Multifocal Multiphoton Microscopy for Neurobiological Imaging

Abstract

Observing neuronal structures and monitoring changes in synaptic connectivity with respect to time have a significant impact on understanding the basis of structural brain abnormalities and the development of therapeutics for their correction. Today, multiphoton excitation fluorescence microscopy is the method of choice for in vivo neuronal imaging with its inherent 3D resolution, minimal photo-damage, and excellent penetration depth. The study of neuronal interactions on dendritic arbor remodeling often requires large volume imaging demanding fast imaging speed. One of the methods to improve imaging speed is multifocal multiphoton microscopy (MMM) that parallelizes imaging process with multiple excitation foci. Early MMM had very limited imaging depth due to signal-to-noise ratio (SNR) degradation resulting from the scattering of emission photons in highly turbid biological specimens. The development of descanned MMM with multianode photomultiplier tube has partly alleviated this problem, but it still suffers from greater signal loss and the presence of image artifacts compared with conventional single focus multiphoton microscopes. In this thesis, adaptive optics compensation, image post processing for emission photon reassignment, and a novel non-descanned MMM have been investigated for SNR improvement. In addition, spectral resolved MMM has been developed for simultaneous fast imaging and spectral detection.