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Optics Express

  • Editor: C. Martijn de Sterke
  • Vol. 19, Iss. 14 — Jul. 4, 2011
  • pp: 13351–13357
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Total internal reflection STED microscopy

Travis J. Gould, Jordan R. Myers, and Joerg Bewersdorf  »View Author Affiliations


Optics Express, Vol. 19, Issue 14, pp. 13351-13357 (2011)
http://dx.doi.org/10.1364/OE.19.013351


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Abstract

Stimulated emission depletion (STED) microscopy achieves diffraction-unlimited resolution in far-field fluorescence microscopy well below 100 nm. As common for (single-lens) far-field microscopy techniques, the lateral resolution is better than the axial sectioning capabilities. Here we present the first implementation of total internal reflection (TIR) illumination into STED microscopy which limits fluorophore excitation to ~70 nm in the vicinity of the cover slip while simultaneously providing ~50 nm lateral resolution. We demonstrate the performance of this new microscope technique with fluorescent bead test samples as well as immuno-stained microtubules. Total internal reflection STED microscopy provides superior axial sectioning capabilities with the potential to reduce photo-bleaching and photo-damage in live cell imaging.

© 2011 OSA

1. Introduction

STED microscopy utilizes two laser beams in a laser scanning microscope configuration. The first beam excites fluorophores residing in the diffraction-limited focus. The second so-called ‘STED’ beam is tuned to a typically red-shifted wavelength where it can deplete the excited state population through stimulated emission and thereby quench fluorescence. By focusing the STED beam to a diffraction-limited donut shape with a zero-intensity center which is superimposed onto the excitation focus, fluorescence emission is constrained to the immediate vicinity of the intensity zero. Increasing the STED-laser intensity saturates the depletion process and narrows the remaining fluorescent spot to full-width-at-half-maximum (FWHM) values down to <10 nm [9

9. 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]

].

STED microscopy has extensively used a phase mask to create a depletion beam with a donut-profile that enhances the lateral (x-y) resolution but not the axial resolution [10

10. J. Keller, A. Schönle, and S. W. Hell, “Efficient fluorescence inhibition patterns for RESOLFT microscopy,” Opt. Express 15(6), 3361–3371 (2007). [CrossRef] [PubMed]

]. Lateral resolution of 25 to 60 nm is typically achieved in this configuration with fluorescent proteins or organic dyes in one, two, or more color imaging [11

11. 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]

14

14. J. Bückers, D. Wildanger, G. Vicidomini, L. Kastrup, and S. W. Hell, “Simultaneous multi-lifetime multi-color STED imaging for colocalization analyses,” Opt. Express 19(4), 3130–3143 (2011). [CrossRef] [PubMed]

]. To improve the axial resolution, alternative phase masks can be used [10

10. J. Keller, A. Schönle, and S. W. Hell, “Efficient fluorescence inhibition patterns for RESOLFT microscopy,” Opt. Express 15(6), 3361–3371 (2007). [CrossRef] [PubMed]

]. This approach, however, usually results in a compromised lateral resolution by diverting laser power to confine the focal volume in the axial direction. Alternatively, STED can be implemented into a 4Pi-microscope geometry utilizing two opposing objectives [15

15. M. Dyba, S. Jakobs, and S. W. Hell, “Immunofluorescence stimulated emission depletion microscopy,” Nat. Biotechnol. 21(11), 1303–1304 (2003). [CrossRef] [PubMed]

,16

16. R. Schmidt, C. A. Wurm, S. Jakobs, J. Engelhardt, A. Egner, and S. W. Hell, “Spherical nanosized focal spot unravels the interior of cells,” Nat. Methods 5(6), 539–544 (2008). [CrossRef] [PubMed]

], although this approach suffers from high experimental complexity.

Total internal reflection fluorescence (TIRF) microscopy provides another way to achieve sub-diffraction axial sectioning by limiting excitation to the immediate vicinity of the sample-substrate interface. This is achieved by illuminating the interface at an angle beyond the critical angle at which total internal reflection occurs [17

17. D. Axelrod, “Cell-substrate contacts illuminated by total internal reflection fluorescence,” J. Cell Biol. 89(1), 141–145 (1981). [CrossRef] [PubMed]

]. The resulting evanescent wave penetrates the sample only by ~60 to ~200 nm depending on the illumination angle and the refractive index mismatch between the substrate and sample. TIRF is commonly used in widefield microscopy but has also been introduced to confocal laser scanning microscopy. In its first realization [18

18. J. W. M. Chon and M. Gu, “Scanning total internal reflection fluorescence microscopy under one-photon and two-photon excitation: image formation,” Appl. Opt. 43(5), 1063–1071 (2004). [CrossRef] [PubMed]

], the center of a confocal illumination focus was blocked by an annular aperture limiting excitation to high aperture angles. Since spatial coherence was maintained in illumination, the laser light was still focused into a diffraction-limited focus as opposed to widefield TIRF microscopy. Detection through a pinhole resulted in a confocal microscope with illumination limited to a penetration depth of down to 100 nm and less [18

18. J. W. M. Chon and M. Gu, “Scanning total internal reflection fluorescence microscopy under one-photon and two-photon excitation: image formation,” Appl. Opt. 43(5), 1063–1071 (2004). [CrossRef] [PubMed]

]. Terakado et al. [19

19. G. Terakado, K. Watanabe, and H. Kano, “Scanning confocal total internal reflection fluorescence microscopy by using radial polarization in the illumination system,” Appl. Opt. 48(6), 1114–1118 (2009). [CrossRef]

] demonstrated a refined realization using a pair of axicons for higher transmission efficiency and a liquid crystal cell to create radially polarized illumination light which results in a sharper, rotationally symmetric focus.

Here we describe the first realization of a TIRF-STED microscope. For this purpose, we have modified the excitation beam path of a stage-scanning STED microscope to facilitate TIRF illumination (Fig. 1
Fig. 1 Schematic of TIRF-STED setup. FI – Faraday Isolator; λ/2 – half-wave plate; GLP – Glen Laser Polarizer; GB – Glas block; DS – delay stage; AOM – Acousto-optical modulator; PC- photonic crystal fiber; F1-3 bandpass filters; PMF – polarization-maintaining single mode fibers; PM – phase mask; PBS – polarizing beam splitter cubes; DM1/DM2 – dichroic mirrors; OBJ – objective lens; xyz – 3-axes piezo sample stage; SWP – segmented wave plate; λ/4 – quarter-wave plate; BB – removable beam blocks; AA – annular aperture; BS – beam splitter cube; MMF – multimode fiber; APD – single-photon counting avalanche photodiode. Not shown are a few additional mirrors and the microscope stand.
).

2. Setup

The excitation laser beam emerging from the fiber is collimated, passes through a half-wave plate, is split into two paths by a polarizing beam splitter cube, and merged again with a 50:50 beam splitter cube. One of the two paths is used for conventional illumination of the objective. The other one is used for TIRF illumination and contains a custom manufactured annular aperture (National Aperture) and a segmented wave plate (ZPol4-633, Micro Laser Systems) for producing radial polarization at the sample. The arrangement allows the user to easily switch between conventional (non-TIRF) and TIRF mode by blocking one beam path or the other. Both paths were adjusted to be of equal length so that the pulse arrival time at the sample relative to the STED pulse remains the same for either imaging mode.

The STED laser beam is collimated after emerging from its fiber by an objective lens (M-20X, Newport) and directed through a bandpass filter (FF01-785/62, Semrock), a vortex phase mask (RPC Photonics), a polarizing beam splitter cube to clean up the beam polarization, and a half-wave plate for fine adjustment of the polarization direction. The beam is then merged with the excitation beam path by a dichroic mirror (FF01-740-Di01, Semrock).

Both beams are then coupled into the standard side port of a commercial microscope stand (IX71, Olympus) equipped with a 100x/1.49NA oil immersion TIRF objective lens (UAPON 100XOTIRF, Olympus). To achieve an optimal STED donut-profile in the sample, a quarter- wave plate is inserted in front of the side port. In concert with the half-wave plate in the STED beam path, this allows to achieve a STED beam with close to perfect circular polarization in the back aperture of the 100X objective. To obtain radially polarized TIRF excitation, an additional quarter-wave plate is included in the TIRF beam path to cancel the effect of the quarter-wave plate in the common beam path. Lens pairs image the annular aperture mask (producing an obstruction diameter of ~5.12 mm at the objective back aperture) for TIRF as well as the vortex phase mask for STED into the back aperture of the 100X objective lens.

Samples were mounted to an xyz piezo stage (PINano, Physik Instrumente) for scanning. Fluorescence collected by the objective was separated from laser light by the dichroic mirrors, bandpass-filtered (FF01-685/40, Semrock), and focused into a 62.5 µm diameter multimode fiber (corresponding to ~0.6 Airy units) attached to a single photon counting avalanche photodiode (ARQ-13-FC, Perkin Elmer). Image acquisition and instrument control was achieved using custom software written in Labview (National Instruments).

3. Characterization of confocal TIRF performance

To determine the penetration depth of the TIRF illumination, 6 µm surface-labeled fluorescent beads (F14807, Invitrogen; actual bead diameter specified as 5.9 µm by the manufacturer) were spread onto a poly-L-lysine (P8920, Sigma-Aldrich) covered #1.5 cover slip (170-172 µm thickness as determined with a digital micrometer). The sample was then mounted in water on a microscope slide using a spacer to avoid mechanical deformations of the beads and imaged with our microscope. Images were acquired with a pixel size of 70 nm, and the excitation powers measured at the objective back aperture were ~10 µW and ~16 µW for the confocal and TIRF modes, respectively. Figures 2(A)
Fig. 2 Conventional and evanescent field confocal excitation of a 6 µm bead. xz-scans through the center of the same surface-labeled fluorescent bead with conventional (A) and TIRF (B) excitation. An intensity profile analysis (C) of xy-images taken in the plane of the cover slip surface (D) allows the determination of the evanescent field depth.
and 2(B) show xz slices through the center of a bead imaged in conventional confocal mode and in TIRF mode, respectively, demonstrating the limited penetration depth in TIRF mode. While the full bead is visible in conventional confocal mode (featuring image distortions from the high refractive index differences between glass, bead and water), only the portion of the bead in close vicinity of the cover slip is visible in TIRF mode due to evanescent field excitation.

We note that the apparent axial “thickness” of the image in Fig. 2(B) is not a measure for the TIRF penetration depth. The TIRF focus can be best described as a Bessel beam. The lateral excitation profile as well as the excitation penetration depth and intensity therefore do not significantly depend on the axial position of the sample and are constant over the whole axial scan range. The axial signal variation displayed in Fig. 2(B) results from confocal detection which detects fluorescence less efficiently when it is defocused than when the emitting portion of the sample (the part of the bead close to the cover slip) is located in the focal plane.

To quantify the TIRF penetration depth, we analyzed the apparent width of the bead image in an xy-scan as shown in Figs. 2(C) and 2(D). The bead appears brightest where it touches the cover slip and becomes dimmer as the curved surface is retreating from the glass surface. Following the method described by Mattheyses and Axelrod [21

21. A. L. Mattheyses and D. Axelrod, “Direct measurement of the evanescent field profile produced by objective-based total internal reflection fluorescence,” J. Biomed. Opt. 11(1), 014006 (2006). [CrossRef] [PubMed]

], we analyzed the radial intensity distribution of 25 xy images taken of 10 beads. After converting the radial distance to an axial distance using the known radius of the beads, we fit a double exponential to each profile and extracted the decay constants. The first exponential, comprising the majority of the signal (~85%), represents the evanescent excitation field while the second exponential represents excitation light scattering in the objective [21

21. A. L. Mattheyses and D. Axelrod, “Direct measurement of the evanescent field profile produced by objective-based total internal reflection fluorescence,” J. Biomed. Opt. 11(1), 014006 (2006). [CrossRef] [PubMed]

]. A representative intensity profile and its fit are shown in Fig. 2(D). The excitation intensity dropped to half its maximum at a depth of 73 +/− 7 nm (1/e penetration depth 106 +/− 10 nm). Similar values were obtained fitting the lateral intensity distribution with a two-dimensional Gaussian and calculating the penetration depth, d, of the evanescent field from:
d=rr2(a/2)2
(1)
where r is the radius of the bead and a is the full with half maximum of the Gaussian [18

18. J. W. M. Chon and M. Gu, “Scanning total internal reflection fluorescence microscopy under one-photon and two-photon excitation: image formation,” Appl. Opt. 43(5), 1063–1071 (2004). [CrossRef] [PubMed]

].

4. Characterization of STED and TIRF-STED performances

In the following, we switched from conventional to TIRF excitation mode as described above. Since the annular aperture is blocking a significant fraction of our laser light, we increased the incoming excitation power by removing a neutral density filter in front of the single mode fiber. TIRF illumination resulted, however, in slightly reduced resolution values in the confocal imaging mode (Fig. 3(D)) which we account to aberrations associated with the extreme aperture angles used with the TIRF objective lens. Deviations from true radial polarization are also likely to contribute to this effect. In the TIRF-STED image (Fig. 3(E)) the resolution (52 nm FWHM measured from line profile shown in Fig. 3(F)) is not affected by this effect since the resolution improvement depends primarily on the dimensions and quality of the STED donut which is not influenced by the TIRF mode. Within measurement accuracy, the measured resolutions for STED and TIRF-STED are in reasonable agreement.

5. Application of TIRF-STED microscopy

To demonstrate the superior optical sectioning of our TIRF-STED setup over conventional (2D) STED microscopy, we imaged immuno-stained microtubules in PtK2 cells. Cells were grown to approximately 50% confluence on #1.5 coverslips (171 µm thickness as determined with a digital micrometer). Cells were briefly methanol-fixed and immuno-stained following procedures detailed by Wurm et al. [22

22. C. A. Wurm, D. Neumann, R. Schmidt, A. Egner, and S. Jakobs, “Sample preparation for STED microscopy,” Methods Mol. Biol. 591, 185–199 (2010). [CrossRef] [PubMed]

]. Anti-beta tubulin primary (1:500 dilution, MAB3408, Millipore) and Atto647N secondary antibodies (1:1000, CAT 15058, Active Motif) were utilized to stain cells at room temperature. Samples were then mounted in 1X sterile phosphate-buffered saline onto glass microscope slides. Nail polish was used to seal the coverslips to the glass slides.

For best comparison, we imaged the same 5.12 µm x 5.12 µm area in the following sequence of imaging modes: TIRF, TIRF-STED, confocal, and STED. The imaging parameters were the same as with the 40 nm fluorescent beads with the exception that the pixel size was set to 20 nm and the power of the STED beam (tuned to 770 nm) was ~75 mW at the objective back aperture. Figure 4
Fig. 4 Biological application of TIRF-STED microscopy. Immuno-stained microtubules in a PtK2 cell were imaged in conventional confocal (A) and STED (B) mode as well as in confocal TIRF (D) and TIRF-STED (E) mode. (C) and (F) show linearly deconvolved versions of (B) and (E), respectively. The arrow heads highlight tubules that are well visible in STED mode but nearly disappear in TIRF-STED mode indicating their position deeper in the cell outside the evanescent excitation field. G - Overlay of the deconvolved STED (shown in green) and TIRF-STED (shown in purple) images. H - profile as depicted by the box shown in (G). Data has been averaged along the short side of the box.
shows conventional confocal (Fig. 4(A)), conventional STED (Fig. 4(B)), TIRF (Fig. 4(D)) and TIRF-STED mode (Fig. 4(E)) images. As expected, both STED modes show dramatic resolution improvement over the non-STED imaging modes. Image quality was further enhanced by linear deconvolution with theoretical 2D Lorentzian-shaped PSFs using the image processing program Imspector (written by Dr. Andreas Schoenle, Max Planck Institute for Biophysical Chemistry, Goettingen, Germany, available via Max-Planck-Innovation GmbH, Munich, Germany) (Figs. 4(C) and 4(F)). Comparing TIRF with conventional modes clearly shows that some microtubules disappear when imaging with TIRF (arrow heads in Figs. 4(C) and 4(F)) while others seem to be unaffected. This is further emphasized by merging the TIRF and non-TIRF STED images using different pseudo-colors (Figs. 4(G) and 4(H)).

6. Conclusions

TIRF-STED microscopy combines the lateral resolution improvement achieved by stimulated emission depletion with the optical section capabilities of total internal reflection. This combination of axially constrained excitation with laterally constrained fluorescence emission enables observation volumes of sub-100 nm diameter in all three dimensions. This decoupling of axial from lateral resolution improvement provides additional flexibility in instrument design: STED can be optimized solely for best resolution in the focal plane while TIR takes care of limiting observation to the immediate vicinity of the cover slip.

One of the potential advantages of TIRF-STED microscopy lies in its application to live cell imaging. STED microscopy has successfully been used to image living cells [23

23. B. Hein, K. I. Willig, and S. W. Hell, “Stimulated emission depletion (STED) nanoscopy of a fluorescent protein-labeled organelle inside a living cell,” Proc. Natl. Acad. Sci. U.S.A. 105(38), 14271–14276 (2008). [CrossRef] [PubMed]

,24

24. 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]

], but bleaching and phototoxicity remain major limitations of fluorescence microscopy in general. In STED microscopy, it is apparent that the dominant path to photobleaching is excitation of fluorophores first into the excited singlet state and then, by the STED laser, into higher singlet states or long-lived triplet states [11

11. 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]

]. By design, the STED wavelength is chosen such that significant excitation from the ground state to the first excited state is only achieved by the excitation laser beam. Therefore, the STED beam itself should not significantly contribute to photobleaching. Rather, it is the combination of excitation with STED light, or excitation light alone, which is primarily responsible for bleaching and the corresponding photo-damage to the sample. As with widefield TIRF microscopy, TIRF-STED microscopy limits excitation to a small portion of the sample. TIRF-STED microscopy therefore has the potential to reduce photo-bleaching of fluorophores and phototoxicity to living samples. Future investigations of this potential will be of significant interest. We expect TIRF-STED to become an important technique for nanoscale imaging of biological structures at the sample-substrate interface.

Acknowledgments

The authors thank Dr. Vladimir Polejaev for helpful discussions. T.J.G. has been supported by a James Hudson Brown—Alexander Brown Coxe Postdoctoral Fellowship. J.B. declares significant financial interest in Vutara, Inc.

References and links

1.

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

2.

D. Toomre and J. Bewersdorf, “A new wave of cellular imaging,” Annu. Rev. Cell Dev. Biol. 26(1), 285–314 (2010). [CrossRef] [PubMed]

3.

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]

4.

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

5.

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]

6.

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]

7.

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U.S.A. 97(15), 8206–8210 (2000). [CrossRef] [PubMed]

8.

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]

9.

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]

10.

J. Keller, A. Schönle, and S. W. Hell, “Efficient fluorescence inhibition patterns for RESOLFT microscopy,” Opt. Express 15(6), 3361–3371 (2007). [CrossRef] [PubMed]

11.

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]

12.

L. Meyer, D. Wildanger, R. Medda, A. Punge, S. O. Rizzoli, G. Donnert, and S. W. Hell, “Dual-color STED microscopy at 30-nm focal-plane resolution,” Small 4(8), 1095–1100 (2008). [CrossRef] [PubMed]

13.

K. I. Willig, R. R. Kellner, R. Medda, B. Hein, S. Jakobs, and S. W. Hell, “Nanoscale resolution in GFP-based microscopy,” Nat. Methods 3(9), 721–723 (2006). [CrossRef] [PubMed]

14.

J. Bückers, D. Wildanger, G. Vicidomini, L. Kastrup, and S. W. Hell, “Simultaneous multi-lifetime multi-color STED imaging for colocalization analyses,” Opt. Express 19(4), 3130–3143 (2011). [CrossRef] [PubMed]

15.

M. Dyba, S. Jakobs, and S. W. Hell, “Immunofluorescence stimulated emission depletion microscopy,” Nat. Biotechnol. 21(11), 1303–1304 (2003). [CrossRef] [PubMed]

16.

R. Schmidt, C. A. Wurm, S. Jakobs, J. Engelhardt, A. Egner, and S. W. Hell, “Spherical nanosized focal spot unravels the interior of cells,” Nat. Methods 5(6), 539–544 (2008). [CrossRef] [PubMed]

17.

D. Axelrod, “Cell-substrate contacts illuminated by total internal reflection fluorescence,” J. Cell Biol. 89(1), 141–145 (1981). [CrossRef] [PubMed]

18.

J. W. M. Chon and M. Gu, “Scanning total internal reflection fluorescence microscopy under one-photon and two-photon excitation: image formation,” Appl. Opt. 43(5), 1063–1071 (2004). [CrossRef] [PubMed]

19.

G. Terakado, K. Watanabe, and H. Kano, “Scanning confocal total internal reflection fluorescence microscopy by using radial polarization in the illumination system,” Appl. Opt. 48(6), 1114–1118 (2009). [CrossRef]

20.

E. Auksorius, B. R. Boruah, C. Dunsby, P. M. Lanigan, G. Kennedy, M. A. Neil, and P. M. French, “Stimulated emission depletion microscopy with a supercontinuum source and fluorescence lifetime imaging,” Opt. Lett. 33(2), 113–115 (2008). [CrossRef] [PubMed]

21.

A. L. Mattheyses and D. Axelrod, “Direct measurement of the evanescent field profile produced by objective-based total internal reflection fluorescence,” J. Biomed. Opt. 11(1), 014006 (2006). [CrossRef] [PubMed]

22.

C. A. Wurm, D. Neumann, R. Schmidt, A. Egner, and S. Jakobs, “Sample preparation for STED microscopy,” Methods Mol. Biol. 591, 185–199 (2010). [CrossRef] [PubMed]

23.

B. Hein, K. I. Willig, and S. W. Hell, “Stimulated emission depletion (STED) nanoscopy of a fluorescent protein-labeled organelle inside a living cell,” Proc. Natl. Acad. Sci. U.S.A. 105(38), 14271–14276 (2008). [CrossRef] [PubMed]

24.

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]

OCIS Codes
(180.2520) Microscopy : Fluorescence microscopy
(260.6970) Physical optics : Total internal reflection
(350.5730) Other areas of optics : Resolution

ToC Category:
Microscopy

History
Original Manuscript: May 16, 2011
Revised Manuscript: June 15, 2011
Manuscript Accepted: June 15, 2011
Published: June 27, 2011

Virtual Issues
Vol. 6, Iss. 8 Virtual Journal for Biomedical Optics

Citation
Travis J. Gould, Jordan R. Myers, and Joerg Bewersdorf, "Total internal reflection STED microscopy," Opt. Express 19, 13351-13357 (2011)
http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-19-14-13351


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References

  1. S. W. Hell, “Microscopy and its focal switch,” Nat. Methods 6(1), 24–32 (2009). [CrossRef] [PubMed]
  2. D. Toomre and J. Bewersdorf, “A new wave of cellular imaging,” Annu. Rev. Cell Dev. Biol. 26(1), 285–314 (2010). [CrossRef] [PubMed]
  3. 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]
  4. S. T. Hess, T. P. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J. 91(11), 4258–4272 (2006). [CrossRef] [PubMed]
  5. 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]
  6. 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]
  7. T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U.S.A. 97(15), 8206–8210 (2000). [CrossRef] [PubMed]
  8. 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]
  9. 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]
  10. J. Keller, A. Schönle, and S. W. Hell, “Efficient fluorescence inhibition patterns for RESOLFT microscopy,” Opt. Express 15(6), 3361–3371 (2007). [CrossRef] [PubMed]
  11. 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]
  12. L. Meyer, D. Wildanger, R. Medda, A. Punge, S. O. Rizzoli, G. Donnert, and S. W. Hell, “Dual-color STED microscopy at 30-nm focal-plane resolution,” Small 4(8), 1095–1100 (2008). [CrossRef] [PubMed]
  13. K. I. Willig, R. R. Kellner, R. Medda, B. Hein, S. Jakobs, and S. W. Hell, “Nanoscale resolution in GFP-based microscopy,” Nat. Methods 3(9), 721–723 (2006). [CrossRef] [PubMed]
  14. J. Bückers, D. Wildanger, G. Vicidomini, L. Kastrup, and S. W. Hell, “Simultaneous multi-lifetime multi-color STED imaging for colocalization analyses,” Opt. Express 19(4), 3130–3143 (2011). [CrossRef] [PubMed]
  15. M. Dyba, S. Jakobs, and S. W. Hell, “Immunofluorescence stimulated emission depletion microscopy,” Nat. Biotechnol. 21(11), 1303–1304 (2003). [CrossRef] [PubMed]
  16. R. Schmidt, C. A. Wurm, S. Jakobs, J. Engelhardt, A. Egner, and S. W. Hell, “Spherical nanosized focal spot unravels the interior of cells,” Nat. Methods 5(6), 539–544 (2008). [CrossRef] [PubMed]
  17. D. Axelrod, “Cell-substrate contacts illuminated by total internal reflection fluorescence,” J. Cell Biol. 89(1), 141–145 (1981). [CrossRef] [PubMed]
  18. J. W. M. Chon and M. Gu, “Scanning total internal reflection fluorescence microscopy under one-photon and two-photon excitation: image formation,” Appl. Opt. 43(5), 1063–1071 (2004). [CrossRef] [PubMed]
  19. G. Terakado, K. Watanabe, and H. Kano, “Scanning confocal total internal reflection fluorescence microscopy by using radial polarization in the illumination system,” Appl. Opt. 48(6), 1114–1118 (2009). [CrossRef]
  20. E. Auksorius, B. R. Boruah, C. Dunsby, P. M. Lanigan, G. Kennedy, M. A. Neil, and P. M. French, “Stimulated emission depletion microscopy with a supercontinuum source and fluorescence lifetime imaging,” Opt. Lett. 33(2), 113–115 (2008). [CrossRef] [PubMed]
  21. A. L. Mattheyses and D. Axelrod, “Direct measurement of the evanescent field profile produced by objective-based total internal reflection fluorescence,” J. Biomed. Opt. 11(1), 014006 (2006). [CrossRef] [PubMed]
  22. C. A. Wurm, D. Neumann, R. Schmidt, A. Egner, and S. Jakobs, “Sample preparation for STED microscopy,” Methods Mol. Biol. 591, 185–199 (2010). [CrossRef] [PubMed]
  23. B. Hein, K. I. Willig, and S. W. Hell, “Stimulated emission depletion (STED) nanoscopy of a fluorescent protein-labeled organelle inside a living cell,” Proc. Natl. Acad. Sci. U.S.A. 105(38), 14271–14276 (2008). [CrossRef] [PubMed]
  24. 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]

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