WO2000079251A1 - Elimination d'empreinte magnetique dans des systemes d'imagerie confocale en fluorescence de jeu ordonne de microechantillons - Google Patents

Elimination d'empreinte magnetique dans des systemes d'imagerie confocale en fluorescence de jeu ordonne de microechantillons Download PDF

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Publication number
WO2000079251A1
WO2000079251A1 PCT/US2000/016807 US0016807W WO0079251A1 WO 2000079251 A1 WO2000079251 A1 WO 2000079251A1 US 0016807 W US0016807 W US 0016807W WO 0079251 A1 WO0079251 A1 WO 0079251A1
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WO
WIPO (PCT)
Prior art keywords
dye
optical signal
total
excited
bands
Prior art date
Application number
PCT/US2000/016807
Other languages
English (en)
Inventor
Herman Deweerd
Thomas F. Mcnall
Original Assignee
Virtek Vision Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Virtek Vision Corporation filed Critical Virtek Vision Corporation
Priority to AU56233/00A priority Critical patent/AU5623300A/en
Publication of WO2000079251A1 publication Critical patent/WO2000079251A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell plates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N2021/6417—Spectrofluorimetric devices
    • G01N2021/6419—Excitation at two or more wavelengths
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N2021/6417—Spectrofluorimetric devices
    • G01N2021/6421—Measuring at two or more wavelengths
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
    • G01N2021/6441—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks with two or more labels

Definitions

  • the subject invention relates generally to an improved scanner of the type that scans specimens for performing subsequent computer analysis on the specimens.
  • Micro array biochips are being used by several biotechnology companies for scanning genetic DNA samples applied to biochips into computerized images. These chips have small substrates with thousands of DNA fragments that represent the genetic codes of a variety of living organisms including human, plant, animal, and pathogens. They provide researchers with information regarding the DNA properties of these organisms. Experiments can be conducted with significantly higher throughput than previous technologies offered by using these biochips. Biochip technology is used for genetic expression, DNA sequencing of genes, food and water testing for harmful pathogens, and diagnostic screening. Biochips may be used in pharmacogenomics and proteomics research aimed at high throughput screening for drug discovery.
  • DNA fragments are extracted from a sample and are tagged with a fluorescent dye having a molecule that, when excited by a laser, will emit light of various colors. Typically, the DNA fragments are tagged with more than one dye. Each dye includes fluorescent spectral band that is excitable by a laser of a particular wavelength. These fluorescently tagged DNA fragments are then spread over the chip. A DNA fragment will bind to its complementary (cDNA) fragment at a given array location.
  • cDNA complementary
  • a typical biochip is printed with a two-dimensional array of thousands of cDNA fragments, each one unique to a specific gene. Once the biochip is printed, it represents thousands of specimens in an area usually smaller than a postage stamp.
  • a microscope collects data through a scanning lens by scanning one pixel of a specimen at a time.
  • the scanning lens projects emitted light from the specimen onto a scanner that is manipulated along a predetermined pattern across the chip scanning an entire biochip one pixel at a time.
  • the pixels are relayed to a controller that sequentially connects the pixels to form a complete, computerized biochip image.
  • Inaccurate computerized biochip images are obtained when the spectral band of a first dye overlaps with the spectral band of a second dye. Often, a laser having a wavelength formulated to excite the spectral band of the first dye will excite the overlapping spectral band from the second dye. When this occurs the pixels relayed to the controller will be blurred potentially rendering the connected pixels unreadable.
  • the present invention provides an improved method for scanning a specimen with an optical instrument.
  • a first dye having an excitation wavelength with multiple fluorescence bands that are excited when contacted by a first optical signal is applied to the specimen.
  • a second dye having an excitation wavelength with multiple fluorescence bands that are excited when contacted by a second optical signal is also applied to the specimen.
  • the overlapping spectral bands of the first dye and of the second dye are predetermined and programmed into a computer.
  • the first optical signal and the second optical signal are directed onto the specimen.
  • the specimen is scanned for emitted wavelengths from the first dye with a first detector for creating a first data set.
  • the first data set is relayed to the computer to create the biochip image.
  • the computer is programmed for removing from the data set the band of wavelength from the second dye excited by the first optical signal.
  • the computer can be programmed to remove the overlap from the relayed data sets. Therefore, the problem of producing unreliable computerized biochip images resulting from overlapping spectral bands from multiple dyes is addressed.
  • Figure 1 is a detailed perspective view of an optical instrument of the present invention.
  • the optical instrument assembly of the present invention is generally shown in Figure 1 at 10.
  • the assembly includes a transmitter 12 for emitting an optical signal 14.
  • the transmitter 12 comprises a laser.
  • Figure 1 shows two transmitters 12a,b, each emitting an optical signal 14a,b having a different wavelength. Additional transmitters 12 may be introduced to the assembly 10 as needed.
  • a reflector 30 directs the optical signal 14 onto a specimen 90.
  • the reflector 30 includes a plurality of turn mirrors 32.
  • Figure 1 shows two turn mirrors 32a,b corresponding to the same number of transmitters 12a,b.
  • Each optical signal 14a,b is reflected by the turn mirrors 32a,b into corresponding beam combiners 34a,b.
  • the beam combiners 34a,b known as dichroic filters, transmit light of one wavelength while blocking other wavelengths.
  • the beam combiners 34a,b collect the individual optical signals 14a,b into a combined beam along a single path and direct the beam towards a beam splitting mirror 20.
  • the beam splitting mirror 20 includes an opening 22 through which the combined optical signals 14a,b travel.
  • the combined optical signals 14a,b reflect off a ninety degree fold mirror 36 located immediately above a scanning objective lens 52, which focuses the combined optical signals 14a,b onto a section of the specimen 90.
  • a first drive mechanism 50 varies the position of the combined optical signal 14a,b on the specimen 90 as will be explained further herein below.
  • the specimen 90 is treated with dyes having fluorescent properties when subjected to the optical signal 14a,b.
  • dyes having fluorescent properties when subjected to the optical signal 14a,b.
  • at least a first dye having an excitation wavelength with multiple fluorescence spectral bands that are excitable by the first optical signal 14a is applied to the specimen.
  • a second dye having an excitation wavelength with multiple fluorescence spectral bands that are excitable by the second optical signal 14b is applied to the specimen 90.
  • the dyes are selected to examine different specimen 90 properties. Utilizing multiple dyes allows different properties of the same specimen 90 to be examined simultaneously.
  • the first and second dyes will have at least one spectral band in common. Therefore, it is possible for the first optical signal 14a to excite a spectral band from the second dye causing inaccurate fluorescence to occur. Likewise, the second optical signal 14b can excite a spectral band from the first dye.
  • the spectral bands that are in common between the two dyes are predetermined, the reason for which will be explained further hereinbelow.
  • the assembly 10 includes a detector 40 with a sensor 42 for detecting an emitted optical signal 44 from the specimen 90.
  • the emitted optical signal 44 reflects off the opposite side of the beam splitting mirror 20 through a plurality of beam splitters 38a,b to separate the emitted optical signal 44 into individual signals 44a,b corresponding to the different dyes.
  • Each individual signal 44a,b passes though an emission filter 46a,b and is focused by a detector lens 48a,b into a pinhole.
  • the individual signals 44a,b proceed through the pinhole to contact the individual sensors 42a,b.
  • the sensors 42a,b are in communication with a computer 80.
  • the first sensor 42a relays a first data set emitted from the first dye and the second dye to the computer 80.
  • the second sensor 42b relays a second data set emitted from the second dye and the first dye to the computer 80.
  • the computer 80 is programmed for removing from the first data set the spectral band from the second dye excited by the first optical signal 14a.
  • the computer is also programmed for removing from the first data set the spectral band emitted from the first dye that is excited by the second optical signal 14b.
  • the computer 80 is programmed for removing from second data set the spectral band from the first dye that is excited by the second optical signal 14b.
  • the computer is programmed for removing from the second data set the spectral band emitted from the second dye that is excited by the first optical signal 14a.
  • An algorithm is programmed into the computer 80 to facilitate the filtering of the unwanted spectral band that are emitted from the various dyes. First, a ratio of the total spectral bands from the first dye excited by the first optical signal to the band of wavelength from the second dye excited by the first optical signal is calculated. Further, a ratio of the total spectral bands from the second dye excited by the second optical signal to the spectral band from the second dye excited by the first optical is calculated.
  • a first algorithm is programmed into the computer 80 to remove from the first data set the spectral band from the second dye excited by the first optical signal and to remove the spectral band from the first dye excited by the second optical signal.
  • Dyel(scan) Scanl(total) - (Scan2(total) - Scanl(total) x Ratiol) x Ratio2 wherein:
  • Dye 1 (scan) - the spectral band from the first dye excluding the spectral bands detected from the second dye; Scan 1 (total) - the total bands of wavelength detected by the first detector;
  • Scan2(total) the total bands of wavelength detected by the second detector;
  • Ratiol the ratio of the total spectral bands from the first dye excited by the first optical signal to the spectral band from the first dye excited by the first optical signal detected by the second detector; and
  • Ratio2 the ratio of the total spectral bands from the second dye excited by the second optical signal to the spectral band from the second dye excited by the first optical signal detected by the first detector
  • a second algorithm is programmed into the computer 80 to remove from the second data set the spectral band from the first dye excited by the first optical signal and to remove the spectral band from the second dye excited by the first optical signal.
  • Dye2(scan) Scan2(total) - (Scanl (total) - Scan2(total) x Ratio2) x Ratiol wherein:
  • Dye2(scan) the spectral band from the second dye excluding the spectral bands detected from the first dye; Scan2(total) - the total bands of wavelength detected by the second detector;
  • Ratio2 the ratio of the total spectral bands from the second dye excited by the second optical signal to the spectral band from the second dye excited by the second optical signal detected by the first detector; and Ratiol - the ratio of the total spectral bands from the first dye excited by the first optical signal to the spectral band from the first dye excited by the second optical signal detected by the second
  • the algorithms perform concurrent calculations utilizing both the predetermined values and the detected wavelength bands to remove unwanted wavelength bands that blur the computerized image of the specimen 90.

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  • Health & Medical Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Physics & Mathematics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

Cet instrument optique comprend un émetteur (12) servant à l'émission d'un premier et d'un second signal optique sur un échantillon. On dépose sur cet échantillon un premier colorant ayant une longueur d'onde réfléchissante à plusieurs bandes excitables par un premier signal optique ainsi qu'un second colorant ayant une longueur d'onde réfléchissante à plusieurs bandes excitables par un second signal optique. L'échantillon est soumis à un balayage au moyen d'un premier détecteur (40) aux fins de la lecture de longueurs d'ondes réfléchies émanant du premier colorant et ce, afin de constituer un premier jeu de données. Ce premier jeu de données est transmis à un ordinateur (80). Cet ordinateur est programmé pour ôter du jeu de données la bande de longueur d'onde émanant du second colorant excité par le premier signal optique.
PCT/US2000/016807 1999-06-18 2000-06-16 Elimination d'empreinte magnetique dans des systemes d'imagerie confocale en fluorescence de jeu ordonne de microechantillons WO2000079251A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU56233/00A AU5623300A (en) 1999-06-18 2000-06-16 Elimination of crosstalk in confocal epifluorescent microarray imaging systems

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US13999299P 1999-06-18 1999-06-18
US60/139,992 1999-06-18

Publications (1)

Publication Number Publication Date
WO2000079251A1 true WO2000079251A1 (fr) 2000-12-28

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Country Status (2)

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AU (1) AU5623300A (fr)
WO (1) WO2000079251A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7585463B2 (en) 2003-10-24 2009-09-08 Aushon Biosystems, Inc. Apparatus and method for dispensing fluid, semi-solid and solid samples

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5091652A (en) * 1990-01-12 1992-02-25 The Regents Of The University Of California Laser excited confocal microscope fluorescence scanner and method
US5578832A (en) * 1994-09-02 1996-11-26 Affymetrix, Inc. Method and apparatus for imaging a sample on a device
US5805342A (en) * 1995-10-31 1998-09-08 Gravely; Benjamin T. Imaging system with means for sensing a filtered fluorescent emission

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5091652A (en) * 1990-01-12 1992-02-25 The Regents Of The University Of California Laser excited confocal microscope fluorescence scanner and method
US5578832A (en) * 1994-09-02 1996-11-26 Affymetrix, Inc. Method and apparatus for imaging a sample on a device
US5805342A (en) * 1995-10-31 1998-09-08 Gravely; Benjamin T. Imaging system with means for sensing a filtered fluorescent emission

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7585463B2 (en) 2003-10-24 2009-09-08 Aushon Biosystems, Inc. Apparatus and method for dispensing fluid, semi-solid and solid samples
US9527085B2 (en) 2003-10-24 2016-12-27 Aushon Biosystems, Inc. Apparatus and method for dispensing fluid, semi-solid and solid samples

Also Published As

Publication number Publication date
AU5623300A (en) 2001-01-09

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