Journal
NEURON
Volume 63, Issue 4, Pages 429-437Publisher
CELL PRESS
DOI: 10.1016/j.neuron.2009.07.011
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Funding
- NINDS NIH HHS [R01 NS046579, T32 NS007484, R01 NS046579-06A1] Funding Source: Medline
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Two-photon laser scanning microscopy (2PLSM) has allowed unprecedented fluorescence imaging of neuronal structure and function within neural tissue. However, the resolution of this approach is poor compared to that of conventional confocal microscopy. Here, we demonstrate supraresolution 2PLSM within brain slices. Imaging beyond the diffraction limit is accomplished by using near-infrared (NIR) lasers for both pulsed two-photon excitation and continuous wave stimulated emission depletion (STED). Furthermore, we demonstrate that Alexa Fluor 594, a bright fluorophore commonly used for both live cell and fixed tissue fluorescence imaging, is suitable for STED 2PLSM. STED 2PLSM supraresolution microscopy achieves approximately 3-fold improvement in resolution in the radial direction over conventional 2PLSM, revealing greater detail in the structure of dendritic spines located similar to 100 microns below the surface of brain slices. Further improvements in resolution are theoretically achievable, suggesting that STED 2PLSM will permit nanoscale imaging of neuronal structures located in relatively intact brain tissue.
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