Optics and experimental resolution of the Heidelberg slit-scan flow fluorometer

Michael Hausmann; Burkhard Wickert; Michael Vogel; Michael Schurwanz; Juergen Doelle; Dietmar Wolf; Klaus Aldinger; Christoph G. Cremer

doi:10.1117/12.229509

8 January 1996 Optics and experimental resolution of the Heidelberg slit-scan flow fluorometer

Michael Hausmann, Burkhard Wickert, Michael Vogel, Michael Schurwanz, Juergen Doelle, Dietmar Wolf, Klaus Aldinger, Christoph G. Cremer

Author Affiliations +

Proceedings Volume 2629, Biomedical Optoelectronics in Clinical Chemistry and Biotechnology; (1996) https://doi.org/10.1117/12.229509
Event: BiOS Europe '95, 1995, Barcelona, Spain

Abstract

Slit-scan flow fluorometry is a laser-technological approach for accelerated screening and sorting of fluorescence labelled metaphase chromosomes. Details of the optics of the Heidelberg slit-scan sorter are presented. In a fluid stream the fluorescence labelled chromosomes rapidly pass one at a time by a scanning laser beam. The laser can be focused by a less complex optic consisting of only a few commercially available lenses. The laser intensity distribution around the focus was measured for 488 nm for two lens configurations. Although the light distribution obtained by such an optic is normally not aberration free, the requirements of a 'ribbonlike' shape in the center of the fluid stream can be fulfilled. Since the chromosomes are oriented perpendicularly to the laser beam by hydrodynamic focusing of the fluid stream, the fluorescence intensity along the chromosome axis can be measured time (equals spatially) resolved. According to their intensity profiles the chromosomes can be classified. Signal processing of the profiles can be performed in less than 600 microseconds, so that in the order of hundred chromosomes per second can be sorted out by a computer controlled electro-acoustic sorting unit. The final spatial resolution of a slit-scan flow sorter is not only affected by the focusing optics of the laser but also by the fluid stream, the detection optics and electronics, as well as by the computer analysis algorithm. Calculations often consider only the optics under ideal conditions. Here, a method is shown how to estimate the overall resolution of a slit-scan flow fluorometer experimentally. According to this criterion the resolution of the Heidelberg slit-scan sorter for 488 nm fluorescence excitation was estimated to be 2.4 micrometer in its basic optical configuration and 1.7 micrometer with additional correction of chromatic aberration effects.

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Michael Hausmann, Burkhard Wickert, Michael Vogel, Michael Schurwanz, Juergen Doelle, Dietmar Wolf, Klaus Aldinger, and Christoph G. Cremer "Optics and experimental resolution of the Heidelberg slit-scan flow fluorometer", Proc. SPIE 2629, Biomedical Optoelectronics in Clinical Chemistry and Biotechnology, (8 January 1996); https://doi.org/10.1117/12.229509

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