Depth of field and improved resolution of slit-scan flow systems

Michael Hausmann; Martin Crone; Christoph G. Cremer

doi:10.1117/12.260808

6 December 1996 Depth of field and improved resolution of slit-scan flow systems

Michael Hausmann, Martin Crone, Christoph G. Cremer

Author Affiliations +

Proceedings Volume 2926, Optical Biopsies and Microscopic Techniques; (1996) https://doi.org/10.1117/12.260808
Event: BiOS Europe '96, 1996, Vienna, Austria

Abstract

In a slit-scan flow cytometer particles specifically labelled by fluorochromes (e.g., cells, chromosomes) are aligned coaxially in a flow stream. One by another they pass a ribbon-like shaped laser beam with a diameter smaller than the particle length. Although several slit-scan flow systems have been developed during the last two decades, a complete description of the theory of optical resolution under the real experimental conditions used as well as a description how to overcome experimental limitations are missing. Often, resolution values are estimated under the assumption of ideal Gaussian beam propagation. These estimates suffer from a discrepancy to practical implementation, Here, some of these effects in slit-scan optics are discussed from a more theoretical point of view. In order to obtain an acceptable depth of field, a focal width around 2 micrometer appears to be an optimum under the regime of Gaussian beam propagation. However, in practice, effects due to thick lenses, finite apertures, chromatic aberrations, or the ellipticity of the laser beam overshadow this result and influence the laser beam shape. To further improve the resolution with a high depth of field, new concepts are required. Therefore, a combination of an interference fringe pattern of two coherent laser beams for excitation (fringe-scanning) with a slit-scan detection of the incoherent fluorescence light is introduced. Preliminary experiences of the first experimental realization are discussed.

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Michael Hausmann, Martin Crone, and Christoph G. Cremer "Depth of field and improved resolution of slit-scan flow systems", Proc. SPIE 2926, Optical Biopsies and Microscopic Techniques, (6 December 1996); https://doi.org/10.1117/12.260808

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