US5631734A
Expired / Lapsed
US
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Method and apparatus for detection of fluorescently labeled materials
Assignee: Affymetrix Inc
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Filing: 1994-02-10
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Granted: 1997-05-20
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Expires: 2014-02-10
Abstract
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.
Inventors
- 👤 David Stern
- 👤 Peter Fiekowsky
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-06 19:35
CPC Classifications
G; G01; G01N; G01N21/00; G01N21/62; G01N21/63; G01N21/64; G01N21/645; G01N21/6456; G01N21/6458; G01N21/6428; B; B01; B01J; B01J2219/00; B01J2219/00274; B01J2219/00583; B01J2219/00603; B01J2219/00605; B01J2219/00612; B01J2219/00659; B01J2219/0068; B01J2219/00702; G01N21/17; G01N2021/1738; G01N2021/174
Claims Analysis
Total Claims: 20
Claim 1
Independent Claim
304 words
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.
Claim 2
Independent Claim
304 words
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.
Claim 3
Dependent Claim
31 words
The apparatus as recited in claim 1, further comprising a video display for displaying said values representing the intensity of said fluoresced light as a function of location on said substrate.
Claim 4
Dependent Claim
78 words
The apparatus as recited in claim 1, wherein said focusing means comprises: a photodiode for generating a voltage representing an intensity of said light reflected from said substrate; a focusing lens for focusing said reflected excitation light from said substrate, from said optical train at said photodiode; and a means for moving said substrate relative to a microscope objective until said intensity of said reflected excitation light focused at said photodiode from said substrate substantially reaches a maximum.
Claim 5
Dependent Claim
20 words
The apparatus as recited in claim 3, wherein a confocal pinhole is located between said focusing lens and said photodiode.
Claim 6
Dependent Claim
32 words
An apparatus as recited in claim 1, wherein said detecting means comprises: a photomultiplier tube; and a lens for focusing said fluoresced light separated by said optical train, at said photomultiplier tube.
Claim 7
Dependent Claim
21 words
An apparatus as recited in claim 5, wherein a confocal pinhole is located between said focusing lens and said photomultiplier tube.
Claim 8
Dependent Claim
47 words
An apparatus as recited in claim 5, wherein said photomultiplier tube is coupled to a means for collecting pulses generated by said photomultiplier tube in response to said fluoresced light, said means for collecting pulses being connected to a programmable computer for storing and analyzing said pulses.
Claim 9
Dependent Claim
37 words
An apparatus as recited in claim 1, further comprising means for controlling temperature in said flow cell, said means for controlling temperature including a recirculating bath device for circulating water through channels disposed in said flow cell.
Claim 10
Independent Claim
204 words
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.
Claim 11
Independent Claim
204 words
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.
Claim 12
Dependent Claim
22 words
The method as recited in claim 9 wherein said body further comprises a temperature controller for controlling the temperature in said cavity.
Claim 13
Dependent Claim
33 words
The method as recited in claim 9, wherein said step of detecting comprises the steps of: collecting said fluoresced light through optics; and directing said fluoresced light from said optics onto a detector.
Claim 14
Dependent Claim
32 words
The method as recited in claim 9, wherein said step of exciting said subsequent region comprises the step of translating said substrate to allow said excitation light to excite said subsequent region.
Claim 15
Dependent Claim
38 words
The method as recited in claim 9, wherein said auto-focusing step comprises the steps of: a) focusing a first surface of said substrate; b) focusing a second surface of said substrate; and c) finely focusing said second surface.
Claim 16
Dependent Claim
129 words
The method as recited in claim 13, wherein said step of focusing said first surface comprises the steps of: directing said excitation light at said substrate, said excitation light being reflected by said substrate; focusing said reflected excitation light from said substrate through a confocal pinhole; detecting an amount of said reflected excitation light passing through said confocal pinhole, said confocal pinhole configured such that said amount of said reflected excitation light is at substantially a maximum when said first surface is located in substantially a focal plane of said excitation light; moving said substrate closer to said excitation light and repeating the directing, focusing, detecting and moving steps until said amount of reflected excitation light passing through said confocal pinhole and detected in said detecting step has peaked.
Claim 17
Dependent Claim
127 words
The method as recited in claim 14, wherein said step of focusing said second surface comprises the steps of: after focusing said first surface, first moving said substrate closer relative to said excitation light, a distance which said substrate is moved being equal to about half a thickness of said substrate; directing said excitation light at said substrate, said excitation light being reflected by said substrate; focusing said reflected excitation light through said confocal pinhole; detecting said amount of reflected excitation light passing through said confocal pinhole; second moving said substrate closer relative to said excitation light and repeating the second moving, directing, focusing, and detecting steps until said amount of reflected excitation light passing through said confocal pinhole and detected in said detecting step has peaked.
Claim 18
Dependent Claim
85 words
The method as recited in claim 15, wherein said step of finely focusing said second surface comprises the steps of: directing said excitation light at said substrate; focusing said reflected excitation light through said confocal pinhole; detecting said amount of reflected excitation light passing through said confocal pinhole; and moving said substrate farther relative to said excitation light and repeating the directing, focusing, detecting, determining, and moving steps until said amount of reflected excitation light passing through said confocal pinhole has reached a desired value.
Claim 19
Dependent Claim
19 words
The method as recited in claim 13, wherein said steps a-c are repeated for each corner of said substrate.
Claim 20
Dependent Claim
30 words
The method as recited in claim 17, further comprising the step of interpolating a focus position of each corner to determine said focus position of each region of said substrate.
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USPTO Annuity & Maintenance Windows
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| 3.5 Years (Year 4) | 2000-05-19 | 2000-11-18 | 2001-05-20 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2004-05-19 | 2004-11-18 | 2005-05-20 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2008-05-19 | 2008-11-18 | 2009-05-20 | $7700.00 | Window Closed / Lapsed |