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.
Hennink
E J
EJ
Department of Cytochemistry and Cytometry, Leiden University, The Netherlands.
de Haas
R
R
Verwoerd
N P
NP
Tanke
H J
HJ
D004526:Efficiency; D005069:Evaluation Studies as Topic; D015200:Fluoroimmunoassay / Q000295:instrumentation* / Q000379:methods*; D007091:Image Processing, Computer-Assisted / Q000295:instrumentation*; D008163:Luminescent Measurements; D008856:Microscopy, Fluorescence / Q000295:instrumentation*; D008863:Microspheres; D011166:Porphyrins / Q000737:chemistry; D012680:Sensitivity and Specificity
D011166:Porphyrins