Use of ferro-electric 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 microseconds to several milliseconds. The modification consists of synchronized operation of a mechanical shutter, positioned in an aperture plane in the excitation pathway, simultaneously with a ferro-electric liquid crystal (FLC) shutter on the emission side. Operation of the microscope is through a microprocessor interfaced keypad by which all timing parameters can be adjusted for optimal suppression of fast decaying luminescence. Accuracy of the timing was within 1 microsecond. Prompt fluorescence was suppressed up to 10(6) times, as determined for bright prompt fluorescing microspheres. The use of the FLC shutter 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, and IR blocking filters). No significant image degradation due to shutter operations was observed. The modified microscope was successfully used for the visualization of delayed luminescing immunolabels, such as inorganic phosphor particles and lanthanide chelates, as well as naturally phosphorescing materials.