University of Minnesota Multi-User Total Internal Reflection Fluorescence Microsc

University Of Minnesota, Minneapolis MN

Investigators

Abstract

DESCRIPTION (provided by applicant): All biological functions are dependent on molecular interactions. Therefore, by studying when, where and how molecules interact, we can elucidate the fundamental principles of biology. The Principle Investigators on this grant come from three different departments at the University of Minnesota and study a wide variety of topics, ranging from the dynamics of phospholipid receptor activation to the mechanisms that control amoeboid cell motility. These research projects each use different model systems and require their own specialized reagents. Yet, these projects are unified by the fact that each would benefit immensely from the use of Total Internal Reflection Fluorescence Microscopy (TIRFM). The long term goal of this proposal is to enhance basic and translational research at the University of Minnesota by providing state-of-the art, shared TIRFM equipment facilities with technical and scientific support. TIRFM is a specialized type of fluorescent microscopy in which only fluorescent molecule(s) within approximately 100 nm of the glass coverslip are excited (i.e. activated to fluoresce). With conventional epifluorescence microscopy, fluorescent excitation occurs though the entire depth of the sample;this generates out of focus background fluorescence, creating a poor signal-to-noise ratio and obscuring visualization. In addition, the sample is exposed to a considerable amount of fluorescent light, which can cause photobleaching (which leads to loss of imaging capacity) and, in the case of live cell imaging, phototoxicity and cell death. With laser confocal microcopy, the excitation beam is focused on a considerably narrower region (diameter: 250 -800 nm and depth: 500-1500 nm), improving the signal to noise ratio and reducing phototoxicity;however, with continuous imaging photobleaching and phototoxicity can still be a problem. All of the projects described in this proposal involve live cell, high speed or time-lapse imaging of fluorescently tagged proteins in order to follow the transport, assembly and/or processing of individual proteins within the cell. These experiments require the extremely narrow region of fluorescent excitation made possible by TIRFM;in each case, the investigators have attempted standard epifluorescence or confocal microscopy and found these techniques insufficient because they failed to provide the necessary resolution and/or sample preservation. Data from these experiments will elucidate the mechanisms that govern cell differentiation, motility and signaling, processes that are fundamental to all living organisms and, when mis-regulated, can deleteriously affect the health and lifespan of the individual. The specific aims of this proposal are: Aim 1) to provide access to specialized and cutting-edge resources not available to individual investigators. Having access to these recourses will extend the research capabilities and expertise of NIH-funded investigators. Aim 2) to foster new collaborations, particularly those that expand the research directions and impact of NIH-funded investigators.