In Vivo Superresolution Imaging of Neuronal Structure in the Mouse Brain.

OBJECTIVE

this study proposes and evaluates a technique for in vivo deep-tissue superresolution imaging in the light-scattering mouse brain at up to a 3.5 Hz 2-D imaging rate with a 21×21 μm field of view.

METHODS

we combine the deep-tissue penetration and high imaging speed of resonant laser scanning two-photon (2P) microscopy with the superresolution ability of patterned excitation microscopy. Using high-frequency intensity modulation of the scanned two-photon excitation beam, we generate patterned illumination at the imaging plane. Using the principles of structured illumination, the high-frequency components in the collected images are then used to reconstruct images with an approximate twofold increase in optical resolution.

RESULTS

using our technique, resonant 2P superresolution patterned excitation reconstruction microscopy, we demonstrate our ability to investigate nanoscopic neuronal architecture in the cerebral cortex of the mouse brain at a depth of 120 μm in vivo and 210 μm ex vivo with a resolution of 119 nm. This technique optimizes the combination of speed and depth for improved in vivo imaging in the rodent neocortex.

CONCLUSION

this study demonstrates a potentially useful technique for superresolution in vivo investigations in the rodent brain in deep tissue, creating a platform for investigating nanoscopic neuronal dynamics.

SIGNIFICANCE

this technique optimizes the combination of speed and depth for improved superresolution in vivo imaging in the rodent neocortex.