2P-miniscope

DEVELOPMENT AND VALIDATION OF TWO-PHOTON MICROSCOPY IN FREELY-MOVING MICE

Summary

Two-photon imaging is a nonlinear microscopy technique that allows high-contrast, functionallarge-depth 3D tissue imaging with excellent axial-sectioning capability. Within the context of liveanimalimaging, the technique is currently limited to head-fixed preparations, due to the bulkydesign of a typical 2-photon microscopes. Wide-field one-photon imaging, a technique commonlyused in Prof. Diester's work, is possible in freely-moving animals but the resolution and penetrationas well as contrast is limited. In this project, we will combine the best of the two worlds, anddevelop a miniaturized two-photon microscope that can be used in freely-moving mice and rats.This technique will be used to characterize neuronal activities in head-fixed and freely-movingconditions. In head-fixed animals, we use virtual reality to induce the impression of freemovements. It is unclear by how much the VR is perceived as naturalistic by the animals. Bycomparing the neuronal activities under comparable imaging parameters (e.g. resolution, imagingdepth,field-of-view, etc.) in both head-fixed and freely moving animals we can for the first timeaddress this question.

Two-photon microscopy is a scanned-imaging technique, where a tightly-focused fs-laser beaminduces nonlinear absorption by an endogenous or an exogeneous fluorescent molecule, resultingin an extremely localized emission. Thus, it can provide not only a high lateral resolution, but alsoan efficient depth-sectioning capability. Furthermore, it relies on near-IR illumination allowingconsiderably larger penetration depth compared to one-photon microscopy, due to lowerscattering in this wavelength regime. All these advantages, however, can only be exploitedthrough a complex opto-mechanical arrangement, which is challenging to miniaturize. En faceimaging requires a 2D scanning of the excitation beam, and depth-scanning requires either focustuningor sample translation. Separation of the excitation and collection paths, on the other hand,needs to be spectrally separated through dichroic optics, and the former needs to be focusedthrough a high-NA (>0.7) objective onto the tissue for efficient two-photon fluorescence emission.Dr. Ataman's group has focused on addressing similar miniaturization challenges in their work inendomicroscopy. Based on the methods developed in this line of work, we aim to demonstrate atwo-photon miniscope prototype that can perform 3D volumetric imaging of GCamp8f/GCamp8m stained neural tissue within a field-of-view of 350x350 μm2, submicron lateral resolution, and ameasurement depth range of ~400 μm.