CALCIUM IMAGING WITH THREE-PHOTON MICROSCOPY IN THE MOUSE BRAIN
Calcium imaging posed both opportunities and challenges for three-photon microscopy. With the development of genetically encoded calcium indicators, optical imaging has enabled in vivo activity recordings from increasingly large neuronal populations with high spatial and temporal resolution. The synergy between calcium imaging and three-photon microscopy can potentially allow functional imaging beyond the depth limit of two-photon microscopy. However, the transition of three- photon microscopy from structural to functional imaging of high temporal resolution requires orders of magnitude stronger signal. The challenge was eventually overcome by the optimization of three-photon microscopy and the improvement of calcium indicators and their genetic expression. Currently, the technology has been adopted by many researchers for biological and neuroscience studies. In this thesis, the performance of three-photon microscopy with 1300-nm excitation and two-photon microscopy with 920-nm excitation are quantified and compared for in vivo imaging of GCaMP6s-labeled neurons. 1300-nm three-photon microscopy is more suited for imaging in the deep cortex of the mouse brain or beyond because it is more power-efficient for signal generation and has significantly higher signal-to-background ratio. These advantages are the results of the reduced light attenuation at the longer excitation wavelength and the improved three- dimensional confinement by high order nonlinear excitation. Furthermore, we demonstrated three-photon calcium imaging of ~ 150 GCaMP6s-labeled neurons in the mouse hippocampus at ~1-mm depth within an intact mouse brain. The imaging achieved 8.5 frame/s speed and 200 × 200 μm field-of-view. With ~50 mW average power at 800 kHz repetition rate, we were able to image the same regions in the hippocampus of several animals over multiple days, which manifested the safety and consistency of the method. Three-photon microscopy also finds another surprising application in imaging through the intact mouse skull. Compared to two-photon- excitation, even with the same long excitation wavelength and imaging system, three- photon-excitation significantly improves optical sectioning in presence of the skull- induced scattering and aberration. Through the intact adult mouse skull, we demonstrated three-photon imaging of vasculature at >500 μm depth, and GCaMP6s- calcium imaging over weeks in cortical layers 2/3 and 4 in awake mice, with 8.5 frames/s and hundreds of micrometers field-of-view.
brain imaging; calcium imaging; in vivo imaging; multi-photon microscopy; three-photon microscopy; Biophysics; Optics
Wise, Frank William; Nishimura, Nozomi
Ph. D., Applied Physics
Doctor of Philosophy
dissertation or thesis