Julie Auger and Robert Nordon both recently asked about using luciferin/luciferase and/or detecting bioluminescence in flow cytometry. There was a paper on this subject some years back (Lindqvist C, Karp M, Åkerman K, Oker-Blom C: Flow cytometric analysis of bioluminescence emitted by recombinant baculovirus-infected insect cells. Cytometry 15:207-212, 1994), which reported detecting emission in the appropriate region and with the appropriate kinetics, from cells infected with baculoviruses encoding luciferase genes. However, the cells were also passing through a 100 mW, 488 nm beam at the time, and fluorescence may have been involved. Most bioluminescence measurements, while sensitive (single photon counting is often used), are slow. The typical measurement time for cells in flow cytometers with normal flow rates is on the order of a few microseconds; operating at PMT gains between 10,000 and 100,000, it can be calculated that the typical four decade measureement scale goes from tens of photons, at the low end, to hundreds of thousands, at the high end. The background levels measured by Lindqvist et al occupied most of the bottom decade (no more than a few hundred photons); putative luminescence signals (550-590 nm) were no more than ten times this magnitude (a few thousand photons). If it is reasonable to assume that luciferase action within a single cell will account for at least a few thousand photons of bioluminescent emission during the few microseconds used for measurement, then the emission should be detectable. However, one really ought to do this with the laser off, or using a laser at a wavelength longer than the luminescence emission wavelength (e.g., a red diode or He-Ne) for a scatter trigger, to eliminate the possibility that what is being detected is fluorescence or phosphorescence rather than bio[chemi]luminescence. Lower levels of luminescent emission could be detected in a system with a slower flow rate and/or photon counting detectors, like those built for measurement of DNA fragments, viruses, etc. -Howard
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