Use of ferro‐electric liquid crystal shutters for time‐resolved fluorescence microscopy

Abstract

A technique is described to modify a standard fluorescence microscope for time-resolved visualization of delayed luminescing substances with decay times from 50 ps 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 ps. Prompt fluorescence was suppressed up to lo6 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.

Platinum Porphyrins as Phosphorescent Label for Time-resolved Microscopy

“…At present, the ultimate detection sensitivity in fluorescence microscopy is mainly determined by the nonspecific background signals caused by autofluorescence of the biological objects, fixative-induced fluorescence, and the natural fluorescence of components such as microscope objectives, immersion oil, and optical filters (Aubin 1979;Jovin et al 1989). These fast-decaying types of autofluorescence can be suppressed by application of time-resolved microscopy (Zotikov and Polyakov 1977;Seveus et al 1992;Marriott et al 1991Marriott et al ,1994Verwoerd et al 1994;Hennink et al 1996) using a delayed luminescent label (Hemmilä et al 1982;Seveus et al 1992;Hennink et al 1996) as reporter molecule bound to immunospecific reagents or nucleic acid probes (Diamandis et al 1988;Savitski et al 1989;Beverloo et al 1990;Marriott et al 1991Marriott et al ,1994Seveus et al 1992; de Hennink et al 1996). Most of the applications of time-resolved microscopy reported thus far use europium chelates.…”

Evaluation of a time-resolved fluorescence microscope using a phosphorescent Pt-porphine model system

“…It enables measuring the lifetime on a pixel by pixel basis. The method we used, referred to as t h eresolved fluorescence microscopy, takes advantage of a large difference in lifetime of the specific luminescence and the non-specific background fluorescence (2,12,13,17,24,(28)(29)(30)33). In this detection scheme, pulsed excitation is used in combination with gated detection of the emission.…”

The addition of a second lanthanide ion to increase the luminescence of europium(III) macrocyclic complexes

“…Various concentrations of each component were tested; the pH of the final solution was kept in the 5.9-6.4 range. (2) In experiments intended to determine the limit of luminescence detection and the luminescence time-dependence, the concentrations of Eu-Mac or EuCl 3 were varied as appropriate, while the composition of the solution was kept constant.…”