Method of discriminating between dyed particles and background fluorescence of the dye
First Claim
1. Method of distinguishing between two species of fluorescent particles in a mixture of said species, which species have respectively different fluorescent quantum efficiencies and corresponding decay lifetimes, but fluoresce at substantially a common wavelength band, said method comprising the steps ofsubstantially simultaneously irradiating said particles in said mixture with radiation capable of stimulating fluorescent emission from both said species, but not intense enough to cause bleaching;
- reducing the intensity of said radiation so as to permit fluorescent emission from both of said species to decay statistically; and
measuring after a delay determined as a function of the shorter of said decay lifetimes following reduction of said radiation intensity, the intensity of said fluorescent emission at said wavelength band during a predetermined time interval.
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Abstract
A system for discriminating between the fluorescent emission from dye molecules bound to biological particles and the background emission from free molecules of the dye in the solution suspending the particles, where the quantum efficiency of the bound dye molecules because of their bound state, differs from the quantum efficiency of the unbound dye so that the emission from the bound dye exhibits a longer statistical decay lifetime. The system involves irradiating the particles in the solution with a mode locked laser, and measuring fluorescent intensity in delayed synchronism with the mode locking frequency.
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8 Claims
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1. Method of distinguishing between two species of fluorescent particles in a mixture of said species, which species have respectively different fluorescent quantum efficiencies and corresponding decay lifetimes, but fluoresce at substantially a common wavelength band, said method comprising the steps of
substantially simultaneously irradiating said particles in said mixture with radiation capable of stimulating fluorescent emission from both said species, but not intense enough to cause bleaching; -
reducing the intensity of said radiation so as to permit fluorescent emission from both of said species to decay statistically; and measuring after a delay determined as a function of the shorter of said decay lifetimes following reduction of said radiation intensity, the intensity of said fluorescent emission at said wavelength band during a predetermined time interval. - View Dependent Claims (2, 3, 4, 5)
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6. Apparatus for discriminating between two fluorescent particulate species having respectively different fluorescent decay lifetimes, in combination,
a laser for providing radiation capable of stimulating fluorescent emission from said species, and disposed for irradiating said species, means for modulating the intensity of radiation from said laser and including a mode locker coupled to said laser, and a source of oscillating signals for driving said mode locker; - and
means for sampling the intensity of fluorescent emission from said species in delayed synchronism with the modulation of said intensity of radiation. - View Dependent Claims (7, 8)
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Specification