Spatial dependence of the optical collection efficiency in flow cytometry

Cytometry
P M GoodwinR A Keller

Abstract

The sensitive flow cytometric detection of fluorescent species in liquid sample streams requires efficient collection of light from small [approximately 1 picoliter (pl)] sample volumes. This is often accomplished with high numerical aperture (NA) imaging collection optics used in combination with a spatial filter. A method to measure the spatial variation of the optical collection efficiency within the sample volume, using a submicrometer light source, is described. Measurements of the relative optical collection efficiency are presented for two optical collection systems used in our laboratory for single molecule detection. The measurement are in qualitative agreement with relative optical collection efficiency calculations using a simple geometrical optics model. Absolute measurements of the peak collection efficiencies for the two collection systems are also presented. These absolute collection efficiency measurements are in good quantitative agreement with ideal collection efficiencies calculated using measured transmissions and rated NAs of the collection optics. The utility of this information for the characterization and optimization of sensitive fluorescence detection apparatus is discussed.

References

Jan 1, 1992·Cytometry·G van den Engh, C Farmer
Jan 1, 1989·Journal of Inherited Metabolic Disease·J E Seegmiller
Feb 25, 1993·Nucleic Acids Research·P M GoodwinR A Keller
Sep 1, 1994·Journal of Fluorescence·U Mets, R Rigler

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Citations

Oct 26, 2007·Lab on a Chip·Kevin S LeeRajeev J Ram
Jul 2, 2004·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Matthew M FerrisRichard A Keller
Aug 4, 2004·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Robert C Habbersett, James H Jett
Jan 19, 2006·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Gregory GoddardGregory Kaduchak
Aug 1, 1997·Applied Optics·J Enderlein, W P Ambrose
Dec 26, 2001·Chemical Reviews·W P AmbroseR A Keller

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