Apparatus for measuring spatial distribution of fluorescence on a substrate
First Claim
1. Apparatus for measuring the spatial distribution of fluorescence on a substrate having multiple fluorophores each having a different characteristic wavelength comprising:
- scanning means for directing a radiant excitation beam at known locations on a substrate containing a plurality of fluorophore targets, said locations defining a scanning path;
collecting means for simultaneously collecting radiation from the scanning path;
detecting means positioned to receive radiation from said collecting means, said detecting means producing a signal corresponding to the intensity of said collected radiation;
first filter means positioned between said collecting means and said scanning path, said first filter means selectively rejecting background wavelengths contained in said excitation beam;
second filter means positioned in the radiation path between said collecting means and said detecting means, said second filter means selectively rejecting background wavelengths contained in said excitation beam; and
third filter means for interchangeably positioning one of a plurality of band pass filters between said second filter means and said detecting means, each of said band pass filters selectively transmitting a different band of wavelengths, each of said bands being associated with a different fluorophore,whereby the distribution of fluorescence on the substrate at selected wavelengths at said known locations can be measured.
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Abstract
A fluorescence detection scanner and method employing a fiberoptic collector, positioned adjacent to the scanning plane of the excitation beam, with a light collecting surface which is oriented to reject back-scattered excitation light from the incident surface of the sample support. The scanning plane of the excitation beam is off normal relative to the incident surface of the sample support and the light collecting surface is located within the area defined by the resulting acute angle of incidence. The light collecting surface is angled away from the location at which the incident excitation beam intersects the surface of the sample support so that back-scattered excitation light does not enter the optical fibers. The orientation of the light collector results in a four to five-fold decrease in excitation-light background without attenuation of the fluorescence emitted by the sample. Long-pass interference filters, selected to reject the excitation wavelength, are located at the input and output surfaces of the fiberoptic light collector to further reduce background excitation light. Multiple fluorophores are discriminated by sequentially scanning the sample with a different interchangeable narrow bandpass filter for each fluorophore. Internal fluorescence standards in the sample are used to determine filter efficiencies for each label in each filter image and the quantity of each fluorophore is computed by linear analysis. Increased detector sensitivity allows linear quantitation of multiple fluorophores in the femto-mole range.
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Citations
16 Claims
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1. Apparatus for measuring the spatial distribution of fluorescence on a substrate having multiple fluorophores each having a different characteristic wavelength comprising:
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scanning means for directing a radiant excitation beam at known locations on a substrate containing a plurality of fluorophore targets, said locations defining a scanning path; collecting means for simultaneously collecting radiation from the scanning path; detecting means positioned to receive radiation from said collecting means, said detecting means producing a signal corresponding to the intensity of said collected radiation; first filter means positioned between said collecting means and said scanning path, said first filter means selectively rejecting background wavelengths contained in said excitation beam; second filter means positioned in the radiation path between said collecting means and said detecting means, said second filter means selectively rejecting background wavelengths contained in said excitation beam; and third filter means for interchangeably positioning one of a plurality of band pass filters between said second filter means and said detecting means, each of said band pass filters selectively transmitting a different band of wavelengths, each of said bands being associated with a different fluorophore, whereby the distribution of fluorescence on the substrate at selected wavelengths at said known locations can be measured. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)
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Specification