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Evaluation of triple‐band filters for quantitative epifluorescence microscopy
Author(s) -
LOWY R. J.
Publication year - 1995
Publication title -
journal of microscopy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.569
H-Index - 111
eISSN - 1365-2818
pISSN - 0022-2720
DOI - 10.1111/j.1365-2818.1995.tb03601.x
Subject(s) - fluorescence , dichroic glass , absorbance , microscopy , dichroic filter , bodipy , fluorescence microscope , wavelength , microscope , materials science , excitation wavelength , chemistry , analytical chemistry (journal) , optics , optoelectronics , chromatography , physics
SUMMARY The performance characteristics of two sets of triple‐band epifluorescence filters have been evaluated for use with digitally enhanced fluorescence microscopy. Use of such filters, at most, requires movement of the excitation filter, while the dichroic and emission filters remain fixed, allowing multi‐wavelength imaging to be performed on standard microscopes. The dyes appropriate for use with these particular filters include Texas Red (TR), Bodipy (BD), FITC and Cascade Blue (CB), four fluorophores now commonly conjugated to both immunochemical probes and other proteins and lipids of biological interest. Good spectral separation existed for most experimental conditions allowing accurate localization of the different fluorophores during multi‐wavelength imaging. Anomalous responses were observed during near‐UV excitation at high concentration for some dyes. Scanning spectrofluorometry demonstrated that concentration‐dependent spectral shifts occurred, resulting in large increases in near‐UV absorbance. Despite the complexity of concentration and dye‐interaction effects, quantitative measurements of dye concentration could be made, even in regions of multiple dye co‐localization. Therefore, multi‐band pass filters are an additional valuable approach for performing quantitative fluorescence microscopic imaging.