Intensity calibration of a laser scanning confocal microscope based on concentrated dyes.
OBJECTIVE
To find water-soluble fluorescent dyes with absorption in various regions of the spectrum and investigate their utility as standards for laser scanning confocal microscopy.


METHODS
Several dyes were found to have characteristics required for fluorescence microscopy standards. The intensity of biological fluorescent specimens was measured against the emission of concentrated dyes. Results using different optics and different microscopes were compared.


RESULTS
Slides based on concentrated dyes can be prepared in a highly reproducible manner and are stable under laser scanning. Normalized fluorescence of biological specimens remains consistent with different objective lenses and is tolerant to some mismatch in optical filters or imperfect pinhole alignment. Careful choice of scanning parameters is necessary to ensure linearity of intensity measurements.


CONCLUSIONS
Concentrated dyes provide a robust and inexpensive intensity standard that can be used in basic research or clinical studies.
Model
Michael A
MA
Department of Biological Sciences, Kent State University, Kent, Ohio, USA. mmodel@kent.edu
Blank
James L
JL
D000818:Animals; D002138:Calibration; D002417:Cattle; D002460:Cell Line; D004247:DNA / Q000235:genetics; D005456:Fluorescent Dyes / Q000032:analysis*; D018613:Microscopy, Confocal / Q000379:methods*
D005456:Fluorescent Dyes; D004247:DNA