The diffraction limit has been broken by several “super-resolution” microscopy techniques, including saturated structured illumination microscopy (SSIM) (Gustafsson, 2005
); stochastic optical reconstruction microscopy (STORM) (Rust et al., 2006
); photoactivated localization microscopy (PALM) (Betzig et al., 2006
); and STED (Hell and Wichmann, 1994
). Each of these approaches has its own limitations. SSIM, STORM and PALM require mathematical post-processing and image reconstruction as well as prolonged imaging of each specimen. Moreover, these three approaches are degraded by spherical aberrations and scattering of emitted photons, generally limiting their utility to thin specimens. STED microscopy uses a purely physical process and is a scanning imaging approach, allowing repeated examination of moving small objects within living cells (Westphal et al., 2008
). However, STED has thus far only been implemented using 1-photon excitation by visible light, preventing its use within scattering tissues such as the brain. Consequently there currently exists no supraresolution approach applicable to live-cell imaging below the surface of brain tissues.
We have demonstrated that STED 2PLSM is capable of imaging neuronal structures deep in brain tissues with resolution beyond the Abbe diffraction limit. In our experiments, ~3 fold improvement in radial resolution was achieved when imaging at depths of ~100 microns in brain slices. Further improvements in resolution will be achieved by more efficient delivery of the STED laser light, both by use of improved optics and by use of a pulsed depletion laser. The latter requires that the STED laser pulses arrive at the specimen precisely after the excitation pulse for optimal depletion of the excited state. Compared to continuous wave STED described here, pulsed depletion should use STED laser power approximately 10 fold more efficiently. According to the theoretically predicted resolution of STED microscopy (Eq. 1
), this will allow ~10 fold improvements in resolution below the diffraction limit, reaching ~50 nm within brain tissue.
The utility of STED 2PLSM for deep tissue imaging arises from the use of multiphoton excitation and near infrared light. The use of near infrared light allows the formation of a focus deep within tissue that would degrade a visible light focus through scattering and absorption. Multiphoton excitation constrains fluorophore excitation to a small focal volume, eliminating out-of-focus fluorescence (Denk and Svoboda, 1997
). This obviates the need for a descanned pinhole in the fluorescence detection path, thus permitting the use of scattered emission photons for image formation. For these reasons, STED 2PLSM using NIR excitation and depletion light is theoretically useful for deep tissue supraresolution imaging. We demonstrate that, in practice, spherical aberrations due to index of refraction mismatches, forward scatter of excitation photons, and phototoxicity are not significant obstacles to the implementation of STED 2PLSM.
In addition, we find that Alexa 594, a bright fluorophore used commonly in live cell and fixed tissue imaging, is suitable for STED 2PLSM. Alexa 594 has a low Isat
for CW STED such that, with our currently available laser power, Imax
=20 and a 4–5 fold improvement in resolution should be possible. However, in our experimental measurements, we find an approximately 3 fold improvement in resolution (as judged from the FWHM of the thin structures). This apparent underperformance of STED 2PLSM compared to the theoretical limit may result from partial absorption of STED power by the tissue or from image degradation due to forward scatter of the excitation and STED beams. Conversely, it may be difficult to detect a 5 fold improvement in resolution in tissue due to the fact that our test imaging structures (dendritic spines and flilopodia) were large (>100 nm) (Bourne and Harris, 2008
). We also find that STED 2PLSM with Alexa 594 is suitable for repeated time-lapse imaging because the small cytoplasmic fluorophore can readily diffuse and continuously replenish photobleached fluorophores. This is particularly important because, as the volume of excited fluorophores is reduced in STED imaging, the number of emitted photons is decreased and increased excitation laser power or averaging is required to maintain signal-to-noise ratios. Since Alexa 594 is an exceptionally bright and stable fluorophore, high-quality STED 2PLSM images can be achieved with low concentrations of fluorophore that are compatible with live cell imaging.
Several further improvements in STED 2PLSM are possible. First, as discussed above, more efficient use of STED power will continue to improve the resolution to below 100 nm. Second, we have described improved resolution in the image plane but not in the axial plane. Improvements in axial resolution can be achieved through use of an annular, instead of helical, phase mask (Klar et al., 2000
). Ideally both in plane and axial STED 2PLSM would be implemented simultaneously as has been demonstrated for conventional STED microscopy (Willig et al., 2007
). Third, many genetically-encoded red fluorophores, such as tdTomato and dsRed, have long fluorescent life-times (~ 3 ns) and long emission tails into the red spectrum (>700 nm), indicating that, if efficiently depleted, they may be suitable for STEP 2PLSM (Shaner et al., 2004
). Lastly, identification of STED 2PLSM compatible synthetic and genetically-encoded reporters of intracellular biochemical pathways, including calcium-sensitive fluorophores, may allow examination of intracellular signaling within functional microdomains.
With these advances, STED 2PLSM will be used for nanoscale imaging in relatively intact brain tissue to address fundamental questions in neuroscience that have been difficult to address. For example, indirect measurements suggest that the dimensions of each spine neck are rapidly regulated by activity of the associated synapse. However, it has been impossible thus far to accurately measure the spine neck in living neurons (Bloodgood and Sabatini, 2005
; Grunditz et al., 2008
; Tanaka et al., 2008
). Second, activity-dependent regulation of ion channel trafficking and corralling (Choquet and Triller, 2003
; Malinow and Malenka, 2002
) is thought to be a principal mechanism of synaptic plasticity but direct measurements of protein movement at high resolution has not been possible in brain slices or in vivo. Third, spatially restricted biochemical signaling within subcellular microdomains is thought to confer specificity on calcium entry and kinase activation. However, direct measurement of the activity of biochemical pathways using fluorescence reporters has not been possible within microdomains. Lastly, STED 2PLSM will help resolve fine features of non-neuronal cells such as astrocytes, whose complex and intimate morphological relationship within neurons is only maintained in more intact preparations.