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Max Planck Institute for Biophysical Chemistry, Departments of * NanoBiophotonics and Neurobiology, 37070 Göttingen, Germany
Correspondence: Address reprint requests and inquiries to Stefan W. Hell, E-mail: hell{at}4pi.de.
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ABSTRACT |
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5 nm alignment accuracy of the two images establishes the precision with which differently labeled proteins are correlated in space. Colocalized nanoscopy is demonstrated with endosomal protein patterns and by resolving nanoclusters of a mitochondrial outer membrane protein, Tom20, in relation with the F1F0ATP synthase. The joint improvement of resolution and colocalization demonstrates the emerging potential of far-field fluorescence nanoscopy to study the spatial organization of macromolecules in cells.
The commonly used variants of lens-based (far-field) fluorescence microscopy would be ideal for exploring the distribution of proteins in cells if their spatial resolution were not limited by diffraction to about a half of the wavelength of light: 
(1
). Stimulated emission depletion (STED) microscopy (2) is an emerging microscopy technique that, although still using regular lenses, fundamentally overcomes the diffraction barrier. In its single-point scanning variant, STED microscopy uses two coaligned beams: one for excitation, and a second one, with a longer wavelength, to de-excite the fluorophore by stimulated emission. Whereas the excitation beam is regularly focused, the STED beam forms a doughnut in the focal plane, featuring a zero at the center. Superposing the first with the latter confines fluorescence emission toward the center, thus narrowing the spot from which fluorescence may originate. The breaking of the diffraction barrier is based on the fact that the excited state population decreases almost exponentially with the intensity I of the STED beam. Increasing I confines the fluorescent spot continually. If we define Is as the intensity at which the probability of fluorescence is reduced to 1/e, the lateral resolution is approximated by 
, with 
denoting the numerical aperture of the lens (3). Ideally, the spatial resolution can be improved to the molecular scale.
So far, STED microscopy has exhibited a resolution of <20 nm (4,5). Moreover, it has been key to answering several biology questions (6). However, in all these studies, STED was implemented with a single color only, meaning that just a single molecular species could be mapped out in the cell. Investigating the spatial relationship of two or more biomolecules at the nanoscale has been impossible, because this task requires at least two color channels. Although molecular proximities <10 nm can be detected through Förster Resonance Energy Transfer using standard fluorescence microscopes (7), the nanoscale organization of the (colocalizing) proteins remained elusive.
Here, we demonstrate the viability of STED microscopy with two different fluorophores, particularly with green and red fluorescence emission. The excitation of the green emitters is performed at 470 nm using a laser diode providing 100 ps pulses. STED of these dyes is performed by an optic parametric amplifier providing 300 ps pulses at a wavelength of 603 nm. The red dyes are excited at 635 nm with a similar diode, whereas the corresponding STED pulses are extracted from a regenerative Ti:sapphire amplifier operating at 780 nm. In fact, our system is an extension of the one detailed in Donnert et al. (5) (Supplementary Material). A time difference of 4 µs between successive pulse pairs ensures that molecules that are occasionally trapped in a dark state with a lifetime <4 µs can relax in the interim. Dubbed T- or D-Rex, this illumination modality reduces photobleaching mediated by transient dark states, thus allowing for greater I/Is (5). Since the excitation pulses were triggered by the basic frequency of the amplifier (250 kHz), the synchronization of the pulses did not pose additional challenges in realizing two-color STED. Attention required the fact that the STED beam for the short-wavelength dye may excite its long-wavelength complement. This issue has been solved by recording the red emitter first.
Fig. 1 displays the measured excitation and doughnut spots in the focal plane (transverse point-spread function, PSF) of the oil immersion lens used (Planapo 100x, Leica Microsystems, Wetzlar, Germany). The nanoresolution of the system was first verified using green and red fluorescent beads (known as "yellow-green" and "crimson" fluorescent beads from Molecular Probes, Eugene, OR) of 24 ± 4 nm specified diameter, with emission at 500–530 nm and 640–670 nm, respectively. In the absence of STED, the resolution is that of a confocal microscope. Applying intensities 
GW/cm2 and 
at the doughnut crest reduced the extent of the fluorescence spot. Probing the spot with 35 yellow-green and 30 crimson beads, gave a full-width at half-maximum of 25 ± 5 nm and 65 ±11 nm, respectively (Fig. 1, C and F, and Supplementary Material). Since the finite bead size is still contained in these values, they represent an upper bound for the resolution under these conditions (5). The comparatively poorer resolution in the red channel is due to the fact that, in our laser system, the 
of the red STED beam (780 nm) was not tunable and hence not fully adaptable to the fluorophores in use. Even so, the resolution in the red channel is 4.3x beyond the diffraction barrier.
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Next we performed immunofluorescence nanoscopy using the organic fluorophore Atto532 for the green (530–570 nm) and Atto647N for the red channel (660–700 nm). In the first experiment, we prepared a fraction of pheochromocytoma (PC12) cells, which is a neuroendocrine cell line, enriched in early endosomes. Fig. 2 displays two synaptic vesicle proteins in endosomes: synaptophysin (green) and synaptotagmin I (red). STED was performed with 
GW/cm2 and 
. To maximize their information content, both the confocal and the STED data were subject to a linear deconvolution (Supplementary Material). Correct overlay of both channels was ensured by double-stained ("yellow-green plus crimson") fluorescence beads that we added to the sample (arrows, Fig. 2 D). These beads allowed us to overlay both color channels with an accuracy of ±5 nm (Supplementary Material). Whereas the more abundant protein, synaptophysin, forms ring-shaped domains, synaptotagmin I exhibits point-like distributions that largely colocalize with the synaptophysin-containing structures (Fig. 2, B and D). Fig. 2 E displays a line profile through synaptotagmin I and synaptophysin agglomerations with the corresponding centers of gravity being 25 ± 5 nm apart. We surprisingly found that not all synaptic vesicle proteins occupy the same sites on the endosomes.
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-subunit of the F1F0ATP synthase complex and Tom20 in mammalian (PtK2) cells with 
GW/cm2 and . Electron microscopy reportedly revealed that the F1F0ATP synthase complex is densely packed within the mitochondrial inner membrane (8). Tom20 is a component of the translocase of the outer mitochondrial membrane. Mediating the transport of nuclear encoded mitochondrial preproteins across the outer membrane, the TOM complex has been suggested to be enriched where the inner and the outer membranes are in close proximity. Nonetheless, its spatial distribution remained largely elusive. This partly stems from the challenges of applying immunolabeling in electron microscopy and from the inability of standard fluorescence microscopy to resolve on the nanoscale. Although the two-color STED recordings confirm the nearly homogeneous distribution of the F1F0ATP synthase (Fig. 3), they reveal for the first time that Tom20 is localized in nanosized clusters. We have reinforced this finding by reverting the labeling: the TOM complex was tagged with the red-labeled antibody whereas the F1F0ATP synthase with the green one (Fig. 4). Again, the TOM complex appeared in nanosized clusters. We expect the two-color nanoresolution of STED microscopy to help elucidate the TOM20 cluster formation and the correlation of these clusters with other mitochondrial morphologies.
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In conclusion, we have demonstrated the viability of dual-color fluorescence nanoscopy. The progress in biological microscopy reported herein should facilitate the imaging of protein colocalizations in cells with hitherto unprecedented detail. STED microscopy should be a powerful technique to image colocalized protein distributions, because it retains most of the advantages of confocal microscopy, including the ability to image organelles in intact cells.
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ACKNOWLEDGEMENTS |
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Submitted on January 21, 2007; accepted for publication February 6, 2007.
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REFERENCES |
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2. Hell, S. W., and J. Wichmann. 1994. Breaking the diffraction resolution limit by stimulated emission: stimulated emission depletion microscopy. Opt. Lett. 19:780–782.
3. Hell, S. W. 2003. Toward fluorescence nanoscopy. Nat. Biotechnol. 21:1347–1355.[CrossRef][Medline]
4. Westphal, V., and S. W. Hell. 2005. Nanoscale resolution in the focal plane of an optical microscope. Phys. Rev. Lett. 94:143903.[CrossRef][Medline]
5. Donnert, G., J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell. 2006. Macromolecular-scale resolution in biological fluorescence microscopy. Proc. Natl. Acad. Sci. USA. 103:11440–11445.
6. Willig, K. I., S. O. Rizzoli, V. Westphal, R. Jahn, and S. W. Hell. 2006. STED-microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis. Nature. 440:935–939.[CrossRef][Medline]
7. Jares-Erijman, E. A., and T. M. Jovin. 2003. FRET imaging. Nat. Biotechnol. 21:1387–1395.[CrossRef][Medline]
8. Allen, R. D., C. C. Schroeder, and A. K. Fok. 1989. An investigation of mitochondrial inner membranes by rapid-freeze deep-etch techniques. J. Cell Biol. 108:2233–2240.
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