Characterizing a Single Plane Illumination Microscope for Imaging Fluorescence Correlation Spectroscopy

Abstract

In many systems, in vitro or in vivo, it has become important to experimentally obtain dynamical information at many different positions simultaneously. This is a challenge as conventionally, dynamic information in biological systems is probed with a confocal microscope to perform either fluorescence correlation spectroscopy (FCS) or fluorescence recovery after photobleaching (FRAP), which can be damaging due to phototoxicity, and yields information at a single position. Advances in camera sensors have allowed their use in place of single point detectors and the implement of imaging FCS by way of single plane illumination microscopy (SPIM). In this modality, a light sheet with a thickness of only a few microns illuminates the sample and the fluorescence is projected orthogonally onto the camera chip. By imaging small regions of interest at a very high frame rate, we can determine dynamic parameters such as diffusion coefficients and local concentrations in a 2D array of pixels. In this thesis, I discuss the theoretical background, hardware setup, design and characterization of a SPIM which I have built in order to perform imaging FCS.