The Advanced Microscopy & Imaging core facility houses a Total Internal Reflection Fluorescence (TIRF) Microscope (Olympus) coupled to multiple lasers. The TIRF system can be attached to an inverted microscope/imaging system and allows the visualization of fluorescent molecules either in wide-field (conventional) or exclusively at the cell-glass interface. This latter capability allows selective, real-time tracking of single molecule or particle dynamics at the surface of living cells.
TIRF Microscopy using evanescent wave technology:
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| Fig1. An evanescent wave (or field) is produced at the interface of two media having different refractive indices when a beam of light is reflected away. This wave decays exponentially in the z-direction and will only excite fluorescent molecules at a depth slightly greater than the width of a cell membrane | Fig2. Wide-field epifluorescence image of Mitotracker Green-labeled mitochondria in a glial cell. B. TIRF image of A demonstrating some mitochondira are within 200nm of cell surface. C. Wide field image of JC-1 loaded mitochondria. D. TIRF image of JC-1 loaded PC demonstrating submembrane localization of energized (red) mitochondria. |
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Advantages:
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