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Home , Optical microscope, STED microscopy

Single- two-photon excitation– stimulated emission depletion (SW2PE-STED) superresolution imaging

Paolo Bianchinia, Benjamin Harkea, Silvia Galiania,b, Giuseppe Vicidominia, and Alberto Diasproa,b,1

aDepartment of Nanophysics, Istituto Italiano di Tecnologia, 16100 Genoa, Italy; and bDepartment of Physics, University of Genoa, 16100 Genoa, Italy

Edited by David A. Weitz, Harvard University, Cambridge, MA, and approved March 15, 2012 (received for review November 22, 2011)

We developed a new class of two-photon excitation–stimulated Physical Principles and Setup emission depletion (2PE-STED) optical . In this work, The key point of the method lies on finding a wavelength that has we show the opportunity to perform superresolved nonzero cross-section for 2PE and STED processes and avoids imaging, exciting and stimulating the emission of a fluorophore 1PE excitation of a given dye. Then one has the chance to find by means of a single wavelength. We show that a widely used red- a balance, in terms of power and pulse length of the beams, to emitting fluorophore, ATTO647N, can be two-photon excited at a achieve a significant fluorescence on-off contrast that enables wavelength allowing both 2PE and STED using the very same such SW 2PE-STED superresolution imaging method. source. This fact opens the possibility to perform 2PE The molecular cross-sections for the two-photon absorption at four to five times STED-improved resolution, while exploiting are typically very low, so high fluxes approximately equal 1030 −1 −2 the intrinsic advantages of nonlinear excitation. to s m photons are required. In order to reach this requirement, 2PE imaging profits of high-repetition pulsed wo-photon excitation (2PE) fluorescence microscopy (1) is a with high peak power, ultrashort pulses (tens to hundreds of fem- toseconds), whereas average power remains fairly low thus avoid- widely used technique in medicine and (2, 3). Its char- T ing local trapping or heating effects (3). It is worth recalling here acteristics make it particularly suitable for deep tissue and in vivo that a common organic fluorophore follows the Kasha’s rule as imaging applications (4). The use of infrared guar- depicted by the simplified Jablonsky diagram in Fig. 1A. When antees a high penetration depth in scattering samples and a re- −17 the simultaneous (ca. 10 s) absorption of two photons occurs, duced photodamage in living specimens. Moreover, the quadratic the goes to an excited vibrational level of one of the dependence of the excitation allows intrinsical rejection of the singlet excited states. In our case, because the wavelength used out-of- fluorescence background and reduces the FWHM pffiffiffi is close to the visible region, we expect that an S2 singlet state 2 of the focal spot by approximately compared to the could be involved in the absorption process. In general, a fluor- limited spot of excitation light. However, 2PE cannot be consid- ophore very efficiently undergoes internal conversion (IC) from “ ” ered a genuine superresolution technique: The doubling in wa- the S2 state to the lowest vibrational level of the S1 state in about velength causes that the size of the diffraction spot of excitation the same time required to relax from an excited vibrational level −13 −11 light is twice than the one achievable by single-photon excitation of the S1 state to its zeroth one (i.e., 10 to 10 s). Thus fluor- (1PE). In 1994, Hell and Wichmann introduced the stimulated escence and stimulated emission occur between S1 and a vibra- emission depletion (STED) concept as a way to achieve optical tional sublevel of the ground state S0 ( and red arrows, superresolution by breaking the diffraction barrier (5). In general, respectively, in Fig. 1A) (13). Because the S0 decays nonradia- STED imaging is based on the fluorescence switching due to sti- tively within 0.2–5 ps, a long (few hundreds of picoseconds) mulated emission depletion introduced by a second red shifted STED pulse is more effective in the depletion of S1 (14, 15). beam. The fluorescent sample is probed by a focused excitation Moreover, a long STED pulse decreases the peak intensity, beam overlaid with a STED beam commonly shaped to a dough- and thus avoids two-photon excitation from the STED beam. nut-like intensity distribution featuring zero intensity in the Notably, we use the same wavelength also for nonlinear excitation center. Because of the saturation of the stimulated emission de- of the fluorophore, the fact that 2PE pulses are shorter than the pletion, the fluorescence is switched in the whole focal area ex- IC time avoids a possible competing STED effect along with the cept for the small region around the zero of the STED beam. The excitation process. resulting resolution is theoretically unlimited being governed by For the above-mentioned reasons we realized a setup (Fig. 1B) the efficiency of the depletion process (6, 7). Recently, STED has where the laser beam of an ultrafast Ti:sapphire IR laser (Cha- been proposed coupled with 2PE microscopy combining the ad- meleon UltraII; Coherent) has been split by a polarizing beam vantages of 2PE with its superresolution ability (8, 9). In general, splitter (PBS) into two beam paths whose relative powers can continuous wave (CW)-STED (10) is preferred for 2PE imaging be tuned by a half-wave plate in front of the PBS. Now, the due to a simplification of the experimental setup at the expenses one used for two-photon excitation has to dodge stimulated emis- of an increased cost. Unfortunately, the use of two distinct sion depletion whereas the other, quenching the fluorescence wavelengths for excitation and depletion requires mostly a special excited by 2PE, has to avoid priming any linear or nonlinear optical filter design making the setup invariable in terms of the excitation. Therefore, in order to perform superresolved imaging, choice of the marker dye, and potential light beam distortions the two beams have to differ in about three orders of magnitude have to be treated separately. Working with only just one wave- length for both excitation and STED, one directly simplifies the Author contributions: P.B. and A.D. designed research; P.B. performed research; P.B., B.H., image formation scheme. In this paper, we propose a method to S.G., G.V., and A.D. analyzed data; and P.B. wrote the paper. perform 2PE-STED imaging using a single wavelength (SW) and, The authors declare no conflict of interest. consequently, the very same laser source for 2PE and depletion. This article is a PNAS Direct Submission. Such a SW approach is in agreement with early reported spectral Freely available online through the PNAS open access option. evidences for different dyes (11, 12). 1To whom correspondence should be addressed. E-mail: [email protected].

6390–6393 ∣ PNAS ∣ April 24, 2012 ∣ vol. 109 ∣ no. 17 www.pnas.org/cgi/doi/10.1073/pnas.1119129109 Downloaded by guest on September 23, 2021 We evaluated the excitation induced by the STED beam and the efficiency of nonlinear excitation and of the depletion as func- SCIENCES APPLIED PHYSICAL

Fig. 1. (A) Simplified Jablonski diagram showing the states involved in 2PE-STED single wavelength. (B) Scheme of the setup. P, polarizer; DL, delay line; VPP, vortex phase plate.

in pulse widths and of more than two orders of magnitude with respect to the intensity. For these reasons, we pointed most of the power for the beam path which is devoted to act as the STED beam. We stretched its pulses with 60-cm glass rod and 100-m -maintaining , reaching about 250 ps pulse length. On this beam path, before entering the fiber, we placed an optical delay line, made by a retroreflector mounted on a motorized linear stage, in order to temporally overlap the pulses of the two beam patches in the focal area. We chose the final position of the delay line by optimizing the maximum fluorescence depletion obtained in an aqueous dye solution. For the two-photon excitation beam path we kept the time pulses as short as possible (ca. 220 fs, Fig. 1B), monitoring them at the microscope scanning head entrance by an FR-103WS autocorrelator (Femtochrome Research). Two- photon excitation and STED beam pathways are recombined by a PBS before entering the infrared port of the confocal unit of the microscope (Leica TCS STED-CW; ). Results In classical STED microscopy, the wavelength of the STED beam has to be chosen taking into account two competing effects, namely, the efficiency of STED decreases when moving far away from the emission peak and the probability to excite directly the Fig. 2. (A) 1PE spectrum by means of the STED beam (black line), 2PE spec- fluorophore increases when moving close to the emission peak. trum (red dots), and depletion effect on the nonlinear excited fluorescence Because in the SW implementation the excitation beam relies (green triangles) of ATTO647N. In the inset, the complete 1-photon excitation B on the same wavelength, one has to consider that the cross-sec- spectrum of ATTO647N. ( ) Fluorescence intensity versus 2PE laser beam tion of two-photon excitation has to be maximized for the specific power (red dots), effect of the STED beam on the nonlinear excited fluores- cence (green triangle), and ratio between the former and the latter, respec- fluorophore being used in order to provide an adequate bright- tively (blue squares). (C) Depletion of the fluorescence, 2P excited and ness. Thus, we performed experiments overlaying the 2PE and P depleted at 770 nm, as a function of the STED beam power STED, corrected I ∼ the STED beams, both Gaussian shaped, in an aqueous solution for direct excitation of the depletion beam. Measurement of sat of ATTO647N (ATTO-TEC). 10 MW cm−2 is comparable with those already published.

Bianchini et al. PNAS ∣ April 24, 2012 ∣ vol. 109 ∣ no. 17 ∣ 6391 Downloaded by guest on September 23, 2021 tion of wavelength and power. Fig. 2A reports the wavelength de- ging with the same scheme should be possible. The efficiency of pendencies while keeping the 2PE and STED intensities con- the stimulated emission process requires quantitative analysis by I stant. The black line shows the fluorescence generated by the measuring the saturation intensity sat, defined as the intensity STED beam without the presence of the 2PE beam and well re- needed to deplete half of the excited (6). The mea- I ∼ 10 −2 presents the red tail of the one photon excitation spectra of AT- sured value of sat MW cm is in agreement with previously TO647N. The trend of the fluorescence excited by the 2PE beam reported results for ATTO647N (16). shows that, between 720 and 740 nm, the dominating effect is one Under a typical imaging configuration, the focused excitation photon event. After that, the trend deviates from the linear ex- beam is superimposed by a donut-shaped STED beam, hereby citation graph and shows a relative peak around 840 nm. For com- realized by the use of a vortex phase plate (RPC Photonics) in parison we plotted (Fig. 2A, Inset) the linear excitation spectra of the STED path (Fig. 1). We then compared SW 2PE-STED ima- ATTO647N (data taken from ATTO-TEC) that interestingly ging mode (Fig. 3C) with classical confocal (Fig. 3B) and 2PE shows a peak around 420 nm matching the 2PE peak. Within imaging (Fig. 3A) of Potoroo kidney line (PTK-2) cells in the same experiment series, we measured the fluorescence beha- which are immunolabeled with ATTO647N. The vior while overlaying the 2PE and the STED beam (green trian- raw data immediately confirm the benefit of the SW 2PE-STED gles, Fig. 2A). An efficient reduction of fluorescence under STED technique—i.e., the resolution is remarkably enhanced. Measur- condition is observed for wavelengths greater than 750 nm. ing the FWHM of several microtubules, we determined that the Fig. 2B shows the log–log plot of the fluorescence intensity in resolution achieved by the current implementation of SW 2PE- dependence on the 2PE power while keeping STED power and STED is approximately 80 nm. This limit is of experimental ori- STED wavelength constant. The graph for the 2PE, in absence gin, mainly due to the maximum power available in our setup. of any STED beam (red dots), clearly shows the expected slope Further optimization, in terms of the optical STED beam path- for two-photon excitation (slope ¼ 1.7 0.1). Superimposing a way, will increase the STED effect, hence the resolution. It is STED beam to the same measurement (green triangles, Fig. 2B) worth noting that substructures within the cellular complex of shows the limitations of the excitation power value in order to the cytoskeleton can only be resolved using the superresolution perform high-resolution imaging: For excitation powers greater achievable by the SW 2PE STED technique, whereas both con- than 2.5 mW,the fluorescence generated by the STED beam itself focal and 2PE imaging cannot reveal such structures. In fact the is, by way of comparison, so high that STED imaging would not be resolution gain hereby attained is three to four times with respect possible under such conditions. For excitation power values to confocal and up to four to five times with respect to the best greater than 4 mW, the fluorescence value can be sufficiently re- 2PE performances. duced by superimposing the STED beam. For higher excitation powers, the depletion efficiency (blue squares, Fig. 2B) becomes Discussion and Outlook almost constant, although at higher power level saturation of the We have developed a class of 2PE-STED using the very same excited state becomes relevant. wavelength for excitation and depletion. We have demonstrated We analyzed the depletion efficiency by measuring the fluor- that this method allows superresolved imaging on biological spe- escence value depending on the STED beam intensity as reported cimens employing the very common fluorophore ATTO647N, in Fig. 2C. Here, the 2PE power was kept constant. The curve achieving a resolution four to five times better than conventional shows the fluorescence reduction due to the STED effect, repre- 2PE microscopy. The single-wavelength approach simplifies the senting a key experiment for this approach. In fact, because the optical scheme in terms of number of dichroic filters and laser fluorescence value results effectively reduced by using the same sources, allowing an easy coupling to a conventional commercial wavelength for excitation and STED beam, high-resolution ima- confocal microscope.

Fig. 3. 2PE-STED single-wavelength microscopy shown in comparison with confocal and 2PE microscopy (A–C). (A) Confocal (excitation at 633 nm), (B) 2PE (excitation at 770 nm), and (C) 2PE-STED (excitation and depletion at 770 nm) micrographs of microtubules immunostained with the dye ATTO647N. (Scale bars: 5 μm.) The lower panels respectively show the raw data in the corresponding boxed areas and the plots of a line profile (black squares) along the arrows indicated in the zoomed images, together with a multiple peak fit (red). (Scale bars: 1 μm.) We acquired the images with a pixel size of 32 × 32 nm, a pixel dwell time, respectively, of 8 (A), 65 (B), 65 μs(C), and a pinhole size, respectively, of 1 (A), 2 (B), 2 airy unit (C). The powers of 2PE and STED beam were 12 and 168 mW, respectively, measured at the back plane.

6392 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1119129109 Bianchini et al. Downloaded by guest on September 23, 2021 The broadening of the cross-section in the 2PE regime makes trol distortions when imaging thick, highly scattering specimens. the microscope more flexible for multicolor high-resolution Although the resolution achieved at certain depths essentially de- imaging, which allows the simultaneous combination of STED pends on the intensity that can be brought at the focal plane by microscopy with conventional 2PE and second harmonic genera- the STED beam, we believe that this imaging technique will allow tion microscopy. Recently, it has been demonstrated that the envisaging developments toward bioimaging of thick samples at STED microscopy can be applied to the imaging of mouse brain nanoscale resolution. (17, 18). Because scattering mainly depends on the wavelength, using the very same wavelength for excitation and depletion, the ACKNOWLEDGMENTS. We thank Christian Wurm (Department of Nanobio- aberration introduced by the specimen should eventually affect photonics, Max Planck Institute for Biophysical Chemistry) for the staining both beams in the same manner. Therefore the SW 2PE-STED of the specimens. This work was partially funded by the Italian Programmi microscopy seems a promising technique to better actively con- di Ricerca d Rilevante Interesse Nazionale 2008JZ4MLB grant.

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Bianchini et al. PNAS ∣ April 24, 2012 ∣ vol. 109 ∣ no. 17 ∣ 6393 Downloaded by guest on September 23, 2021