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
A new class of microscopy techniques has overcome the century-old diffraction-limit in far-field fluorescence microscopy. By switching fluorescent probe molecules between optically distinct states, images with diffraction unlimited resolution can be generated [1,2]. Next to techniques which stochastically switch fluorescent molecules on and off and record the appearing and disappearing single molecules in the field of view with a camera [3–5], targeted switching has been applied very successfully in stimulated emission depletion (STED) microscopy [6,7] and related techniques using other saturable optical transitions [1,8].
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 .
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 . 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 [11–14]. To improve the axial resolution, alternative phase masks can be used . 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 [15,16], 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 . 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 , 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 . Terakado et al.  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 ).
As illustrated in Fig. 1, a modelocked Ti:Sapphire laser (Chameleon Ultra II, Coherent) passes through a Faraday isolator (Newport) and a half-wave plate before it is split into two beam paths by a Glan laser polarizer (Newport). The transmitted beam which serves as the STED beam is first transmitted through a 19 cm long glass block to stretch the pulse width to >1 picosecond. The beam is then sent through a delay stage for pulse delay adjustment and an acousto-optical modulator (AA Optoelectronics) for laser power adjustment before it is coupled into a 100 m polarization-maintaining single mode fiber (Fibercore) for beam clean-up and additional pulse stretching to a few hundred picoseconds. The beam reflected at the Glan laser polarizer is focused into a photonic crystal fiber (SCG-800, Newport) which creates a white light spectrum ranging from ~500 nm to the near infrared. As implemented in a previous report , this approach provides excitation pulses that are inherently synchronized to the depletion pulses. A bandpass filter (FF01-625/26, Semrock) centered at 625 nm with 26 nm bandwidth was used for selecting the fluorescence excitation band from the white light spectrum. The excitation light is then coupled into a second polarization-maintaining single mode fiber of 5 m length (Thorlabs).
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) 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 , 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 . 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:18].
4. Characterization of STED and TIRF-STED performances
We next characterized the performance of our instrument in conventional STED mode. For this purpose, we attached 40 nm fluorescent beads (F8789, Invitrogen) to poly-L-lysine coated #1.5 cover slips and mounted them in water. Beads were imaged in confocal and STED mode (Figs. 3(A) and 3(B)) with a pixel size of 15 nm. The excitation powers measured at the objective back aperture were ~2 µW and ~45 µW for the confocal and TIRF modes, respectively. The power of the STED beam (tuned to 760 nm) was similarly measured to be ~58 mW. A model function (Lorentzian) was fit to the STED line profile (Fig. 3(C)) resulting in a FWHM of 58 nm. Considering the non-negligible size of the bead (40 nm diameter), the measured FWHM gives an upper bound for the resolution of the system.
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. . 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 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)).
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 [23,24], 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 . 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.
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.
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