An Experimental Study of the Precision of Fluorescence Measurements

Abstract

The precision of fluorescence measurements by the SLM 22 fluorometer was calculated with the use of fluorescein solutions of varying concentrations. Relative standard deviations for 10 replicate fluorescence measurements, each integrated over five seconds, are not observably affected by the changing of bandpasses from 1 to 8 nm, by the changing of cell orientation, or by fluorescein concentration at levels of 1.0 × 10−9M and above. Fluctuations in source intensity are shown to be the primary source of noise. Statistical fluctuations in photon and photoelectron flux do not seem to be a significant source of variability. Because of this, the precision of single-channel intensity measurements is poorer than for intensity measurements ratioed directly to source intensity. However, the precisions of the ratio of fluorescence intensities measured by dual detectors on opposite sides of the cell were often found to be better than for intensity measurements ratioed to the source. It is hypothesized that this is observed because the measurements of source intensity and fluorescence are not identically affected by fluctuations in the source. At fluorescein concentrations in the 10−9M range and above, percent RSDs for single channel measurements typically fall in the range of 0.2% to 0.5%, although occasionally larger values are observed. The percent RSDs for the ratio of simultaneous fluorescence measurements are typically in the range of 0.1 to 0.2% at higher concentrations.