Howard, We've also had an interest in doing this with luciferase. All of our attempts have flopped. The data from the paper you cite has been difficult to reproduce. We've even tried using cell-permeable luciferin. We can't see it in a scope either, but know that its there by checking with a luminonmeter. If one could really accomplish this, it would be quite useful. Luciferase systems are extremely powerful because of their excellent signal:noise ratios (backgrounds are zip) in part due to the complete elimination of autofluorescence issues. We would like to use flow cytometry to sort positive luciferase transfectants. I hope somebody can get this system to work reliably some day. Phil Marder --- Howard Shapiro <hms@shapirolab.com> wrote: > > > > > >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 > > > > > __________________________________________________ Do You Yahoo!? Talk to your friends online with Yahoo! Messenger. http://im.yahoo.com
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