The University of Toledo

Tirf Microscopy

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:

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.

 

Advantages: No contribution from out of focus fluorescence from deeper in the sample. Better z-axis resolution than confocal microscopy (100 nm vs. 500 nm). Can track events exclusively at or just below cell membrane of live cells.