Parole chiave: phototoxicity, lasers, confocal, time-lapse imaging, AOTF, fluorescence, Fluorophore, AOM, live-cell imaging, immunofluorescence
Definizione:A means of inactivating specific proteins in living cells (with high spatial and temporal resolution) using pulsed laser light
TECHNOLOGY:
In CALI (also known as fluorophore-assisted laser inactivation - FALI) specific proteins are targeted with non-function blocking antibodies conjugated to a dye, such as malachite green. The dye is selectively activated by focused laser irradiation (for example, 620 nm in the case of malachite green), which does not significantly affect surrounding cellular components. Irradiation of the dye results in the generation of highly reactive hydroxyl radicals that damage the antibody-bound protein. In micro-CALI, microscope optics focus the laser beam so that only a micron-scale spot in a cell is photoactivated. Technologies such as AOM and AOTF can help to precisely target laser power to defined areas of interest. CALI can inactivate proteins within minutes (depending on laser power, number of laser pulses and dye concentration) allowing researchers to study the effects of the inactivated protein on cellular processes. Recovery of function requires synthesis of new proteins - a process that may take several hours.
APPLICATIONS:
CALI has been used to inactivate, and study the function of, a variety of cellular proteins including enzymes, cytoskeletal proteins, receptors, signal transduction molecules and transcription factors. CALI has the advantage that it is a relatively easy knock-out strategy to perform and has very high spatial and temporal resolution. In addition, genetic compensation, which could confound observations in other knock-down methods, is unlikely to affect this technique in the short-term.
MICROSCOPE CONFIGURATION:
CALI can be carried out on widefield epi-fluorescence microscopes and confocal microscopes, although confocal provides greater opportunity for better fluorescence resolution and 3-D imaging.
RECOMMENDED SYSTEM:
TE2000 with environmental control configured with C1 confocal system.
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