Fluorescence microscopy
First Claim
1. A multi-chronal fluorescence imaging apparatus for spatial differentiation of a multitude of separate biological, organic and inorganic components that includesa sample holder for containing a sample with a plurality of fluorescent dyes bonded to specific sample components,said dyes each reaching a different maximum output intensity upon being excited and having substantially different decay times with the dye of greatest maximum output intensity reaching final decay first and the remaining dyes decaying in the order of their respective maximum output intensities such that the maximum output of each dye can be detected in an ordered sequence with little interference from the other dyes,a pulsed light source for periodically irradiating a sample in said holder with a pulsed excitation radiation wherein each dye emits fluorescent radiation at about its maximum output before reaching final decay.a spatially sensitive detector means positioned in an optical light path for viewing the radiation emitted by said dyes,a gate means having a shutter movable between a closed position wherein radiation emitted by said sample is prevented from reaching the detector means and an open position wherein radiation emitted by said sample is transmitted to the detector means,means for intermittently energizing the light source to produce a light pulse having a duration that is less than the shortest decay time of said dyes, andcontrol means to open the shutter at periodic intervals to permit the detector to view time resolved images produced by each dye prior to its decay.
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Abstract
A multi-chronal fluorescence imaging technique for spatial differentiation and correlation of a plurality of separate sample components tagged with site specific dyes is described. The dyes have decay times that are widely separated and vary by approximately a factor of ten from one dye to the next. These are added to the sample and are excited with a short pulse of ultraviolet light. Between the sample and a detector is placed an adjustable shutter or gate which is opened and closed at predetermined intervals so that the detector can see the approximate maximum intensity output of each dye in a shown ordered sequence without significant interference from the other dyes.
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Citations
18 Claims
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1. A multi-chronal fluorescence imaging apparatus for spatial differentiation of a multitude of separate biological, organic and inorganic components that includes
a sample holder for containing a sample with a plurality of fluorescent dyes bonded to specific sample components, said dyes each reaching a different maximum output intensity upon being excited and having substantially different decay times with the dye of greatest maximum output intensity reaching final decay first and the remaining dyes decaying in the order of their respective maximum output intensities such that the maximum output of each dye can be detected in an ordered sequence with little interference from the other dyes, a pulsed light source for periodically irradiating a sample in said holder with a pulsed excitation radiation wherein each dye emits fluorescent radiation at about its maximum output before reaching final decay. a spatially sensitive detector means positioned in an optical light path for viewing the radiation emitted by said dyes, a gate means having a shutter movable between a closed position wherein radiation emitted by said sample is prevented from reaching the detector means and an open position wherein radiation emitted by said sample is transmitted to the detector means, means for intermittently energizing the light source to produce a light pulse having a duration that is less than the shortest decay time of said dyes, and control means to open the shutter at periodic intervals to permit the detector to view time resolved images produced by each dye prior to its decay.
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10. A method of resolving spatial relations among different structural and functional components of a biological, organic and inorganic nature that includes
selecting a plurality of dyes that reach different maximum output intensities upon being excited and which have substantially different decay times such that the dye of greatest maximum output intensity reaches final decay first and the remaining dyes decay in the order of their respective maximum output intensities, incorporating the dyes into a sample so that the dyes attach to components contained in the sample, illuminating the dyes in the sample with a short pulse of radiation to excite the dyes so that the dyes emit a maximum amount of radiation before decaying in an ordered sequence. sequentially detecting the radiation emitted by the sample after the illumination pulse is terminated in a sequence corresponding to the decay sequence of the dyes to view a maximum radiation image produced by each dye before it finally decays.
Specification