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Electro-optical Wavelength Selection Enables Confocal Ratio Imaging at Low Light Levels

Published online by Cambridge University Press:  08 August 2003

Roland Nitschke
Affiliation:
Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health Bldg. 10, Room 6N307, Bethesda, MD 20892, USA Present address: Albert-Ludwigs-Universitat Freiburg, Physiologisches Institut, Hermann-Herder-Strasse 7, D-79104 Frieburg, Germany.
Kenneth R. Spring
Affiliation:
Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health Bldg. 10, Room 6N307, Bethesda, MD 20892, USA
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Abstract

A confocal attachment (Odyssey) to an inverted microscope was modified to better study living cultured epithelial cells stained with fluorescent dyes. Improvements to the instrument included elimination of light leaks, improved electronic shielding, reduction of thermal effects, and use of low dark current detectors. In addition, rapid changes in illumination wavelength and power were accomplished by replacing the original mechanical filter changer by an acousto-optic tunable filter attached to the argon laser light source. The addition of a liquid crystal tunable filter to one of the two photomultiplier detectors also permitted rapid spectral scanning of the fluorescence emission. High-resolution, differential interference contrast transmitted light images were formed simultaneously by replacement of the photodiode-based transmitted light detector with a photomultiplier tube and dichroic mirror assembly. An illumination intensity of only 40 μW/cm2 at the back focal plane of the microscope objective allowed high-quality fluorescence and transmitted light images of living cells at video rates with minimal bleaching and photodynamic damage. Both excitation ratio imaging and emission spectral scanning of living epithelial cells were accomplished. The system performance was evaluated by optical sections of fluorescent beads and thin films.

Type
Research Article
Copyright
© 1995 Microscopy Society of America

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