Chemical Analysis and Flow Cytometry

The difficulties of intensity-based flow cytometry are illustrated by the present difficulties of cell-by-cell measurements of intracellular calcium. This can be accomplished using the calcium probe Indo-l,(34 38) but requires an ultraviolet (UV) laser source which is not routinely available in flow cytometry (Indo-1 is an emission wavelength ratiometric probe). Flow cytometers routinely have argon ion laser sources with outputs of 488 or 514 nm. Measurement of intracellular ions other than Ca2+ is nearly impossible. (The SNAFL and SNARF probes should allow pH measurement from the wavelength-ratiometric data.(15)) 

The problem has now been solved, and it is possible to measure the phase angle of the probe as the cells pass through the laser beam.09,40) While these measurements have not yet been applied to Ca2+, the method has been validated with standard beads and stained cells. In our opinion, this new flow cytometry parameter, and our increasing knowledge of lifetimes of probes, will result in the increased use of flow cytometry for studies of intracellular physiology, in addition to the current emphasis in immunology, cell activation, and ploidy. 

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