University of Massachusetts

Joan C Politz

Robert H Singer

Krishan L Taneja

Disclosed are methods for removing autofluorescence background from microscopic images or from flow cytometer measurements. The microscopic images can be fluorescent in situ hybridization images or images of living cells. The methods involve the steps of: (1) detecting fluorescence from a cell before 'uncaging' a caged fluorochrome label on a probe; (2) uncaging the fluorochrome; (3) detecting fluorescence from the cell; and (4) subtracting fluorescence detected before uncaging from fluorescence detected after uncaging. Also disclosed is a method for tracking the movement of a target molecule in a living cell. Also disclosed is a fluorochrome-uncaging flow cytometer. The fluorochrome-uncaging flow cytometer includes a first fluorescence excitation light beam, an uncaging light beam, a second fluorescence excitation light beam, an electronic data system, a first photodetector operably linked to the electronic data system, and a second photodetector operably linked to the electronic data system.

Caged fluorochrome-labeled probes and subtraction of autoflourescence

A method of nucleic acid hybridization in living cells is described, which is useful for detecting, quantitating and locating a specific nucleic acid in a cell or tissue, for selecting cells based on the expression or presence of a specific nucleic acid, and for monitoring the amount and location of a specific nucleic acid over time or under various inducing or inhibiting conditions.

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Holden, MA, United States of America

Joan C Politz