Intensity calibration and flat-field correction for fluorescence microscopes.

Intensity calibration and flat-field correction for fluorescence microscopes. Curr Protoc Cytom. 2014 Apr;68:10.14.1-10.14.10 Authors: Model M 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. PMID: 24692055 [PubMed - in process]
Source: Current Protocols in Cytometry - Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research