WO1994018547B1 - Apparatus for quantitative imaging of multiple flurophores - Google Patents

Abstract

A quantitative fluorometer for multiple fluorophores having a separate time-modulated beam of excitation light for each fluorescent target (26). Each beam is synchronized with one or more detectors (114 and 116) and separate lock-in amplifiers (30 and 32). The fluorophores are simultaneously excited and the combined fluorescent emission is resolved into components corresponding to each fluorophore. Confocal scanning means (24) are used to excite and detect fluorescent emission from locations throughout a volume. The location specific output of each amplifier is stored in a computer (34) which resolves the emission into the components corresponding to each fluorophore. The location specific data may be further processed or visually displayed to provide simultaneously fluorescence lifetime images of two fluorophores with this frequency domain method. Multiple amplifiers for each detector channel allow phase discrimination in each channel so that prompt and delayed fluorescence may be measured, allowing use of multiple fluorophores in each detector channel.

Claims

- 30 -AMENDED CLAIMS[received by the International Bureau on 2 August 1994 (02.08.94); original claims 1-3 amended; remaining claims unchanged (1 page)]

1. A device for quantifying fluorescent targets by excitation with electromagnetic radiation of multiple discrete wavelengths comprising: means for directing dual beams of continuous electromagnetic radiation, each beam having a discrete wavelength, said beams impinging together in a scanning manner at a plurality of individual locations where fluorescent targets are present causing excitation thereof; means for modulating the intensity of each of said beams at separate modulation frequencies to give each of said beams a specific time-modulated waveform; means for detecting the combined fluorescent emission from said targets in response to said excitation, including at least two lock-in amplifiers, each associated with a different modulation frequency; and demodulation means for extracting the contributions corresponding to each of said targets from said detected combined fluorescent emission in the dual detector means, thereby quantifying said targets.

2. The device of claim 1 further defined by confocal means for collecting light from said fluorescent targets.

3. The device of claim 2 wherein said confocal means for collecting light includes volumetric scanning means for pixel-by-pixel scanning of a stack of planes. - 31 -

STATEMENT UNDER ARTICLE19

In the International Search Report, certain references were cited against the claims, particularly claims 1 and 3. Claim 1 has been amended to point out that at least two lock-in amplifiers are employed with the detection means in order to separately sense fluorescent contributions from dual beams modu¬ lated at separate wavelengths. Separation of modulation informa¬ tion with the lock-in amplifiers allows quantitation of multiple fluorescent targets, responding differently to the combined exci¬ tation of the two separately modulated excitation wavelengths.

Claim 2 has been amended to point out that the device of claim 1 also incorporates a confocal type light collector. Claim 3 has been amended to point out that the confocal collector of claim 2 includes a volumetric scanning means for pixel by pix¬ el scanning of a stack of planes.