USE OF FERROELECTRIC LIQUID-CRYSTAL SHUTTERS FOR TIME-RESOLVED FLUORESCENCE MICROSCOPY

A technique is described to modify a standard fluorescence microscope for time-resolved visualization of delayed luminescing substances with decay times from 50 mu s to several milliseconds. The modification co nsists of synchronized operation of a mechanical shutter, positioned i n an aperture plane in the excitation pathway, simultaneously with a f erro-electric liquid crystal (FLC) shutter on the emission side. Opera tion of the microscope is through a microprocessor interfaced keypad b y which all timing parameters can be adjusted for optimal suppression of fast decaying luminescence. Accuracy of the timing was within 1 mu s. Prompt fluorescence was suppressed up to 10(6) times, as determined for bright prompt fluorescing microspheres. The use of the FLC shutte r resulted in a reduction in emission intensity by a factor of 8 (due to the use of polarizers, the lower transmission of the FLC devices, a nd IR blocking filters). No significant image degradation due to shutt er operations was observed. The modified microscope was successfully u sed for the visualization of delayed luminescing immunolabels, such as inorganic phosphor particles and lanthanide chelates, as well as natu rally phosphorescing materials. (C) 1994 Wiley-Liss, Inc.