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

A time-resolved fluorescence microscope is presented that allows the sensitive detection of delayed luminescent labels with decay times from one microsecond to several milliseconds. The microscope utilizes an argon ion laser chopped with an acoustooptical modulator as excitation light source in combination with a gated multichannel plate image intensifier in the image plane. A theoretical model for the detection efficiency of practically any time-resolved fluorescence microscope is verified using phosphorescent Pt-porphine-stained Sephadex beads. The detection efficiency of the presented setup was shown to be 42%, which is near the theoretical limit of 50% for non-saturated luminescent dyes. The suppression of prompt fluorescence signals was found to be 1:5,500. The Pt-porphine beads proved to be an excellent model system for time-resolved fluorescence microscopy, showing a high extinction coefficient and high phosphorescence quantum yield in aqueous environment under room temperature conditions. Furthermore, for the microscope described the decay time of the Pt-porphine beads of 47 microseconds is long enough to enable efficient suppression of the prompt fluorescence while maintaining a high excitation and emission duty cycle. This is considered to be of vital importance in order not to saturate the luminescence with the excitation intensities commonly used in fluorescence microscopy.

Photobleaching kinetics of fluorescein in quantitative fluorescence microscopy.

An investigation on the photobleaching behavior of fluorescein in microscopy was carried out through a systematic analysis of photobleaching mechanisms. The individual photochemical reactions of fluorescein were incorporated into a theoretical analysis and mathematical simulation to study the photochemical processes leading to photobleaching of fluorescein in microscopy. The photobleaching behavior of free and bound fluorescein has also been investigated by experimental means. Both the theoretical simulation and experimental data show that photobleaching of fluorescein in microscopy is, in general, not a single-exponential process. The simulation suggests that the non-single-exponential behavior is caused by the oxygen-independent, proximity-induced triplet-triplet or triplet-ground state dye reactions of bound fluorescein in microscopy. The single-exponential process is a special case of photobleaching behavior when the reactions between the triplet dye and molecular oxygen are dominant.

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.

White blood cell differentiation using a solid state flow cytometer.

A flow cytometer using a solid state light source and detector was designed and built. For illumination of the sample stream two types of diode lasers (670 nm and 780 nm) were tested in a set-up designed to differentiate human leukocytes by means of light scattering. The detector is an avalanche photodiode, which was used to detect the weak scattered light in the orthogonal direction. The new flow cytometer set-up is very small, relatively cheap and yields similar results as a standard flow cytometer set-up using a helium-neon laser and photomultipliers.