A method is described for measuring optical properties and deriving chromophore concentrations from diffuse reflection measurements at the surface of a turbid medium. The method uses a diffusion approximation model for the diffuse reflectance, in combination with models for the absorption and scattering coefficients. An optical fibre-based set-up, capable of measuring nine spectra from 400 to 1050 nm simultaneously, is used to test the method experimentally. Results of the analyses of phantom and in vivo measurements are presented. These demonstrate that in the wavelength range from 600 to 900 nm, tissue scattering can be described as a simple power dependence of the wavelength and that the tissue absorption can be accurately described by the addition of water, oxy- and deoxyhaemoglobin absorption.

Doornbos, R.M.P.; Schaeffer, M.; Hoekstra, A.G.; Sloot, P.M.A.; de Grooth, B.G.; Greve, J.

This report describes a feasibility study concerning the use of a visible diode laser for two important fluorescence applications in a flow cytometer. With a 3 mW 635 nm diode laser, we performed immunofluorescence measurements using the fluorophore allophycocyanin (APC). We have measured CD8 positive lymphocytes with a two-step labeling procedure and the resulting histograms showed good separation between the negative cells and the dim and the bright fluorescent subpopulations. As a second fluorescence application, we chose DNA analysis with the recently developed DNA/ RNA stains TOTO-3 and TO-PRO-3. In our setup TO-PRO-3 yielded the best results with a CV of 3.4%. Our results indicate that a few milliwatts of 635 nm light from a visible diode laser is sufficient to do single color immunofluorescence measurements with allophycocyanin and DNA analysis with TO-PRO-3. The major advantages of using a diode laser in a flow cytometer are the small size, the low price, the high efficiency, and the long lifetime. 0 1994 Wiley-Liss, Inc.

We investigated the fluorescence emission from three fluorophores commonly used for labeling cells in flow cytometry. We have demonstrated that the fluorescence emission from cells labeled with fluorescein-isothiocyanate (FITC), phycoerythrin (PE), and allophycocyanin (APC) is considerably saturated and bleached in standard flow cytometric conditions. Therefore, for optimization of fluorescence detection in a flow cytometer, it is important to know the emission kinetics in detail. We made a mathematical model of the optical processes involved: absorption, fluorescence emission, nonradiative decay, photodestruction, and triplet state occupation. The validity of the model was experimentally tested with a set of averaged fluorescence pulses, measured in a large range of intensities and illumination times. The fluorescence of APC could be completely described by the model and produced the following rate constants: photodestruction rate k b1 ‫؍‬ 6 • 10 3 s ؊1 , triplet state population rate k 12 ‫؍‬ 2 • 10 5 s ؊1 , and depopulation rate k 20 ‫؍‬ 5 • 10 4 s ؊1 . The fluorescence kinetics of FITC-and PE-labeled cells could not be fitted with only three parameters over the entire range, indicating that other optical processes are involved. We used the model to determine the sensitivity of our flow cytometer and to calculate the optimum conditions for the detection of APC. The results show that in principle a single APC molecule on a cell can be detected in the presence of background, i.e., autofluorescence and Raman scattering by water.

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