Total internal reflection fluorescence microscopy (TIRFM) is a form of near-field illumination in which the difference of refractive index between the sample and coverslip is used to generate an evanescent wave of illumination that penetrates to a very shallow depth (on the order of 200 nm). This illumination strategy is ideal for imaging events at or very near the cell membrane without contributions from out-of-focus blur.
Many researchers have utilized this technique to image endocytic and exocytic events as well as protein interactions at the cell membrane. EMCCD technology is well suited to TIRFM owing to the superb temporal resolution and sensitivity such cameras provide in the context of dynamic imaging studies. Photometrics EMCCD cameras have been used to extremely good effect under highly demanding TIRF imaging conditions.
One of the goals of modern microscopy is to correlate the spatial and temporal data-gathering ability of fluorescence microscopy to the functional activity of biochemical events. When imaging molecular interactions and signaling processes in space and time, camera sensitivity and the ability to acquire images at a high rate of speed can have an appreciable impact on the quality of results obtained. Advanced CCD imaging solutions from Photometrics have been demonstrated to produce significant and impressive results in this context.
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