R.M.P. Doornbos

Elastic Light Scattering Measurements of Single Biological Cells in an Optical Trap

We have developed an instrument for determination of the angular light scattering of beads and biological cells. The instrument uses radiation pressure for levitation of particles inside a cuvette. The setup consists of two 780-nm diode lasers in a vertical double-beam trapping configuration. In the horizontal direction a weakly focused 633-nm probe beam is used to illuminate the trapped particle. One can detect scattered light over the range of from - 150 to 150 deg with an angular resolution of 0.9 deg using an avalanche photodiode. With this setup light scattering from polystyrene beads was measured, and the obtained scattering patterns were compared with theoretical scattering patterns from Lorenz-Mie theory. The results show that the setup is stable, gives reproducible patterns, and qualitatively agrees with the calculations. Trapping of biological cells is more difficult than trapping of beads, because smaller forces result from smaller refractive indices. We present an angular scattering pattern measured from a human lymphocyte measured from 20 to 60 deg.

Experimental and model investigations of bleaching and saturation of fluorescence in flow cytometry

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 kb1 = 6 x 10(3) s(-1), triplet state population rate k12 = 2 x 10(5) s(-1), and depopulation rate k20 = 5 x 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.

Another face of Lorenz-Mie scattering: monodisperse distributions of spheres produce Lissajous-like patterns

The complete scattering matrix S of spheres was measured with a flow cytometer. The experimental equipment allows simultaneous detection of two scattering-matrix elements for every sphere in the distribution. Two-parameter scatterplots with x and y coordinates determined by the S(ll) + S(ij) and S(ll)-S(ij) values are measured. Samples of spheres with very narrow size distributions (< 1%) were analyzed with a FlowCytometer, and they produced unexpected two-parameter scatterplots. Instead of compact distributions we observed Lissajous-like loops. Simulation of the scatterplots, using Lorenz-Mie theory, shows that these loops are due not to experimental errors but to true Lorenz-Mie scattering. It is shown that the loops originate from the sensitivity of the scattered field on the radius of the spheres. This paper demonstrates that the interpretation of rare events and hidden features in flow cytometry needs reconsideration.

Interleukin-2 Postremission Therapy in Acute Myeloid Leukemia (AML): In Vitro and In Vivo Effects of a Five Day Continuous Infusion of IL-2 on Phenotype and Function of Peripheral Lymphocytes

In vitro studies have demonstrated the anti-leukemic effect of IL-2 stimulated cytotoxic lymphocytes on allogeneic leukemic blasts prompting clinical trials with IL-2 in vivo. We initiated a phase II study with IL-2 in 6 patients with acute myeloid leukemia. The patients were in second or third remission after successful salvage treatment for relapse. All patients had received a standardized first line treatment according to the protocol of the German AML cooperative group. Salvage treatment consisted of sequential high dosed cytosine-arabinoside and mitoxantrone (sHAM). the postremission IL-2 therapy consisted of continuous intravenous infusion of IL-2 in a dosis of 3 × 106 IU/m/day over 5 days, four weeks later the next cycle was given. Lymphocyte subsets in the peripheral blood and their IL-2 receptor expression of these patients were studied on day 0, 2 and 5 of the IL-2 cycle using multiparameter flow cytometry. 13 IL-2 cycles in 6 patients were analysed.

Manipulation and characterization of cells by laser light

Both for medical and biotechnological purposes it is very important to be able to analyze and manipulate cellular populations on a single cell basis. Moreover it is often important to be able to sort a subfraction of cells from the total population with defined specific characteristics. Since the retainment of biological viability is of importance in such cases the use of monoclonal antibodies for cell characterization cannot always be applied. The use of light scattering offers an attractive alternative. One of the manipulations which is at the beginning of many biotechnological processes is the fusion between two well characterized and differing cell types. In the present paper we concentrate on the use of flowcytometry for light scattering characterization and fusion of cells.