title: Intensity calibration and flat-field correction for fluorescence microscopes.
abstract: Standardization in fluorescence microscopy involves calibration of intensity in reproducible units and correction for spatial nonuniformity of illumination (flat-field or shading correction). Both goals can be achieved using concentrated solutions of fluorescent dyes. When a drop of a highly concentrated fluorescent dye is placed between a slide and a coverslip it produces a spatially uniform field, resistant to photobleaching and with reproducible quantum yield; it can be used as a brightness standard for wide-field and confocal microscopes. For wide-field microscopes, calibration can be further extended to absolute molecular units. This can be done by imaging a solution of known concentration and known depth; the latter can be prepared by placing a small spherical lens in a diluted solution of the same fluorophore that is used in the biological specimen.
authors.affiliation: Department of Biological Sciences, Kent State University, Kent, Ohio.
authors.forename: Michael
authors.initials: M
authors.lastname: Model
mesh_terms: D000818:Animals; D002138:Calibration; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods / Q000592:standards
chemical_list: D005456:Fluorescent Dyes
keywords: Acid Blue 9; Acid Fuschin; Rose Bengal; calibration; confocal microscopy; fluorescence microscopy; shading correction; sodium fluorescein; standardization