> >Andy Oberyszyn writes: > > >We are attempting to measure luminescence from yeast cells (4-6 um > >spheroids/ellipsoids) expressing the calcium-sensitive photoprotein > >aequorin. Aequorin luminescence is maximum at 470 nm. We are using a > >Beckman-Coulter Elite instrument with a HeNe laser (633 nm) beam for > >forward and side scatter, a single 600 nm dichroic shortpass filter to > >reflect the red side scatter, allowing the transmitted (300-595 nm) > >luminescence to be collected by PMT 4. > > > >Since luminescence under these conditions is quite weak, we are asking for > >suggestions on how to increase and optimize sensitivity and improve the > >signal to noise. Suggestions on configuration, ways to reduce stray light > >reaching the detector and details or references to previous studies > >measuring bioluminescence would be appreciated. > > The only paper on this subject which comes immediately to mind is: 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 These authors were looking at emission from a luciferin-luciferase system, but measurements were made as the cells passed through a 488 nm laser beam, which could potentially have been exciting fluorescence as emission was at a longer wavelength. Peak fluorescence was near the boundary between the lowest and next lowest decades on a four-decade log scale. Whether it is feasible to measure emission from the aequorin system can be determined from measurements in a static apparatus, preferably a microscope or instrument capable of examining single cells and using a photon counting detector. This would allow you to estimate how many luminescent photons will be emitted by a single cell and collected by microscope optics in the time period required for the cell to traverse the laser beam in a flow cytometer. It would be harder to estimate this figure from bulk luminescence measurements, but not impossible. If the number is 100 or more, you'll probably be able to detect the luminescence in the Elite, which has fairly efficient light collection optics. If it is less than a few dozen, it's unlikely you'll be able to make the measurements. You will get the best sensitivity by using the slowest flow rate the instrument will tolerate; you also want to make sure no red laser light is getting through your emission filter. Since you're talking about detecting 300-595 nm, it sounds as if you're not using an emission filter; that will almost certainly give you problems with stray light. Try a wideband (40-50 nm) filter centered near the emission maximum of aequorin - if and only if the feasibility exercise suggests you ought to bother proceeding. A slow-flow Cytomutt to measure luminescence, using a diode laser, a diode scatter detector (for triggering), and a PMT for luminescence detection, could probably be built fairly cheaply, but your user would have to be really hot to do the measurements to justify going along that route. -Howard
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