal of the lobe artifacts alone and does not 2. Material and methods lead to an increase of resolution per se, because it does not widen the spatial frequency bandwidth of 2.1. 4Pi-confocal microscope the optical system. Secondly, we applied an iterative image restoration algorithm with a non- The employed two-photon excitation 4Pi-con- negativity constraint. We chose the Richardson– focal microscope of type A has been described in Lucy (RL) algorithm [10,11] which encompasses Ref. [9]; here we summarize the physical para- maximum likelihood estimation (MLE) for images meters of operations and expound on the method dominated by Poisson noise. This algorithm has with which we reduced chromatic aberrations. The been shown to be a good choice for images H. Kano et al. / Ultramicroscopy 90 (2002) 207–213 209 recorded by a fluorescence microscope. The RL medium (4% Dapco, Serva, Heidelberg, Germany algorithm has been modified by Conchello and in 96% glycerol) and placed between two cover- McNally [12], so as to include Tikhonov regular- slips. ization as well. For our restorations, we used this modified algorithm [6]. Suitable regularization 2.5. Labeling of E. coli parameters were determined by simulated imaging of a known test object and subsequent restoration. E. coli K-12 DH5a were grown overnight to For a thorough illustration of the resolution steady state in LB-medium (10 g/l tryptone, 5 g/l improvement, we also recorded the data stack in yeast extract and 10 g/l NaCl) at 371C. Membrane the confocal mode, which can be accomplished by labellingwas accomplished by addingthe vital obstructingone of the highaperture lenses. stain FM 1–43 (Molecular Probes) to a final concentration of 0.1 mM from a 2.5 mM EtOH 2.3. Eliminating longitudinal chromatic aberrations stock solution to the bacterial culture. Staining was performed at room temperature for 1–2 h. Two-photon excitation enabled the excitation of Nucleoids were labeled by adding30 mm DAPI to both fluorophores in the same focal volume, so the cells 15 min prior to fixation. For fixation the that longitudinal chromatic aberration on the cells were incubated for 1 h in 3.5% paraformal- excitation side cannot occur. Chromatic aberra- dehyde. tion associated with the two detection color To mount the bacteria, glass cover slips were channels is equivalent to an axial offset of the first coated with poly-l-lysine (Sigma, St. Louis, detection PSF defined by the confocal spatial MO). The coverslips were briefly incubated with openings of the two fibers. We eliminated this a diluted suspension of 110 nm green spheres offset by imaging a monomolecular fluorescent (Molecular Probes). These spheres attached to polydiacetylene layer mounted on a cover slip by the glass and facilitated adjustment of the wave- the Langmuir–Blodgett technique [9,13]. Two- fronts to constructive interference and the control photon excitation of these thin layers results in a of the relative phase of the wavefronts during broad fluorescence coveringa wide rangeof the image acquisition. After a washing step the cover- visible spectrum. Therefore the fluorescence pro- slips were anew coated with poly-L-lysine. Finally duced by the same excitation spot could be the fixed labeled bacteria were re-suspended in recorded in both channels. The matchingof the Dapco mountingmedium and placed between two two detection PSFs was accomplished by translat- coverslips each covered by fluorescent beads. ingeach detection fiber openingalongthe optic axis, so that both detection PSFs matched in space with the common excitation spot.
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