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Virtual Journal for Biomedical Optics

Virtual Journal for Biomedical Optics

| EXPLORING THE INTERFACE OF LIGHT AND BIOMEDICINE

  • Editors: Andrew Dunn and Anthony Durkin
  • Vol. 9, Iss. 5 — Apr. 29, 2014
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Large parallelization of STED nanoscopy using optical lattices

Bin Yang, Frédéric Przybilla, Michael Mestre, Jean-Baptiste Trebbia, and Brahim Lounis  »View Author Affiliations


Optics Express, Vol. 22, Issue 5, pp. 5581-5589 (2014)
http://dx.doi.org/10.1364/OE.22.005581


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Abstract

As a scanning microscope, STimulated Emission Depletion (STED) nanoscopy needs parallelization for fast wide-field imaging. Using well-designed optical lattices for depletion together with wide-field excitation and a fast camera for detection, we achieve large parallelization of STED nanoscopy. Wide field of view super-resolved images are acquired by scanning over a single unit cell of the optical lattice, which can be as small as 290 nm * 290 nm. Optical Lattice STED (OL-STED) imaging is demonstrated with a resolution down to 70 nm at 12.5 frames per second.

© 2014 Optical Society of America

1. Introduction

Recent developments in super-resolution microscopy techniques [1

1. S. W. Hell, “Microscopy and its focal switch,” Nat. Methods 6(1), 24–32 (2009). [CrossRef] [PubMed]

5

5. M. G. L. Gustafsson, “Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy,” J. Microsc. 198(2), 82–87 (2000). [CrossRef] [PubMed]

] achieved nanometer scale resolution and showed great potential in live cell imaging. Techniques based on single molecule localization [3

3. M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (2006). [CrossRef] [PubMed]

,4

4. E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006). [CrossRef] [PubMed]

,6

6. S. T. Hess, T. P. K. Girirajan, and M. D. Mason, “Ultra-High resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J. 91(11), 4258–4272 (2006). [CrossRef] [PubMed]

10

10. J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell, “Fluorescence nanoscopy by ground-state depletion and single-molecule return,” Nat. Methods 5(11), 943–945 (2008). [CrossRef] [PubMed]

] and structured illumination (SIM) [5

5. M. G. L. Gustafsson, “Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy,” J. Microsc. 198(2), 82–87 (2000). [CrossRef] [PubMed]

,11

11. R. Heintzmann, T. M. Jovin, and C. Cremer, “Saturated patterned excitation microscopy--a concept for optical resolution improvement,” J. Opt. Soc. Am. A 19(8), 1599–1609 (2002). [CrossRef] [PubMed]

,12

12. M. G. L. Gustafsson, “Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution,” Proc. Natl. Acad. Sci. U. S. A. 102(37), 13081–13086 (2005). [CrossRef] [PubMed]

] are intrinsically parallelized because they use wide-field illumination and cameras for detection. The first techniques require a low density of simultaneously emitting single molecules and therefore need a large number of frames for super-resolved image reconstruction, which limits their imaging speed. The second rely on sophisticated data post-processing, and consequently require high signal to noise camera frames accumulated over long integration times. Because it requires fewer raw data images, linear SIM has showed its potential for video rate imaging but it can only improve the resolution by a factor two of the diffraction limit [13

13. P. Kner, B. B. Chhun, E. R. Griffis, L. Winoto, and M. G. L. Gustafsson, “Super-resolution video microscopy of live cells by structured illumination,” Nat. Methods 6(5), 339–342 (2009). [CrossRef] [PubMed]

]. Nonlinear SIM can achieve better resolutions but at the expense of imaging speed since it requires a larger number of raw images per time frame [14

14. E. H. Rego, L. Shao, J. J. Macklin, L. Winoto, G. A. Johansson, N. Kamps-Hughes, M. W. Davidson, and M. G. L. Gustafsson, “Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution,” Proc. Natl. Acad. Sci. U. S. A. 109(3), E135–E143 (2012). [CrossRef] [PubMed]

].

STED [2

2. S. W. Hell and J. Wichmann, “Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy,” Opt. Lett. 19(11), 780–782 (1994). [CrossRef] [PubMed]

,15

15. E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, and S. W. Hell, “STED microscopy reveals crystal colour centres with nanometric resolution,” Nat. Photonics 3(3), 144–147 (2009). [CrossRef]

18

18. V. Westphal, S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, and S. W. Hell, “Video-rate far-field optical nanoscopy dissects synaptic vesicle movement,” Science 320(5873), 246–249 (2008). [CrossRef] [PubMed]

] nanoscopy is a scanning technique requiring only a limited number of fluorescence photons, which can be acquired at short pixel exposure times, to locate the position of the emission. Since it is a coordinate-targeted method, it does not need sophisticated data processing. However, STED and more generally RESOLFT [19

19. S. W. Hell and M. Kroug, “Ground-state-depletion fluorescence microscopy: A concept for breaking the diffraction resolution limit,” Appl. Phys. B 60(5), 495–497 (1995). [CrossRef]

21

21. T. Grotjohann, I. Testa, M. Reuss, T. Brakemann, C. Eggeling, S. W. Hell, and S. Jakobs, “rsEGFP2 enables fast RESOLFT nanoscopy of living cells,” eLife 1, e00248 (2012).

] (REversible Saturable OpticaL Fluorescence Transitions) need parallelization in order to fully benefit from this temporal resolution for fast wide-field imaging.

A straightforward approach to STED parallelization is based on focused beam multiplication. A configuration using four pairs of scanning excitation and doughnut-STED beams, together with four avalanche photodiodes has been reported [22

22. P. Bingen, M. Reuss, J. Engelhardt, and S. W. Hell, “Parallelized STED fluorescence nanoscopy,” Opt. Express 19(24), 23716–23726 (2011). [CrossRef] [PubMed]

]. However, scaling the parallelization further up with this approach is mainly limited by experimental complexity and by the power of the available laser sources. Another approach for parallelization is based on structured illumination pattern. RESOLF parallelization has been proposed using 1D interference pattern, but the resolution improvement is only obtained along one direction [20

20. M. Hofmann, C. Eggeling, S. Jakobs, and S. W. Hell, “Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins,” Proc. Natl. Acad. Sci. U. S. A. 102(49), 17565–17569 (2005). [CrossRef] [PubMed]

]. Recently methods for massive parallelization of RESOLFT with photo-switchable proteins [23

23. A. Chmyrov, J. Keller, T. Grotjohann, M. Ratz, E. d’Este, S. Jakobs, C. Eggeling, and S. W. Hell, “Nanoscopy with more than 100,000 ‘doughnuts’,” Nat. Methods 10(8), 737–740 (2013). [CrossRef] [PubMed]

] and STED nanoscopy [24

24. B. Yang, F. Przybilla, M. Mestre, J.-B. Trebbia, and B. Lounis, “Massive parallelization of STED nanoscopy using optical lattices,” arXiv:1307.3833 [physics] (2013).

] based on the use of 2D structured illumination have been reported. Larger field of view could be achieved for parallelized RESOLFT using photo-switchable fluorescent proteins, because it requires less intensity to switch. However, protein switching is a relatively slow on-off process (~10 ms), which sets a limit to the imaging acquisition rate. Moreover RESOLFT with photo-switchable fluorescent proteins is constrained in its versatility by the need for genetic modification and transfection.

In this article, we show how well-designed optical lattices [25

25. A. Hemmerich and T. W. Hänsch, “Two-dimesional atomic crystal bound by light,” Phys. Rev. Lett. 70(4), 410–413 (1993). [CrossRef] [PubMed]

28

28. M. P. MacDonald, G. C. Spalding, and K. Dholakia, “Microfluidic sorting in an optical lattice,” Nature 426(6965), 421–424 (2003). [CrossRef] [PubMed]

] created by multi-beam interference can provide efficient depletion patterns with moderate laser power and can be used for large parallelization of STED, so far the most important and widely used RESOLFT technique. The stimulated emission depletion being an ultrafast on-off switching process (~1 ns), its imaging speed is therefore only limited by the number of detected photons and fast large field of view super-resolution imaging can be achieved.

2. Experimental setup and the generation of optical lattices

Our OL-STED microscopy setup, sketched in Fig. 1
Fig. 1 OL-STED experimental setup. A depletion beam (red) is split with an SLM into 3 or 4 beams, or with a combination of two Wollaston prisms into 4 beams. The beams sent through an objective interfere at the sample plane to form an optical lattice. An excitation beam (green) of ~10 µm * 10 µm size is overlaid with the lattice. A CMOS camera conjugated with the sample plane records the wide-field fluorescence images.
, is based on two synchronized laser sources delivering excitation and depletion pulses, akin to a standard STED microscope [29

29. B. Harke, J. Keller, C. K. Ullal, V. Westphal, A. Schönle, and S. W. Hell, “Resolution scaling in STED microscopy,” Opt. Express 16(6), 4154–4162 (2008). [CrossRef] [PubMed]

]. The fluorescence excitation beam (wavelength 571 nm and pulse duration 2 ps) is delivered by a frequency doubled optical parametric oscillator (Mira-OPO, Coherent) pumped by picosecond Ti-sapphire laser (Mira 900, Coherent) at a repetition rate of 76 MHz. A second Ti-sapphire active mode-locked laser (Tsunami, Spectra Physics) emitting at 760 nm, provides the depletion beams. It is synchronized with the excitation laser and delivers Fourier transform limited pulses of ~100 ps duration. A Spatial Light Modulator (SLM LCOS, Hamamatsu) or a set of two Wollaston prisms, conjugated with the sample plane, is used to generate multiple depletion beams. The laser beams are focused on the back focal plane of a high numerical aperture objective (Nikon Apo TIRF 60x NA = 1.49) and illuminate a wide-field region of the sample (~3 µm). The total magnification of the system is 225x, with extra 1.5x and 2,5x added after the tube lens of the microscope. An optical lattice is produced by the interference of the depletion beams at the sample plane and is overlaid with a uniform excitation beam. Two dichroic filters are used for illumination (Chroma 605 DCXR and Chroma T700DCSPXXR-UV). The fluorescence emission is collected with the same objective, filtered from excitation and depletion photons using filters (Semrock, Bandpass 641/75 and Shortpass 720) and sent to a fast CMOS camera (ORCA-Flash4.0, HAMAMATSU). A fast piezo-scanner (PI P-733.3DD) is used for sample scanning.

Figure 2
Fig. 2 Design of the optical lattices. Top left panel, 3 beams, parallel to the optical axis of the objective, intersect its back focal plane at the vertices of a centered equilateral triangle. The beams have the same linear polarization, parallel to one of the three sides of the triangle. After passing through the objective they are deviated by an angle towards the focal region where they interfere. (a) Intensity profile of the hexagonal optical lattice calculated for θ = 60° showing a suitable depletion pattern for STED parallelization. (b) Fluorescence depletion pattern recorded with a 100 nm fluorescent bead scanned in the interference pattern (2.8 µm * 2.8 µm) in the presence of the excitation beam. Bottom left panel, The 4 beam configuration obtained with two pairs of beams (one pair of beams polarized along the x axis and the other pair along the y axis) interfering independently. (c) Intensity profile of the obtained square optical lattice calculated for the same θ as in (a). (d) The corresponding depletion pattern.
shows the optical lattice generated by three beams. The beams issued from the SLM with the same polarization are parallel to the optical axis of the objective, and intersect its back focal plane at the vertices of a centered equilateral triangle. The optical lattice depends on the beams’ polarization, and on θ, the angle formed by the optical axis and the beams emerging from the objective [30

30. J. L. Stay and T. K. Gaylord, “Three-beam-interference lithography: contrast and crystallography,” Appl. Opt. 47(18), 3221–3230 (2008). [CrossRef] [PubMed]

]. If the beams’ polarization is parallel to one of the three sides of the triangle, their interference produces a hexagonal lattice with a periodicity 2λ/(3n sinθ), where λ is the depletion laser wavelength and n~1.5 the sample refractive index. The intensity profile of the optical lattice, calculated for θ = 60° and displayed in Fig. 2(a), shows a suitable depletion pattern for STED parallelization: an array of zero intensity minima, each of which being surrounded by a nearly uniform high intensity region.

To probe the depletion pattern, we scan a big fluorescent bead (>100 nm) with a piezo-stage over the field of illumination in presence of both depletion and excitation beams, while recording its fluorescence with the CMOS camera. For each scanning step, an image is acquired and the fluorescence intensity, integrated over the Point Spread Function (PSF) of the bead image, is plotted in Fig. 2(b). The signal maxima of the image of Fig. 2(b) correspond to the regions of minimal depletion, which occur at the zero-intensity positions of the optical lattice. While this image provides information about the position of the intensity maxima and minima of the depletion pattern, the depth of the minimum can be obtained by scanning a gold nanoparticle [31

31. D. Wildanger, J. Bückers, V. Westphal, S. W. Hell, and L. Kastrup, “A STED microscope aligned by design,” Opt. Express 17(18), 16100–16110 (2009). [CrossRef] [PubMed]

] in the depletion pattern and recording its intensity scattering with an avalanche photodiode (data not shown). Intensity ratios between the minima and maxima less than 3% can be obtained.

As expected, we obtain a hexagonal lattice with periodicity of 390 nm. Our calculations show that for θ = 71°, the interference contrast is maximum (~1) and the intensity distribution around the minima is nearly isotropic in the sample plane [30

30. J. L. Stay and T. K. Gaylord, “Three-beam-interference lithography: contrast and crystallography,” Appl. Opt. 47(18), 3221–3230 (2008). [CrossRef] [PubMed]

]. However, at this incidence angle, the transmittance of our objective is low (few percent only). We choose θ = 60°, a compromise maximizing the transmittance of the objective (~30%) and the contrast of optical lattice (~0.98). The elongated shape observed on the bright spots of Fig. 2(b) can be explained by anisotropy of the intensity distribution around the minima when the pattern is constructed at θ deviating from the optimal value of θ = 71°.

3. Results and discussions

OL-STED images of various samples are obtained as follows (Fig. 3(a)
Fig. 3 OL-STED Images Acquisition. (a) a stack of a CMOS camera frames is acquired while the sample is scanned over a 290 nm x 290 nm optical lattice unit cell. A binary mask is applied to the frames. The “point-detectors” correspond to the white crosses on the mask, each of which records a unit cell image of the sample. (b) diffraction limited image of 20 nm fluorescent beads recorded without the depletion beam at an excitation power of 2 mW (15 x 15 points per unit cell). (c) Super-resolved OL-STED image of the same region in the presence of the depletion beam (total depletion power ~400 mW). (d) Normalized fluorescence intensity profiles measured from a single bead. (e) diffraction limited image of microtubules in a fixed cell (10 x 10 points per scan area, integration time 800 µs per point). (f) OL-STED image of the same region. (g) Normalized fluorescence intensity profiles (Cut along the dashed lines).
): First we scan the sample over a unit cell in the presence of the wide field excitation and the depletion pattern, while acquiring with the sCMOS camera a fluorescence image (128 * 128 pixels) for each scanning step. We then overlay a binary mask on these images, the transparent parts corresponding to the minima positions of the depletion pattern. Therefore, the CMOS camera together with the digital mask act as an array of parallelized “point detectors” (100 detectors, each giving the integrated signal of 13 camera pixels and recording an image of the size of the lattice unit cell). The complete OL-STED image is then obtained by assembling all the unit cell images together. We choose the total magnification of the microscope to be 225, so that the PSF spreads over more than 100 pixels (a pixel of the camera corresponds to ~29 nm at the sample plane). This increases the overall detection dynamics (higher detection saturation) and provides a better spatial discretization of the PSF. The number of camera pixels forming the point detector is chosen such that it optimizes the detection efficiency of a point emitter, minimizes the reading noise and ensures a low cross talk between two adjacent detectors. The ratio of the number of photons measured by the central detector to that measured by a neighboring one is less than 2%. This cross talk can be corrected while reconstructing the final STED images.

Figure 3(b) and 3(c) display 2.9 µm * 2.9 µm images of a sample containing 20 nm fluorescent beads spin-coated on a glass coverslip without and with the 4 beam depletion pattern. Figure 3(b) clearly shows that the PSF of the beads are diffraction limited ~290 nm, while the resolution of the OL-STED image of Fig. 3(c) is well below this limit (typically ~70 nm, see Fig. 3(d)). Since this resolution depends on the depletion intensity, it is better in the center of the pattern where the intensity is maximal. The resolution is ~1.5 less at the edges of the images where the intensity maxima are two times lower (the resolution scales as the inverse square root of the STED beam intensity [1

1. S. W. Hell, “Microscopy and its focal switch,” Nat. Methods 6(1), 24–32 (2009). [CrossRef] [PubMed]

]).

We apply OL-STED microscopy to image microtubules in fixed COS cells. The microtubules were stained using a standard immunofluorescence protocol involving a primary antibody (anti beta-tubulin) and a secondary antibody labeled with fluorescent Dyes (Atto647N). As shown in Figs. 3(e)3(g), the OL-STED gives super-resolved images of tubulin fibers. The resolution is below 100 nm and microtubules distant by less than the diffraction limit are clearly distinguished. Importantly, the acquisition time of an OL-STED image ~80 ms is only limited by the CMOS camera readout time and can therefore be further shortened using faster cameras.

4. Theoretical comparison of the resolutions of OL-STED and multi-doughnut STED

The OL-STED setup provides many advantages compared to the multi-doughnut configuration [22

22. P. Bingen, M. Reuss, J. Engelhardt, and S. W. Hell, “Parallelized STED fluorescence nanoscopy,” Opt. Express 19(24), 23716–23726 (2011). [CrossRef] [PubMed]

]. First, with few interfering beams and a single chip detector, we can easily obtain a large number of intensity minima and their corresponding “point detectors”. Secondly, for the same number of local minima and the same target resolution, the OL-STED setup requires up to six-fold less depletion power than a multi-doughnut STED setup (see calculations below). This is due to the fact that with interference one can achieve depletion intensities higher than that obtained for isolated donuts, and better-confined zero-intensity regions. Finally, the periodicity of the optical lattice is much smaller than the distance between the two neighbor doughnuts (several microns), therefore small scan regions and shorter acquisition times are required.

We theoretically compare the resolution obtained in the OL-STED and in the multi-doughnut STED setups. An analytical expression of the doughnut STED resolution Δrdoug (Full Width at Half Maximum) has been derived in [34

34. V. Westphal and S. W. Hell, “Nanoscale resolution in the focal plane of an optical microscope,” Phys. Rev. Lett. 94(14), 143903 (2005). [CrossRef] [PubMed]

].

For high STED intensities this expression reads:
Δrdoug2λπNA1Idougmax/Isat
(1)
where Isat is the saturation intensity of the depletion transition, Idougmax>>Isatthe doughnut peak intensity, NAthe numerical aperture of the objective, λ the optical wavelength and d=λNArepresents the diameter of doughnut profile.

We use the same theoretical approach to establish an analytical expression for 4 beam OL-STED resolution ΔrOL. We first assume that all the beams (excitation and depletion) have uniform intensity profiles. The optical lattice created by the interference of the depletion beams has an intensity distribution IOL(x,y) at the sample plane:
IOL(x,y)=IOLmax2[sin2(2πnxsinθλ)+sin2(2πnysinθλ)]
(2)
where IOLmax is the maximal intensity of the interference pattern, and θ the angle formed by the optical axis and the beams emerging from the objective (Fig. 2).

The resolution along the x axis (for y = 0) is given by:
ΔrOLλπnsinθ1IOLmax/Isat
(3)
This resolution depends linearly onp=λ2nsinθ, the period of the depletion pattern, and is inversely proportional to the square root of IOLmax.

To compare the performance of OL-STED to multi-doughnut STED, we calculate the power needed to achieve a given resolution for same number N of intensity minima for the two experimental configurations. For this purpose, we first express the intensity maxima function of the total depletion powers Pdoug andPOLused for each configuration. In the doughnut configuration, using a Laguerre-Gauss mode intensity profileIdoug(r)=Idougmax4er2d2e4r2d2, one obtains Idougmax=4eπd2NPdoug, while in the case of an optical lattice of Fig. 2(c) IOLmax=2cosθNp2POL. Equaling Eqs. (1) and (3), we obtainPdougPOL=eπ4(dp)4cosθ which clearly shows that OL-STED is more efficient than multi-doughnut STED. For θ=60°,p=290nmand d=450nm, the multi-doughnut STED microscope requires 6.2 times more depletion power than the OL-STED microscope.

5. Conclusion

We showed that tailored optical lattices allow large parallelization of standard STED microscopy. Super-resolved 2.9 µm x 2.9 µm images with ~70 nm resolution are obtained up to a rate of 12.5 frames per second, limited only by the CMOS camera readout time. A larger field of view can be achieved using a depletion laser with a lower repetition rate [35

35. D. Wildanger, E. Rittweger, L. Kastrup, and S. W. Hell, “STED microscopy with a supercontinuum laser source,” Opt. Express 16(13), 9614–9621 (2008). [CrossRef] [PubMed]

], which would provide higher intensity pulses for depletion.

Acknowledgments

We warmly thank Philippe Tamarat for his valuable help with the laser sources, Olivier Rossier and Grégory Giannone for providing the cell culture and staining, and Laurent Cognet for helpful discussions. We acknowledge financial support from the Agence Nationale de la Recherche, Région Aquitaine, the French Ministry of Education and Research, the European Research Council and FranceBioImaging (Grant N° ANR-10-INSB-04-01).

References and links

1.

S. W. Hell, “Microscopy and its focal switch,” Nat. Methods 6(1), 24–32 (2009). [CrossRef] [PubMed]

2.

S. W. Hell and J. Wichmann, “Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy,” Opt. Lett. 19(11), 780–782 (1994). [CrossRef] [PubMed]

3.

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (2006). [CrossRef] [PubMed]

4.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006). [CrossRef] [PubMed]

5.

M. G. L. Gustafsson, “Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy,” J. Microsc. 198(2), 82–87 (2000). [CrossRef] [PubMed]

6.

S. T. Hess, T. P. K. Girirajan, and M. D. Mason, “Ultra-High resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J. 91(11), 4258–4272 (2006). [CrossRef] [PubMed]

7.

M. Heilemann, S. van de Linde, M. Schüttpelz, R. Kasper, B. Seefeldt, A. Mukherjee, P. Tinnefeld, and M. Sauer, “Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes,” Angew. Chem. Int. Ed. Engl. 47(33), 6172–6176 (2008). [CrossRef] [PubMed]

8.

A. Sharonov and R. M. Hochstrasser, “Wide-field subdiffraction imaging by accumulated binding of diffusing probes,” Proc. Natl. Acad. Sci. U.S.A. 103(50), 18911–18916 (2006). [CrossRef] [PubMed]

9.

G. Giannone, E. Hosy, F. Levet, A. Constals, K. Schulze, A. I. Sobolevsky, M. P. Rosconi, E. Gouaux, R. Tampé, D. Choquet, and L. Cognet, “Dynamic superresolution imaging of endogenous proteins on living cells at ultra-high density,” Biophys. J. 99(4), 1303–1310 (2010). [CrossRef] [PubMed]

10.

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell, “Fluorescence nanoscopy by ground-state depletion and single-molecule return,” Nat. Methods 5(11), 943–945 (2008). [CrossRef] [PubMed]

11.

R. Heintzmann, T. M. Jovin, and C. Cremer, “Saturated patterned excitation microscopy--a concept for optical resolution improvement,” J. Opt. Soc. Am. A 19(8), 1599–1609 (2002). [CrossRef] [PubMed]

12.

M. G. L. Gustafsson, “Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution,” Proc. Natl. Acad. Sci. U. S. A. 102(37), 13081–13086 (2005). [CrossRef] [PubMed]

13.

P. Kner, B. B. Chhun, E. R. Griffis, L. Winoto, and M. G. L. Gustafsson, “Super-resolution video microscopy of live cells by structured illumination,” Nat. Methods 6(5), 339–342 (2009). [CrossRef] [PubMed]

14.

E. H. Rego, L. Shao, J. J. Macklin, L. Winoto, G. A. Johansson, N. Kamps-Hughes, M. W. Davidson, and M. G. L. Gustafsson, “Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution,” Proc. Natl. Acad. Sci. U. S. A. 109(3), E135–E143 (2012). [CrossRef] [PubMed]

15.

E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, and S. W. Hell, “STED microscopy reveals crystal colour centres with nanometric resolution,” Nat. Photonics 3(3), 144–147 (2009). [CrossRef]

16.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U. S. A. 103(31), 11440–11445 (2006). [CrossRef] [PubMed]

17.

U. V. Nägerl, K. I. Willig, B. Hein, S. W. Hell, and T. Bonhoeffer, “Live-cell imaging of dendritic spines by STED microscopy,” Proc. Natl. Acad. Sci. U.S.A. 105(48), 18982–18987 (2008). [CrossRef] [PubMed]

18.

V. Westphal, S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, and S. W. Hell, “Video-rate far-field optical nanoscopy dissects synaptic vesicle movement,” Science 320(5873), 246–249 (2008). [CrossRef] [PubMed]

19.

S. W. Hell and M. Kroug, “Ground-state-depletion fluorescence microscopy: A concept for breaking the diffraction resolution limit,” Appl. Phys. B 60(5), 495–497 (1995). [CrossRef]

20.

M. Hofmann, C. Eggeling, S. Jakobs, and S. W. Hell, “Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins,” Proc. Natl. Acad. Sci. U. S. A. 102(49), 17565–17569 (2005). [CrossRef] [PubMed]

21.

T. Grotjohann, I. Testa, M. Reuss, T. Brakemann, C. Eggeling, S. W. Hell, and S. Jakobs, “rsEGFP2 enables fast RESOLFT nanoscopy of living cells,” eLife 1, e00248 (2012).

22.

P. Bingen, M. Reuss, J. Engelhardt, and S. W. Hell, “Parallelized STED fluorescence nanoscopy,” Opt. Express 19(24), 23716–23726 (2011). [CrossRef] [PubMed]

23.

A. Chmyrov, J. Keller, T. Grotjohann, M. Ratz, E. d’Este, S. Jakobs, C. Eggeling, and S. W. Hell, “Nanoscopy with more than 100,000 ‘doughnuts’,” Nat. Methods 10(8), 737–740 (2013). [CrossRef] [PubMed]

24.

B. Yang, F. Przybilla, M. Mestre, J.-B. Trebbia, and B. Lounis, “Massive parallelization of STED nanoscopy using optical lattices,” arXiv:1307.3833 [physics] (2013).

25.

A. Hemmerich and T. W. Hänsch, “Two-dimesional atomic crystal bound by light,” Phys. Rev. Lett. 70(4), 410–413 (1993). [CrossRef] [PubMed]

26.

G. Grynberg, B. Lounis, P. Verkerk, J.-Y. Courtois, and C. Salomon, “Quantized motion of cold cesium atoms in two- and three-dimensional optical potentials,” Phys. Rev. Lett. 70(15), 2249–2252 (1993). [CrossRef] [PubMed]

27.

J. I. Cirac and P. Zoller, “Physics. How to manipulate cold atoms,” Science 301(5630), 176–177 (2003). [CrossRef] [PubMed]

28.

M. P. MacDonald, G. C. Spalding, and K. Dholakia, “Microfluidic sorting in an optical lattice,” Nature 426(6965), 421–424 (2003). [CrossRef] [PubMed]

29.

B. Harke, J. Keller, C. K. Ullal, V. Westphal, A. Schönle, and S. W. Hell, “Resolution scaling in STED microscopy,” Opt. Express 16(6), 4154–4162 (2008). [CrossRef] [PubMed]

30.

J. L. Stay and T. K. Gaylord, “Three-beam-interference lithography: contrast and crystallography,” Appl. Opt. 47(18), 3221–3230 (2008). [CrossRef] [PubMed]

31.

D. Wildanger, J. Bückers, V. Westphal, S. W. Hell, and L. Kastrup, “A STED microscope aligned by design,” Opt. Express 17(18), 16100–16110 (2009). [CrossRef] [PubMed]

32.

G. Donnert, C. Eggeling, and S. W. Hell, “Major signal increase in fluorescence microscopy through dark-state relaxation,” Nat. Methods 4(1), 81–86 (2007). [CrossRef] [PubMed]

33.

J. Vogelsang, R. Kasper, C. Steinhauer, B. Person, M. Heilemann, M. Sauer, and P. Tinnefeld, “A Reducing and oxidizing system minimizes photobleaching and blinking of fluorescent dyes,” Angew. Chem. Int. Ed. Engl. 47(29), 5465–5469 (2008). [CrossRef] [PubMed]

34.

V. Westphal and S. W. Hell, “Nanoscale resolution in the focal plane of an optical microscope,” Phys. Rev. Lett. 94(14), 143903 (2005). [CrossRef] [PubMed]

35.

D. Wildanger, E. Rittweger, L. Kastrup, and S. W. Hell, “STED microscopy with a supercontinuum laser source,” Opt. Express 16(13), 9614–9621 (2008). [CrossRef] [PubMed]

OCIS Codes
(100.6640) Image processing : Superresolution
(180.0180) Microscopy : Microscopy
(180.2520) Microscopy : Fluorescence microscopy

ToC Category:
Microscopy

History
Original Manuscript: December 2, 2013
Revised Manuscript: February 17, 2014
Manuscript Accepted: February 18, 2014
Published: March 4, 2014

Virtual Issues
Vol. 9, Iss. 5 Virtual Journal for Biomedical Optics

Citation
Bin Yang, Frédéric Przybilla, Michael Mestre, Jean-Baptiste Trebbia, and Brahim Lounis, "Large parallelization of STED nanoscopy using optical lattices," Opt. Express 22, 5581-5589 (2014)
http://www.opticsinfobase.org/vjbo/abstract.cfm?URI=oe-22-5-5581


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References

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  20. M. Hofmann, C. Eggeling, S. Jakobs, S. W. Hell, “Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins,” Proc. Natl. Acad. Sci. U. S. A. 102(49), 17565–17569 (2005). [CrossRef] [PubMed]
  21. T. Grotjohann, I. Testa, M. Reuss, T. Brakemann, C. Eggeling, S. W. Hell, S. Jakobs, “rsEGFP2 enables fast RESOLFT nanoscopy of living cells,” eLife 1, e00248 (2012).
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  25. A. Hemmerich, T. W. Hänsch, “Two-dimesional atomic crystal bound by light,” Phys. Rev. Lett. 70(4), 410–413 (1993). [CrossRef] [PubMed]
  26. G. Grynberg, B. Lounis, P. Verkerk, J.-Y. Courtois, C. Salomon, “Quantized motion of cold cesium atoms in two- and three-dimensional optical potentials,” Phys. Rev. Lett. 70(15), 2249–2252 (1993). [CrossRef] [PubMed]
  27. J. I. Cirac, P. Zoller, “Physics. How to manipulate cold atoms,” Science 301(5630), 176–177 (2003). [CrossRef] [PubMed]
  28. M. P. MacDonald, G. C. Spalding, K. Dholakia, “Microfluidic sorting in an optical lattice,” Nature 426(6965), 421–424 (2003). [CrossRef] [PubMed]
  29. B. Harke, J. Keller, C. K. Ullal, V. Westphal, A. Schönle, S. W. Hell, “Resolution scaling in STED microscopy,” Opt. Express 16(6), 4154–4162 (2008). [CrossRef] [PubMed]
  30. J. L. Stay, T. K. Gaylord, “Three-beam-interference lithography: contrast and crystallography,” Appl. Opt. 47(18), 3221–3230 (2008). [CrossRef] [PubMed]
  31. D. Wildanger, J. Bückers, V. Westphal, S. W. Hell, L. Kastrup, “A STED microscope aligned by design,” Opt. Express 17(18), 16100–16110 (2009). [CrossRef] [PubMed]
  32. G. Donnert, C. Eggeling, S. W. Hell, “Major signal increase in fluorescence microscopy through dark-state relaxation,” Nat. Methods 4(1), 81–86 (2007). [CrossRef] [PubMed]
  33. J. Vogelsang, R. Kasper, C. Steinhauer, B. Person, M. Heilemann, M. Sauer, P. Tinnefeld, “A Reducing and oxidizing system minimizes photobleaching and blinking of fluorescent dyes,” Angew. Chem. Int. Ed. Engl. 47(29), 5465–5469 (2008). [CrossRef] [PubMed]
  34. V. Westphal, S. W. Hell, “Nanoscale resolution in the focal plane of an optical microscope,” Phys. Rev. Lett. 94(14), 143903 (2005). [CrossRef] [PubMed]
  35. D. Wildanger, E. Rittweger, L. Kastrup, S. W. Hell, “STED microscopy with a supercontinuum laser source,” Opt. Express 16(13), 9614–9621 (2008). [CrossRef] [PubMed]

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