Development of High-speed Two-photon Microscope for In vivo Deep Tissue Imaging and Biological Applications

Abstract

This thesis describes the implementation and characterization of a high-speed two-photon microscope (TPM) and its biological applications. The goal of this thesis work is implementation of higher performance TPM than previous reported TPM. The key performance of TPM is an imaging depth which depends on fluorescence signal collection and is affected by aberration from objective lens within turbid sample. First, result from measurement of signal decay coefficient and ex vivo brain imaging shows that constructed TPM has high imaging depth over 400 μm that is same level of world class. Second, the aberration effect of the objective lens is not much significant. TPM is a three dimensional (3D) fluorescence imaging technique suitable for studying live cells within in vivo tissue. For preliminary imaging of real biological sample before in vivo , this paper shows ex vivo immune cells imaging within lymph node and small intestine.Although TPM has good contrast in visualizing fluorescence cells, in formation of surrounding tissue such as non-fluorescent cells and tissue structure is not shown. Optical coherence microscopy (OCM) is a 3D imaging technique based on low coherence interference. OCM can show unlabeled cells and tissue structure. Thus, the combined TPM and OCM will simultaneously provide functional and morphological information of tissue.