Multifocal plane microscopy for the study of cellular dynamics in three dimensions

Fluorescence microscopy is an invaluable tool to investigate cellular trafficking pathways in live cells. For example, Total Internal Reflection Fluorescence (TIRF) microscopy has enabled high sensitivity visualization of trafficking events on the plasma membrane of a cell. Similarly, epifluorescence microscopy is a widely used imaging technique to study cellular dynamics inside a cell. However, tracking of cellular components that move in three dimensions, i.e. between different focal planes, is problematic. To enable tracking in three dimensions, typically multiple focal planes of a sample are sequentially imaged by changing the objective focus using a focusing device. However, the speed of image acquisition is limited with such focusing devices. Additionally, only one focal plane can be visualized at a given time. Therefore, important events that take place outside this focal plane can be missed. To facilitate the simultaneous visualization of multiple focal planes of a sample, multifocal plane microscopy has been developed during this research project. This has been achieved by a modification of the emission light path of a conventional microscope and by utilizing a multi-camera image acquisition format. A design of the excitation light path of a fluorescence microscope that allows for simultaneous TIRF and epifluorescence illumination is also presented in this thesis. This excitation scheme together with multifocal plane microscopy enables the simultaneous visualization of the events on the plasma membrane and inside a cell. To demonstrate the capabilities of this new imaging modality, a study has been conducted at the cellular level to understand the mechanism of antibody trafficking in three dimensions, specifically of immunoglobulin G as mediated by the neonatal Fc receptor, FcRn. This study has provided insight into the intracellular trafficking events on the recycling pathway preceding exocytosis of FcRn. Software tools were also developed for automated image acquisition and image processing. Developed using the object-oriented design philosophy, the image acquisition software package provides a flexible environment to integrate hardware components. The software tools for image processing utilize image pointers to organize and process the multifocal plane microscopy data.