From: Marty Bigos (BIGOS@Darwin.Stanford.EDU)
Date: Mon Jun 06 1994 - 12:45:27 EST
David- Several factors influence bead cv measurements. One is sample flow rate, which dstermines sample core size in the jet (or cuvette). The smaller the sample core, the more likely each bead will traverse the same path in the jet through the excitation, and the more uniform the measurements should be. So, for most flow instruments, one way to achieve better cv's is to slow down the sample flow. The illumination and collection optical geometry also can have a great effect on the measured cv; most people will have to assume that it is a given, since they are not going to redisgn their setup. However, insuring the cleanliness of all these parts can result in a stronger signal which would reduce measurement error. Lasers (if used for excitation) are another place where error can be reduced. If you have control over the laser aperture, adjusting for a gausian profile is important. Also, in general, the higher the excitation power, the less measurement error there will be due to statistical variation in the signal. However, with real dyes there is a trade-off between higher autofluorescence of cells (depending on excitation/emission wavelengths and cell type) and dye saturation (depending on dye type). Lastly, here is a method we have used to estimate both the number of photons measured for a given bead (very useful for comparing machines) and an estimate of the intrinsic cv of the signal source (which includes bead cv). 1) Under normal operating conditions, measure bead signal strength and cv. 2) Using various neutral density filters in the light collection path, measure the cv and signal levels at the same laser, flow rate, and PMT settings as (1). You might have to increase the gain of the linear amp used in the measurements in order to do this. 3) Plot the data as 1/(signal level) on X and CV**2 on Y. The y-intercept is an estimate of the cv**2 of the source, which includes the bead cv, and the slope yields an estimate of the number of photoelectrons the source is producing, using the formula: EVENTS = 1/(cv**2 - CVo**2), where CVo**2 is the y-intercept on the graph. Hope this helps. -Marty Bigos Stanford Shared FACS Facility
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