QUANTITATIVE IMAGING OF NANOPARTICLES AND INTRACELLULAR VESICLE TRAFFICKING USING TOTAL INTERNAL REFLECTION FLUORESCENT MICROSCOPY (TIRFM)

摘要

Total internal reflection fluorescent microscopy (TIRFM) is a relatively well known tool used to examine the near wall region (approximately 1 mm). For years, cellular biologists have used TIRFM in a variety of experiments to examine multiple cell lines. However, much of the research has been somewhat static in nature, considering only initial and final states. With the increased ability to stain specific organelles within cells through the use of green fluorescent protein (GFP), dynamic imaging is becoming a viable solution to previously difficult problems. Of particular interest in cellular studies is protein transport to different cellular regions. Often, these proteins are trafficked through the use of vesicles. Vesicles are generally tubular in structure and are able to move relatively large quantities of proteins to different locations. When cells are grown on coverslips, TIRFM is particularly adept at examining how vesicles interact with the plasma membrane. An excellent paper examining the delivery of proteins through the fusion of a vesicle with the plasma membrane is given by Toomre et. al. [1]. Previous work by the MiNSFET lab at The University of Tennessee has dealt with the tracking of polystyrene nanoparticles undergoing Brownian motion in solutions of differing salinity [2]. These particles are of the same order in size as several of the vesicles involved in protein trafficking within multiple cell lines. The goal of this presentation is to demonstrate methods that have been developed from this previous work and to demonstrate how it is being applied to current cellular studies.