Multiple spectral parameter imaging in quantitative fluorescence microscopy. I: Quantitation of bead standards.
Digitized images from fluorescence microscopy may suffer from nonlinearity of camera response to illumination intensity, from uneven spatial response of the camera, and from uneven illumination of the microscopical field. Structureless fluorescence standard slides are used to characterize the camera response. This information permits the images to be corrected for all the above errors, in one operation. The computer algorithms are described, and also those for thresholding and finding objects. The method is illustrated by tests using fluorescent beads.
Galbraith
W
W
Allegheny-Singer Research Institute, Pittsburgh, PA 15212.
Ryan
K W
KW
Gliksman
N
N
Taylor
D L
DL
Waggoner
A S
AS
D000465:Algorithms; D002469:Cell Separation; D005434:Flow Cytometry; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000592:standards*; D008863:Microspheres; D008961:Models, Structural; D010781:Photography / Q000592:standards; D013057:Spectrum Analysis