Method and apparatus for detection of fluorescently labeled materials
AssigneeAffymetrix, Inc.
InventorsDavid Stern, Peter Fiekowsky
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Abstract
Fluorescently marked targets bind to a substrate 230 synthesized with polymer sequences at known locations. The targets are detected by exposing selected regions of the substrate 230 to light from a light source 100 and detecting the photons from the light fluoresced therefrom, and repeating the steps of exposure and detection until the substrate 230 is completely examined. The resulting data can be used to determine binding affinity of the targets to specific polymer sequences.
Claims18
An apparatus for detecting fluorescently marked regions on a first surface of a substrate, said apparatus comprising:
- an excitation light source;
- an optical train for directing an excitation light from said excitation light source at said substrate, and separating reflected excitation light from said first surface from fluoresced light from said first surface, said optical train comprising a spatial filter having a first lens and a second lens and a confocal pinhole located between said first lens and said second lens, a beam splitter cube, and a dichroic mirror for passing light having a wavelength of about said fluoresced light and reflecting light having a wavelength of about said excitation light, an optical lens and a microscope objective for directing said light at said substrate;
- a focusing system for determining a focal plane of said excitation light passing through said optical train, said focusing means providing data for locating said first surface at said focal plane;
- a detector for detecting said fluoresced light from said fluorescently marked regions in response to said light;
- an x-y-z translation system for translating said substrate from a first position to a second position;
- a flow cell mounted on said translation system, said flow cell comprising a mounting surface with a cavity therein, said mounting surface including a means for mounting said substrate thereon, and maintaining a sealed relationship with said substrate, whereby said first surface of said substrate is in fluid communication with said cavity, said cavity having an inlet and an outlet, and said inlet being connected to a pump for transferring materials into said cavity through said inlet and out of said cavity through said outlet;
- and
- a storage system for storing a set of values representing an intensity of said fluoresced light as a function of the location on said substrate fluorescing said fluoresced light.
A method for detecting fluorescently marked regions on a substrate, said method comprising the steps of:
- immobilizing said substrate on a body, said body comprising a mounting surface having a cavity disposed therein, said substrate being immobilized on said mounting surface such that a probe array fabricated on said substrate is in fluid communication with said cavity, said cavity comprising a inlet and a outlet for flowing fluids into and through said cavity;
- directing an excitation light from an excitation light source at said substrate;
- auto-focusing said substrate in a focal plane of said excitation light;
- exciting a first region of said substrate with said excitation light from said excitation light source, said excitation light source having a first wavelength;
- detecting fluoresced light from said substrate in response to said excitation light, said fluoresced light having a second wavelength, said detecting comprising separating said light having a first wavelength from said light having a second wavelength and detecting said light having a second wavelength;
- exciting a subsequent region on said substrate;
- repeating steps of detecting and exciting a subsequent region until all regions of said substrate have been excited;
- and
- processing and storing said fluoresced light to generate a 2-dimensional image of said substrate.
Dependencies are inferred from the claim text.