Open AccessSpatial dependence of the optical collection efficiency in flow cytometry
Author(s) -
Goodwin Peter M.,
Ambrose W. Patrick,
Martin John C.,
Keller Richard A.
Publication year - 1995
Publication title -
cytometry
Language(s) - English
Resource type - Journals
eISSN - 1097-0320
pISSN - 0196-4763
DOI - 10.1002/cyto.990210205
Subject(s) - optics , data collection , sample (material) , numerical aperture , aperture (computer memory) , sampling (signal processing) , materials science , physics , statistics , wavelength , mathematics , acoustics , thermodynamics , detector
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.