Resolution of complex fluorescence spectra recorded on single unpigmented living cells using a computerised method

Abstract

The identification and quantification of fluorescent compounds in a complex fluorescence spectra are always difficult, especially in the case of low signaknoise ratio.We propose a computerised method that allows the resolution of low light level complex fluorescence spectra into its components.Based on a linear combination of N possible characteristic fluorescence spectra, and using N weighting functions, this method allows the integration of fluorescence intensities over the entire fluorescence spectra and the generation of n equations with N unknowns.The compounds that participate in complex fluorescence spectra are identified and quantified. Because fluorescence intensities can be integrated we can resolve complex fluorescence spectra presenting a low signaknoise ratio. The reliability and sensitivity of our method are shown through examples of resolution of complex intracellular fluorescence of single living cells pretreated with benzo(a)pyrene. Depending on the cell type and treatment, two, four, or five components can be identified in the complex fluorescence spectra.

Nile red labeling of single living cells for contour delineation to quantify and evaluate the distribution of rhodamine 123 with fluorescence image cytometry.

“…The complex fluorescence spectrum resulting from the accumulation of Rl23 and NR in the cells was resolved into its components with a library of characteristic fluorescence spectra of these compounds recorded and stored before analysis. The fit between the experimental spectrum and the calculated spectrum was monitored by: (a) the statistical distribution of weighted residues (WR) (WR was defined as the ratio of the difference between the measured signal and the corresponding calculated signal versus noise on the difference); and (b) the variance of the distribution of WR (chi-square) (for more details see reference 21) Numerical Image Analysis. The fluorescence image cytometry system has been described elsewhere (25).…”

Microspectrofluorimetric study of the kinetics of cellular uptake and metabolization of benzo(a)pyrene in human T 47 D mammary tumor cells: evidence for cytochrome P1450 induction

“…those of B(a)P and its metabolites) previously recorded under the same experimental conditions from the HPLC fractions collected from cellular extracts. The program is based on a least squares method (following Salmon et al (1988)). The procedure involves a matrix analysis includ- ing all the reference spectra and gives the coefficients of the linear combination which minimize the sum (over the 1024 channels) of the quadratic deviation between the experimental and the calculated spectra.…”

C-SNARF-1 as a Fluorescent Probe for pH Measurements in Living Cells: Two-Wavelength-Ratio Method versus Whole-Spectral-Resolution Method

“…We defined I j (λ) as the characteristic emission spectrum of a solution that contains a known concentration of the chemical j. The whole experimental spectrum I(λ) can be expressed as a linear combination of the characteristic emission spectra of the N components (7),…”