Lasers appropriate fluorochromes

Fluorescence microscope equipped with appropriate filter sets for detection of one or more fluorochromes and image capture system. If cells that grow flat are to be used, a conventional epifluorescence microscope is adequate. For thicker cells, a microscope equipped with a confocal laser scanning system is an advantage. 

For FLIM determinations, cells are plated and transfected as in Subheading 3.7.1, FLIM measurements are performed using a confocal microscope with a High-Speed Lifetime Module and a 60x PlanApo 1.4 objective or equivalent equipment. Fluorescence lifetime is determined after excitation with a pulsed laser (picosecond pulses) and a bandpass emission filter appropriate for the fluorochrome used, and quantitated using LIMO (Nikon) or similar software. Avoid the use of mounting solutions, which increase autofluorescence. 

Specific antibodies conjugated to appropriately selected fluorescent dyes can be used to study fixed cells. For example, although Gy3 as donor and Gy5 as acceptor form a suitable fluorochrome pair, the Gy2 donor/Cy3 acceptor pair is often more convenient, as it permits use of the widely available 488 nm argon laser line. Secondary antibodies are occasionally necessary, although the increased distance between fluorophores complicates FRET detection this can be resolved using dye-labeled F(ab ) fragments. 

Most of the modern flow instruments are capable of multiparameter detection. A series of detectors and filters are positioned appropriately to measure fluorescence at a number of wavelengths. Usual placement for fluorescence detectors is at 90° to the incident laser beam. The presence of multiple detectors allows use of several fluorochromes to detect multiple cellular parameters. Many instruments also use two or more sequential lasers to excite fluorescence in cells at multiple wavelengths. 

Laser scanning confocal microscope or fluorescence microscope equipped with appropriate filter sets for genetically encoded FRP(s) and fluorochrome(s) or fluorescent dyes employed in double/multiple labeling procedures. See Notes 5 and 7. 

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