Confocal fluorescence spectroscopy and anisotropy imaging system.
We report the design and implementation of a laser scanning confocal fluorescence system with spectroscopy and anisotropy imaging capabilities. Confocal spectroscopy is achieved with a fiber pinhole that is inserted into and removed from the detection path as needed. Fluorescence anisotropy imaging is accomplished with a polarizing beam splitter placed after the conventional pinhole. Two orthogonal polarizations are detected simultaneously with balanced photomultiplier tubes. The quality of the axial sectioning that is achieved in the confocal fluorescence spectroscopy mode is demonstrated experimentally, and examples of polarization-sensitive fluorescence imaging are demonstrated in tumor cell monolayers.
Bigelow
Chad E
CE
University of Rochester, Department of Radiology and the Institute of Optics, 601 Elmwood Avenue, Box 648, Rochester, New York 14642, USA. cbig@optics.rochester.edu
Conover
David L
DL
Foster
Thomas H
TH
D000818:Animals; D002477:Cells / Q000378:metabolism; D004867:Equipment Design; D005260:Female; D005454:Fluorescence Polarization / Q000295:instrumentation; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents / Q000493:pharmacokinetics; D008164:Luminescent Proteins / Q000493:pharmacokinetics; D008325:Mammary Neoplasms, Experimental / Q000473:pathology; D051379:Mice; D018613:Microscopy, Confocal / Q000295:instrumentation; D013050:Spectrometry, Fluorescence / Q000295:instrumentation
D007202:Indicators and Reagents; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins