Multichannel high dynamic range scanner

A multiple frequency laser induced fluorescence scanner for scanning a sample labeled with a plurality of luminescence dyes to cause fluorescence emission of light at different wavelengths, comprising:

• at least one laser for forming a first and second beam of light of at least first and second predetermined excitation wavelengths, each excitation wavelength preferentially exciting one of the plurality of luminescence dyes;
• an optical system for focusing the first and second beams of light on respective first and second spatially separated spots, and for collecting and transmitting the resulting emission through respective spatially separated apertures;
• and
• detector means coupled to the spatially separated apertures for measuring the emission signal level of the collected and transmitted emissions;
• wherein the first and second beams focused on the spots have orthogonal polarizations;
• the scanner additionally comprising polarizers each in a corresponding detection path.

A method for detecting fluorescence from a sample labeled with a plurality of fluorescent dyes, comprising:

• generating a plurality of laser beams of a first and second wavelength;
• directing the light of a first wavelength to a first spot and directing the light of a second wavelength to a second spot that is spatially separated from the first spot, to cause the sample to fluoresce and emit light at a plurality of different wavelengths;
• and
• scanning the spots across the sample in one direction and a slow scan direction;
• applying the resulting emission from the dye-labeled sample to a detector to generate an output signal representative of the fluorescence from the sample, wherein each detector only receives light from a spatially defined area illuminated by the laser that preferentially excites the dye to preferentially be detected by that detector;
• wherein a separation of the spots in the slow scan direction is provided to ensure that the excited dyes have fully recovered from any triplet saturation before they get scanned by the next laser beam.

A method for detecting fluorescence from a sample labeled with a plurality of fluorescent dyes, comprising:

• generating a plurality of laser light beams of a first and second wavelength;
• directing light of the first wavelength to a first spot and directing light of a second wavelength to a second spot that is spatially separated from the first spot, to cause the sample to fluoresce and emit light at a plurality of different wavelengths;
• scanning the spots across the sample;
• applying the resulting emission from the dye-labeled sample to a detector to generate an output signal representative of the fluorescence from the sample, wherein crosstalk is minimized as the emission spectra of the dyes detected overlap as little as possible and each detector only receives light from a spatially defined area illuminated by the laser that preferentially excites the dye to preferentially be detected by that detector;
• wherein the longest wavelength of the first and second wavelengths is scanned across the sample first.

A method for detecting fluorescence from a sample labeled with a plurality of fluorescent dyes, comprising:

• generating a plurality of laser beams of a first and second wavelength;
• directing the light of a first wavelength to a first spot and directing the light of a second wavelength to a second spot that is spatially separated from the first spot, to cause the sample to fluoresce and emit light at a plurality of different wavelengths;
• and
• applying the resulting emission from the dye-labeled sample to a detector to generate an output signal representative of the fluorescence from the sample, wherein crosstalk is minimized as the emission spectra of the dyes detected overlap as little as possible and each detector only receives light from a spatially defined area illuminated by the laser that preferentially excites the dye to preferentially be detected by that detector;
• wherein up to two dyes are excited with orthogonal polarizations of light, such that fluorescence that is polarized parallel to the excitation light which preferentially excites a given dye is detected in the absence of depolarization effects.