New developments in STED Microscopy

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1 New developments in STED Microscopy Arnold Giske*, Jochen Sieber, Hilmar Gugel, Marcus Dyba, Volker Seyfried, Dietmar Gnass Leica Microsystems CMS, Am Friedensplatz 3, Mannheim, Germany ABSTRACT STED microscopy has gained recognition as a method to break the diffraction limit of conventional light microscopy. Despite being a new technique, STED is already successfully implemented in life science research. The resolution enhancement is achieved by depleting fluorescent markers via stimulated emission. The performance is significantly dependent on the laser source and the fluorescence markers. Therefore the use of novel fluorescent markers in conjunction with the right laser system was the main focus of our research. We present new developments and applications of STED microscopy, unraveling structural details on scales below 90nm and give an overview of required specifications for the solid state laser systems. Keywords: fluorescence, microscopy, superresolution, imaging 1. INTRODUCTION Fluorescence microscopy features many different advantages, the greatest of all is the ability to look for the desired structure or function inside the cells noninvasively. Usually the mammal cells are transparent in the visible range of the spectrum, thus giving low contrast in light microscopy. This issue can be overcome by using fluorescent markers, where a fluorescent dye molecule is specifically chemically bound to a protein or structure of interest [1]. The intrinsic spectral shift between excitation and emission spectra in the fluorescence, called Stokes shift, allows a clear separation of fluorescence light from any reflection caused by the deployed laser lines by using appropriate spectral blocking filters. The labeling technique to introduce fluorescent dye molecules into the cells often requires a disintegration of the cell membrane and is usually only applicable for fixed cells. The discovery of fluorescent proteins, which are produced inside living cell opened up a new dimension of applications for fluorescence microscopy in living tissue [2]. Living cell staining ability combined with the gentleness of an optical microscope opened up a new field of applications, and allowed observing dynamics of living cells. Unfortunately, the use of light microscopy has a drawback in terms of final resolution. Due to the diffraction, it is impossible to focus light to a smaller spot, than approximately the half of its wavelength. The more general expression of diffraction limited resolution was formulated by Ernst Abbe in following form: λ Δ d = 2NA where λ is the wavelength of light and NA is numerical aperture of used lens [3]. The resolution limit in far-field optical systems, even with highly developed optical lenses, is limited to a value around 200 nm. In past decades, few new approaches have been elaborated in order to break the diffraction limit in optical far-field fluorescence microscopy. These approaches use the fact, that fluorescent markers can be forced into a non fluorescent state, or at least in a distinguishably different state. In STimulated Emission Depletion (STED) microscopy, the excited state is depopulated by forced stimulated emission into the ground state, thus these molecules do not contribute to the detected fluorescence image [4]. In a simple picture, the stimulated emission helps to sharpen the fluorescence spot be turning off the fluorescent markers in the periphery of the initial diffraction limited spot, thus breaking the resolution limit by photophysical properties of the fluorescent dye. The resolution enhancement in STED microscopy is depending on the efficiency of the stimulated emission process and how much STED intensity is available. To gain a resolution increase, we deploy two synchronized lasers with different intensity distributions, called point spread function (PSF). The first laser, well matched to the fluorescence excitation spectrum of the used fluorescent marker, is used in the conventional way with typical spatial distribution featuring a maximum in the center. The second Solid State Lasers XIX: Technology and Devices, edited by W. Andrew Clarkson, Norman Hodgson, Ramesh K. Shori, Proc. of SPIE Vol. 7578, 75781X 2010 SPIE CCC code: X/10/$18 doi: / Proc. of SPIE Vol X-1

2 laser is used as stimulated emission source and has a non-trivial intensity distribution featuring a zero in the center and a homogeneously increasing intensity around zero, resulting in a ring-like structure. Stimulated emission is triggered and the fluorescence is quenched efficiently everywhere outside the zero of the STED PSF. Thus, the remaining fluorescence stems from the position of the zero inside the stimulated emission laser spot, delivering fluorescence images with strongly improved spatial resolution. The key feature of a STED microscope is the resolution enhancement combined with the versatility of a confocal microscope. All applications suitable for a confocal system can be principally performed with enhanced STED resolution. How far the resolution can be squeezed down, is dictated by the interplay of the properties of dyes and used lasers. In last decade, many dyes have been shown to work properly for STED imaging in all parts of the spectrum. So far, the efficiency for stimulated emission for at least well known standard dyes appears to be in the same order of magnitude, so the resolution enhancement nowadays is driven by the properties of the laser. 2. METHODOLOGY There are different factors which determine the efficiency of stimulated emission. First, there must be a spectral overlap between the spontaneous fluorescence and the deployed STED laser line, the efficiency is proportional to the spectrum of the spontaneous fluorescence [5]. Since the effect of resolution enhancement is maximal when STED laser only deplete fluorescence, the STED wavelength must be selected in respect to the excitation spectrum of the molecule: when the cross section of the fluorescence excitation spectrum and STED laser line becomes non zero, STED beam directly excites fluorescence and this process slowly consumes the resolution enhancement. Second, the power of the STED laser line dictates the resolution enhancement. The intensity distribution around the zero inside the STED PSF increases quadratically in spatial dimension, thus using an approximation one can derive the formula for the resolution in STED microscopy Δd 2NA where NA is numerical aperture, λ is the wavelength, I is the intensity of the STED laser line and Isat is proportional to the efficiency of stimulated emission of the used dye [6]. The formula indicates the dependency of the achievable resolution on the intensity of STED laser to be the square root of the intensity I for very high intensities. Therefore nowadays, the resolution is primarily limited by the available STED laser intensity. The basic concept of STED microscopy and its implementation into a confocal system are presented in figure 1. The used fluorescent marker, represented by the chemical structure in subfigure 1a, is excited by incident blue photons and delivers after usual mean fluorescence lifetime a photon of green emission color. In STED microscopy a second photophysical process is introduced, where the green spontaneous fluorescence is efficiently inhibited by induced emission, triggered from STED laser source. This process of stimulated emission can be very efficient for well matched dye and STED wavelength combinations, as presented in subfigure 1b. Here we show a typical fluorescence behavior under illumination with STED laser with sweeping intensity. A schematic of STED mode implementation into a confocal system is shown in subfigure 1c. We are using standard Ar laser with most prominent 488 nm and 514 nm lines for fluorescence excitation of green and green-yellow markers, coupled into a microscope by using appropriate beam splitter systems (dichroic mirrors combination or high-end acousto-optical systems). The STED laser, providing high power 592 nm laser line, is coupled over a dichroic mirror and aligned with respect to the excitation and detection beam path by automatic procedure. The non-trivial ring-like shape of the STED PSF is created by a helical phase distribution plate and circular polarization. This combination ensures the highly symmetrical fluorescence spot shape and features lowest experimentally achievable zero values [7]. The STED light, shaped into a ring-like distribution ensures that the fluorescent markers remain dark in the periphery of the confocal fluorescing volume, thus shaping a much sharper resulting fluorescence intensity distribution. The FWHM of this dynamically achieved fluorescence distribution is the resolving power of the STED microscope. λ 1+ I I sat, Proc. of SPIE Vol X-2

3 Figure 1. The basic concept of STED Microscopy is shown. The subfigure (a) displays the two competing processes in the STED Microscopy, where the usual fluorescence process can be inhibited by induced emission with a second laser source. The two processes can be marked as bright and dark states, when the detection is sensitive for fluorescence light only. Subfigure (b) shows a typical efficiency curve for stimulated emission, implying that the use of more STED intensity will provide higher saturation levels in STED microscopy. Subfigure (c) shows the schematic for the laser implementation into a confocal microscope, where 1 is excitation laser, 2 is the STED laser source, 3 is the helical phase distribution filter, 4 is confocal detection with an appropriate fluorescence filter, 5 is an objective lens, 6 is the fluorescence intensity distribution stemming from single spot in confocal mode, 7 is the ring like STED PSF, inhibiting the fluorescent markers from fluorescing in the periphery of the initial confocal volume and 8 is the resulting fluorescence distribution in STED mode with significantly reduced volume. In principle, a STED microscope can be designed with a pulsed laser as well as with a CW laser, there are however some aspects which have to be taken into account [8]. The event of stimulated emission has to occur immediately after the molecule is in the excited state and before the fluorescent transition. In the case of pulsed lasers, the excitation and STED sources have to be perfectly synchronized in respect to each other, the STED pulse has to follow the excitation pulse almost immediately. For CW lasers, the synchronization is obsolete and excitation and stimulated emission compete with each other all the time. In case of pulsed systems, the intensity of the STED laser is concentrated during one pulse, perfectly depleting the fluorescence immediately after excitation pulse. In CW mode however, the STED intensity is equally distributed over time, thus one excited molecule senses only a fraction of STED intensity. The difference in needed STED intensity for the same resolution enhancement is given by the product of the mean fluorescence lifetime with reciprocal value of the repetition rate. Considering an available pulsed system with 80 MHz repetition rate and Proc. of SPIE Vol X-3

4 mean fluorescence lifetimes between 3 4 ns, CW mode needs up to 3 4 times more average intensity for the same resolution enhancement. In terms of how many fluorescence photons can be harvested during one time period, CW solution becomes more advantageous. Due to the fact, that at usually available repetition rates, the intermediate pulse time is much greater than the mean fluorescence lifetime, there cannot be any fluorescence excited in between the pulses. In case of CW lasers, fluorescent molecules can be excited continuously and thus cycled more often. The empirical difference between the pulsed and the CW modes is a product of fluorescence lifetime with reciprocal value of the repetition rate. As above, the use of CW lasers can deliver up to 3 4 times more signal in the same time period, enabling faster acquisition times. We realized a STED setup with CW lasers, using an Ar gas laser with several excitation lines in the blue green range of the visible spectrum, and a 592nm CW fiber laser as a depletion source. The beam splitter directs the excitation light onto a point scanner, the returned fluorescence light is focused onto a pinhole of the size of 1 Airy, thus providing confocal detection. The 592 nm STED laser is coupled into the beam path via an appropriate beam splitter. The use of a helical phase plate in the STED beam path, combined with a circular polarization distribution of excitation and STED light, guaranties a zero intensity value in the center of the STED PSF. The images are recorded with an oil objective lens with high numerical aperture of 1.4. Immunostained structures inside cells are used for the demonstration of resolution enhancement. Different green-yellow fluorescent dyes have been studied under STED conditions. The major criteria of examination were the achievable resolution and the photostability of dyes. Also few fluorescent proteins, mostly derivatives from YFP, were examined. 3. RESULTS The fluorophores are excited with an Ar line, 488 nm in case of Chromeo 488 and Alexa 488 and 514 nm in case of eyfp. We record two images in parallel line by line acquisition. During the acquisition of the second image STED laser is activated at full power. The overall acquisition time of two images lies around 1-5 seconds, depending on the brightness of the fluorescent staining. We present images acquired with STED CW system with different fluorescent markers. Figure 2 demonstrates the difference in resolution between confocal and STED imaging with different markers. Subfigure 2a shows strong resolution increase on vimentin fibers inside a fixed PtK2 cell, marked with Chromeo 488. The image recorded in STED mode shows much more structural details compared to confocal image. The line profile along the white bar is shown on right hand side indicating the resolving power of STED CW system. The measured FWHM here is 70 nm. Considering the fact that the vimentin fiber has a certain size by itself, the resolution in STED mode must be better than 70 nm. One of findings during this work is that many different fluorescent dyes are working good with STED CW. Subfigure 2b shows a comparison between a confocal and STED image recorded from microtubuli stained with Alexa 488. Here again, the line profile indicates the resolving power being below 75 nm. Similar results could be achieved with other fluorescent dyes, such as Oregon Green, Atto 488 and FITC. Especially the last candidate, FITC, well known for its bleaching behavior, turns out to work quite well with STED CW. All listed dyes show similar resolution enhancement and perform at same level regarding the bleaching behavior. The bleaching rate are sufficiently low, allowing acquisition of several consecutive images, e.g. for recording z-stacks. Especially Chromeo 488 fluorescent dye shows slightly higher photostability under STED condition compared to other listed dyes. Subfigure 2c shows a comparison of confocal and STED mode recorded with a fluorescent protein, which was produced by the cell itself. Here, the genetic code was manipulated and cells produced yellow fluorescent protein (eyfp) at certain structure. Afterwards, the cells were fixed for imaging. The used 592 nm STED laser source spectrally fits to the eyfp and enables imaging in STED mode with strongly enhanced resolution, as shown in subfigure 2c. The STED image clearly reveals structural details which weren t visible in confocal image. The line profile indicates here again that the achievable resolution is around 80 nm. In principle, the eyfp can be used as a marker inside living cells providing a great possibility to observe dynamics in living cells with substantially higher spatial resolution below the diffraction limit. Among other studied yellow fluorescent proteins, the most promising results could be obtained with Citrin and Venus fluorescent proteins. Proc. of SPIE Vol X-4

5 Figure 2. Different examples of fluorescent markers in comparison between confocal and STED mode. Subfigure 2a shows the vimentin fiber network inside a PtK2 cell labeled with Chromeo 488. The image recorded in STED mode clearly unveils more structural details compared to confocal image. Detailed evaluation of the fiber size is shown in a line profile, indicating a high resolution increase. The measured FWHM value shows 70 nm, regarding the fact, that the fiber has a finite size by itself, the resolution must be < 70 nm. The subfigure 2b shows a different kind of fiber network, microtubule labeled with Alexa 488. Here again the STED image shows much more structural details. The line profile indicates a resolution enhancement of < 75 nm. The two presented dye markers are only capable for imaging of fixed cells, the subfigure 2c shows example images with eyfp. eyfp is a protein with capability for live cell imaging, here, however, the cells are fixed. The STED image clearly shows a resolution increase to < 80 nm. Proc. of SPIE Vol X-5

6 4. CONCLUSION The implementation of CW lasers into STED microscope simplifies the technical design due to absence of matching of excitation and depletion pulses. Thus, the use of CW lasers provides a technically most robust solution for implementation of STED concept into a confocal imaging system. The achievable resolution with this design is below 80 nm for all listed dyes. Additionally, STED CW features high acquisition rates due to continuous excitation light distribution. The use of orange STED laser line spectrally fits to standard green dyes, such as Alexa 488 or Oregon Green, providing access to superresolution without changing standard labeling protocols. The ability to image YFP opens up the possibility of imaging inside living cells below the confocal resolution limits [9]. Deployment of fast scanning techniques, a resonant scanning mode, opens up the possibility to observe dynamical processes in living cells as well as providing user with very fast acquisition platform for standard imaging with immonafluorescently labeled cells [10]. The advance of more blue and green fluorescent markers will extend the field of possible applications for STED microscopy at highest level of spatial resolution. ACKNOWLEDGMENTS This work has been supported by the German Ministry for Research and Education (BMBF, Biophotonic III). Samples were kindly provided by Max-Planck-Institute for biophysical Chemistry, Goettingen, Germany and the Institute for molecular and cellular anatomy at RWTH Aachen University, Germany. REFERENCES 1. Osborn, M., W. W. Franke and K. Weber. "Visualization of a system of filaments 7-10nm thick in cultured cells of an epithelioid line (PtK2) by immunofluorescence microscopy" PNAS 74: (1977). 2. Chalfie, M. Y. Tu, G. Euskirchen, W. W. Ward and D. C. Prasher. "The green fluorescent protein as a marker for gene expression" Science 263: (1994). 3. Abbe, E. "Beitraege zur Theorie des Mikroskops und der mikroskopischen Wahrnehmnung " Archiv fuer Mikroskopische Anatomie 9: (1873). 4. Hell, S. W. and J. Wichmann. "Breaking the diffraction resolution limit by stimulated emission" Opt. Lett. 19(11): (1994). 5. Rittweger, E., B.R. Rankin, V. Westphal, S.W. Hell. "Fluorescence depletion mechanisms in super-resolving STED microscopy" Chem. Phys. Lett. 442, (2007). 6. Westphal, V., and S. W. Hell. "Nanoscale Resolution in the Focal Plane of an Optical Microscope" Phys. Rev. Lett. 94: (2005). 7. Harke, B., J. Keller, C. K. Ullal, V. Westphal, A. Schönle, S. W. Hell: "Resolution scaling in STED microscopy". Opt. Expr. 16 (6), (2008) 8. Willig, K. I., B. Harke, R. Medda, S. W. Hell. "STED microscopy with continuous wave beams" Nature Meth. 4 (11), (2007). 9. Hein, B., K. I. Willig, S. W. Hell. "Stimulated emission depletion (STED) nanoscopy of a fluorescent proteinlabeled organelle inside a living cell" PNAS 105 (38), (2008) 10. Moneron, G., R. Medda, B. Hein, A. Giske, V. Westphal, S. W. Hell: "Fast STED microscopy with continuous wave fiber lasers". Opt. Exp. 18 (2), (2010) Proc. of SPIE Vol X-6