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Spatial dependence of the optical collection efficiency in flow cytometry

  1. Peter M. Goodwin1,*,
  2. W. Patrick Ambrose1,
  3. John C. Martin2,
  4. Richard A. Keller1

Article first published online: 8 MAR 2005

DOI: 10.1002/cyto.990210205

Issue

Cytometry

Cytometry

Volume 21, Issue 2, pages 133–144, October 1995

Additional Information

How to Cite

Goodwin, P. M., Ambrose, W. P., Martin, J. C. and Keller, R. A. (1995), Spatial dependence of the optical collection efficiency in flow cytometry. Cytometry, 21: 133–144. doi: 10.1002/cyto.990210205

Author Information

  1. 1

    Chemical Science and Technology Division, Center for Human Genome Studies, Los Alamos National Laboratory, Los Alamos, New Mexico

  2. 2

    Life Sciences Division, Center for Human Genome Studies, Los Alamos National Laboratory, Los Alamos, New Mexico

*Los Alamos National Laboratory, P.O. Box 1663, Mail Stop M888, Los Alamos, NM 87545

Publication History

  1. Issue published online: 23 JUN 2005
  2. Article first published online: 8 MAR 2005
  3. Manuscript Accepted: 6 APR 1995
  4. Manuscript Received: 27 APR 1994

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Keywords:

  • Sensitive fluorescence detection;
  • laserinduced fluorescence;
  • single molecule detection;
  • microscope objective;
  • spatial filter;
  • submicrometer light source

Abstract

The sensitive flow cytometric detection of fluorescent species in liquid sample streams requires efficient collection of light from small [∼1] picoliter (p1) 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 measurements 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. © 1995 Wiley-Liss, Inc.

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