CA2390651A1 - Apparatus and method for calibration of a microarray scanning system - Google Patents
Apparatus and method for calibration of a microarray scanning system Download PDFInfo
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- 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
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
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- C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
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- G01N21/27—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands using photo-electric detection ; circuits for computing concentration
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- 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"
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Abstract
A microarray scanning system (10) for conducting microarray experiments on a planar substrate (101) includes an excitation radiation source (21), a detection system (40), and a computational device (60), the planar substrate supporting a plurality of dilution marks (119) containing a fluorophore and located on the substrate surface at predetermined distances from a fiducial reference mark (106) and/or a microarray. Automatic calibration adjustment of either or both the detection system and the excitation radiation source is achieved via the computational device by irradiating the dilution spots, detecting emission radiation produced by the dilution spot fluorophore material, deriving a series of brightness readings from the levels of emission radiation detected at corresponding dilution spots; analyzing the brightness readings to obtain a fluorophore brightness characteristic as a function of concentration; and adjusting the sensitivity of the detection system and/or the intensity level of the source of excitation radiation in accordance with the fluorophore brightness characteristic.
Description
WO 01/35074 CA 02390651 2002-05-08 pCT/US00/41170 APPARATUS AND METHOD FOR CALIBRATION OF A
MICROARRAY SCANNING SYSTEM
BACKGROUND OF THE INVENTION
Field Of the Invention s This invention in general relates to optical scanning systems and, in particular, to an apparatus and method for calibration of a microarray scanning system.
Description Of The Prior Art The use of excitation radiation to produce fluorescence in a series of scanned genetic samples is known. U.S. Patent No. 5,689,110 issued to Dietz et al., for ~o example, discloses a calibration method and device for a fluorescence spectrometer which uses fluorescence from a homogenous solid state standard as the source of calibration fluorescence. Fluorescent imagers are used to acquire data in experiments that utilize fluorescent labels, or fluorophores, to identify the state of a sample being tested. In some cases the presence of or lack of fluorophores in the sample determines is the experimental result. In other cases the fluorophore concentration, which is a function of the intensity of the emission radiation received from the sample, is the measurement of interest and the experimental result can be inferred by measuring the intensity of the detected radiation.
An example of a process that uses fluorophores is the microarray which is a set zo of experiments utilizing genetic material such as DNA or RNA, bound to a glass substrate. Reference or 'target' DNA is spotted onto a glass substrate -typically a one-by three-inch glass microscope slide - where it chemically binds to the surface. Each spot, or sample, of DNA constitutes a separate experiment. A sample of 'probe' DNA
or RNA, to which has been added the fluorophore material, is subsequently placed on zs the target spots on the surface of the substrate and is allowed to hybridize with the target DNA. Excess probe DNA that does not bind with target DNA is removed from the surface of the slide in a subsequent washing process.
_2_ The experiments measure the binding affinities between the probe DNA and the target DNA to determine the similarity in molecular structure; complementary molecules have a much greater probability of binding than do unrelated molecules. The fluorophore added to the probe DNA emits a range of radiation energy centered about a wavelength ~, when illuminated by incident excitation radiation of a emission particular, shorter wavelength ~. excitation' The brightness of the emitted radiation, measured by the detection system of a microarray scanning system, is a function of the fluorophore concentration present in the illuminated spot. Because the fluorophore concentration is a function of the binding affinity or likeness of the probe molecule to ~o the target molecule, the brightness of a hybridized spot is an indication of the degree of similarity between the probe DNA and the target DNA present in the hybridized spot.
A typical microarray sample may provide for up to tens of thousands of experiments to be performed simultaneously on the probe DNA, thus producing a detailed characterization of a particular gene under investigation.
is In a microarray scanning system, the area of interest is usually divided into an array of discrete elements referred to as 'pixels.' Each pixel is illuminated independently as it is being addressed by the scanning system. The optical radiation source is typically a single-wavelength laser device focused down to form an excitation radiation spot of the desired size. Emission radiation is emitted by the illuminated Zo fluorophore in an outward, spherical beam. A portion of this emission beam is collected by an optical system and transmitted to a detection apparatus. In addition to the emitted radiation, some of the incident excitation radiation scattered from the surface of the sample is also collected by the optical system. To minimize the amount of excitation radiation reaching the detector assembly, the optical system may be Zs designed using filtering components, such as dichroic and band-pass filters, to provide discrimination between excitation and emission radiation wavelengths.
In order to obtain accurate information from the scanning of a microarray, it is important to know which fluorophore materials have been used in order to use the correct wavelengths in illuminating the spots and to filter the correct wavelengths of the so fluorescent emissions. Furthermore, it is advantageous to excite the fluorophores with a high-intensity excitation beam so as to return the maximum signal to the microarray scanning system detector. However, the intensity of the excitation beam must be kept below the level at which the flurorophore becomes saturated or the sample material may degrade.
Furthermore, analysis of raw data collected by the microarray scanning system must be performed in accordance with protocols that may vary in accordance with experiment parameters. In conventional scanning systems, entry of the scanning and analysis protocols is performed manually. This involves significant operator time and, fiu-ther, is a source of errors in the scanning and analysis procedure.
io The sensitivity of the detection system is a critical parameter in a microarray scanning system. The possible range of fluorescence emission varies enormously between samples and often exceeds the dynamic range of the detection system, causing saturation of signals. The occurrence of saturated signals in a data set makes it impossible to quantify the fluorophore brightness emitted from the hybridized spots is exhibiting saturation.
In a conventional microarray scanning system, sensitivity adjustment of the detection system is an iterative procedure. The user performs a partial scan using a particular channel of the system, views the image, and adjusts the excitation radiation power and/or the gain of the detector system accordingly such that the optimal range of ao sensitivity lies within the dynamic range of the detection system. This process is time consuming for the user and, further, degrades the experimental samples by a process of photobleaching the fluorescently-tagged spots on the substrate.
While the relevant art provides iterative procedures for calibration of microarray scanning systems, there remains a need for improvements that offer advantages and as capabilities not found in presently available methods of calibration, and it is a primary object of this invention to provide such improvements.
SUMMARY OF THE INVENTION
In accordance with the present invention a series of dilution spots is imprinted on a microarray sample which includes an array of genetic material samples containing 30 one or more fluorophores. A microarray scanning system, which includes an excitation W~ 01/35074 CA 02390651 2002-05-08 PCT/US00/41170 radiation source, a detection system, and a computational device, is used to analyze the fluorophores in the genetic material samples. Automatic calibration adjustment of either or both the detection system and the excitation radiation source is achieved by i) irradiating the dilution spots with the source of excitation radiation; ii) detecting s emission radiation produced by the dilution spot fluorophore material in response to the irradiation; iii) deriving a series of brightness readings corresponding to the levels of emission radiation detected at corresponding dilution spots; iv) analyzing the brightness readings with the computational device to obtain a fluorophore brightness characteristic as a function of fluorophore concentration; and v) adjusting the sensitivity of the io detection system and/or the intensity level of the source of excitation radiation in accordance with the fluorophore brightness characteristic.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention description below refers to the accompanying drawings, of which:
is Fig. 1 is a diagrammatical view of a microarray scanning system as used in the analysis of a microarray sample;
Fig. 2 is a diagrammatical view of the sample surface of the microarray sample of Fig. 1;
Fig. 3 is a diagram illustrating a fluorophore brightness as a function of zo fluorophore concentration; and Fig. 4 is a diagram illustrating response of fluorophores at various concentrations to a constant level of incident excitation radiation.
DETAILED DESCRIPTION OF AN ILLUSTRATIVE EMBODIMENT
There is shown in Fig. 1 a diagrammatical representation of a microarray is scanning system 10 as can be used in the analysis of a microarray sample 100. The microarray scanning system 10 includes an illumination head 20, an optical system 30, and a detector assembly 40. The illumination head 20 comprises an excitation radiation source 21 producing source radiation 25 of two or more different wavelengths and a shutter assembly 23 which functions to pass any one of the different wavelengths W~ Ol/350~4 CA 02390651 2002-05-08 PCT/US00/411~0 received from the excitation radiation source 21. In the example shown, the excitation radiation source 21 is producing radiation 25 having wavelength 7~, and wavelength ~,2 . The shutter assembly 23 is blocking radiation of wavelength ~,, and is allowing radiation of wavelength 7~z to pass as a beam of single-wavelength excitation radiation s 27. The excitation radiation source 21 may include, for example, two or more single-wavelength coherent optical radiation sources such as lasers, one or more mufti-wavelength coherent optical radiation sources, or one or more broadband sources.
It should thus be understood that operation of the microarray scanning system 10 is not limited to the use of only two wavelengths and that the illumination head 20 may ~o provide excitation radiation of three or more different wavelengths.
The optical system 30 includes an excitation mirror 33 positioned to redirect the excitation radiation beam 27 onto the microarray sample 100 as an incident excitation beam 27'. An objective lens 31 is disposed between the excitation mirror 33 and the microarray sample 100 in the optical path of the incident excitation radiation beam 27'.
is The objective lens 31 serves to focus the incident excitation beam 27' to a desired spot size on the microarray sample 100.
When the incident excitation radiation 27' illuminates a fluorescent label, or fluorophore, present in the microarray sample 100, there is produced a corresponding emission radiation beam 29 of wavelength 7~ , typically 20 to 40 nm longer emission zo than the wavelength (i.e., ~,~ or 7~z ) of the incident radiation beam 27'.
In the configuration shown, the excitation mirror 33 functions as a geometric beamsplitter where the width of the incident excitation beam 27' is much smaller than the width of the emission radiation beam 29. The relatively small excitation mirror 33 thus reflects the incident excitation beam 27' scattered from the microarray sample 100 back to the Zs illumination head 20 while allowing the greater portion of the emission radiation beam 29 to pass upstream of the objective lens 31.
The detector assembly 40 includes a photomultiplier tube 41 and a variable high-voltage reference 43. In an alternative embodiment, an avalanche photodiode or a solid state optical detection device (e.g., a CCD) can be used in place of the so photomultiplier tube 41. The photomultiplier tube 41 outputs a signal to a variable-gain WU ~1/35~74 CA 02390651 2002-05-08 PCT/US00/41170 amplifier 45.
A band-pass or long-pass filter 37, substantially transmissive to the emission radiation beam 29 and substantially non-transmissive to the excitation radiation beam 27 may be disposed in the optical path of the optical system 30 between the objective s lens 31 and a focusing lens 39. In a preferred embodiment, the focusing lens 39 forms a confocal system with the objective lens 31 and images the emission radiation beam 29 onto the photomultiplier tube 41. The optical system 30 may further include a broadband mirror 35 to provide a folded transmission path for the emission radiation beam 29, and an aperture stop 34 may be provided between the focusing lens 39 and io the photomultiplier tube 41. The aperture stop 34 serves to block that portion of the illuminated microarray sample 100 which is not in focus at the photomultiplier tube 41.
As can be appreciated by one skilled in the relevant art, the microarray scanning system may further include a corresponding band-pass or long-pass filter for each of the other excitation-emission wavelength pairs utilized by the microarray scanning system is 10.
The operation of the microarray scanning system 10 can best be described with reference to Fig. 1 and to Fig. 2 which is a diagrammatical plan view of the microarray sample 100. The microarray sample 100 includes a planar substrate 101, such as a one by three-inch glass microscope slide. A sample surface 103 of the planar substrate 1 O1 Zo may, for example, include a marking 105 and/or an etched or 'frosted' region 107 extending from a boundary 108 to the edge of the planar substrate, either or both produced by the substrate manufacturer. The microarray sample 100 includes at least a first microarray 111 comprising a plurality of first target spots 113 (denoted by open circles), containing genetic target material, disposed on the sample surface 103 and may as further include a second microarray 115 comprising a plurality of second target spots 117. The first target spots 113 and second target spots 117 are typically arrayed in rows and columns as shown. As probe material (not shown) containing a predetermined concentration of fluorophore material is added to successive first target spots 113, hybridized spots 114 (denoted by filled circles) remain after excess probe 3o material is removed. Similarly, hybridized spots 118 result from the addition of probe material to second target spots 117.
_7_ The microarray sample 100 is removably secured to a test platform 55, in Fig.
1, such as by mechanical restraint or by a suction device, as is well-known in the relevant art. A positioning system S 1 imparts translational movement in an X-Y plane to the test platform 55, and thus to the microarray sample 100, by means of a mechanical linkage 53. The microarray scanning system 10 further includes a computational device 60, such as a computer, connected to the positioning system S 1 so as to provide control by the computational device 60 via positioning software 61. When the microarray sample 100 has been secured to the test platform 55, the detector assembly 40 can be used to optimize the focus position of the objective lens 31. This can be io done, for example, by imaging the marking 105 or a user-applied fiducial mark 106 with the optical system 30. The focusing procedure is described in greater detail in the related application, incorporated herein in its entirety by reference.
The computational device 60 also receives the signal output of the variable-gain amplifier 45, which provides positional feedback as the microarray sample 100 is is aligned and scanned via the positioning system 51. The positional feedback obtained by illuminating the test surface 103 with the incident excitation radiation 27' and imaging the illuminated portion of the test surface 103 back to the positioning software 61 via the detector 40 as the microarray sample 100 is moved in the X-Y plane.
The sensitivity of the microarray scanning system can be adjusted for a zo particular microarray sample 100 by using a dilution spots 119 provided on the sample surface 103, in Fig. 2. The dilution spots 119 includes a plurality of dilution spots 119a through 119g each having a different fluorophore concentration. The user quantifies the dilution spots 119 on a spot-by-spot basis to obtain a concentration-to-brightness curve for a particular fluorophore. It should be understood that although seven dilution zs spots are shown, a greater or lesser number can be used.
In a preferred embodiment, the first microarray 11 l, the second microarray 115, and the dilution spots 119 are placed at predetermined positions relative to one another by using as a reference feature any of, for example, the marking 1 O5, the etched region 107 and boundary 108, the user-applied fiducial mark 106, or an edge 109 of the 3o substrate 101. This configuration enables use of automated equipment to image the WO 01/35074 CA 02390651 2002-05-08 pCT/US00/4117U
first microarray 111, the second microarray 115, and the dilution spots 119, and to perform subsequent calibration as described in greater detail below.
The microarray scanning system 10 divides the dilution spots 119 into pixels.
As the fluorophore material in each of the dilution spots 119a through 119g is s illuminated by the incident excitation radiation 27', each pixel is successively acquired by the detector assembly 40 and analyzed for the presence of fluorophore material by the computational device 60. Each analysis measurement results in a data point that represents the relative fluorophore concentration of the measured pixel. The pixel data is then reconstructed to produce a quantified description of the scanned dilution spots io 119. A similar procedure is used to analyze the fluorescent emission from the hybridized spots 114 and 118.
It is known in the relevant art that the brightness characteristics of the hybridized spots 114 are typically nonlinear functions of the fluorophore concentration, as shown in Fig. 3. As exemplified by a concentration-to-brightness curve 121, a is fluorophore may have a more useful response within a relatively narrow concentration range (e.g., from about 10 to 1000 '~uor in the example provided), and an essentially pm flat response outside this concentration range. It is important to be able to measure the concentration-to-brightness curve on a known fluorescent sample for the purpose of quantifying the fluorophore concentration in the corresponding hybridized spot 114.
2o Once the characteristic curve of the corresponding fluorescent imager has been determined, operational parameters of the microarray scanning system 10 can be specified.
By way of example, a comparison of various fluorescent dyes is provided in Fig. 4. In a Cy3 dilution fiducial series 123 containing seven individual dilution spots is having fluorophore concentrations ranging from 0.01 '~uor to 10,000 '~uor , a pm pm brightness of 15,155 was measured at a concentration of 100 '~uor and saturation ~m occurred at a concentration of 1000 ~uor for a constant level of incident excitation ~,m WO 01/35074 CA 02390651 2002-05-08 PCT/[J$00/41170 _9_ radiation. The signal values are average pixel values obtained in a 2 millimeter circle.
For an Alexa532 dilution fiducial series 125, a concentration of 100 '~uor produced a ~m measured brightness of 21,280. For an Alexa594 dilution fiducial series 127 and a concentration of 1000 '~uor , the brightness measurement was 38,363, and for a Cy5 ~m s dilution fiducial series 129, saturation was reached at a concentration of 10,000 ~uor .
~m If the sensitivity of the detector system 40 is set too high, saturated signals are produced, reducing the usefulness of the resulting data set. If, on the other hand, the sensitivity of the detector system 40 is set too low, the full resolution of the microarray scanning system 10 is not used and maximum differentiation in fluorescence levels ~o between the hybridized spots 114 is not obtained. Moreover, if two or more channels of the microarray scanning system 10 are being used, the channels need to be balanced such that the dynamic range of the fluorophore sensitivity of each channel lies within the dynamic range of the microarray scanning system 10.
The computational device 60, in Fig. 1, includes dilution software 63, or other ~s machine-readable code, for obtaining a concentration-to-brightness curve from the dilution spots 119. In a preferred embodiment, the dilution spots 119 are set by protocol, and the position and characteristics of the dilution spots 119 are predetermined. Prior to imaging the hybridized spots 114, the microarray scanning system 10 images the dilution spots 119 while adjusting any combination of: i) the Zo emitted power of the excitation radiation source 21, ii) a high-voltage reference 43 in the photomultiplier tube 41, and iii) the gain of the variable-gain amplifier 45, for all applicable channels. The outputs of the excitation radiation source 21 and the photomultiplier tube 41 can typically be adjusted over a range of at least 100:1. This allows the sensitivity of the microarray scanning system 10 to be adjusted over a range is of 10,000:1 or greater. The sensitivity of the microarray scanning system 10 can thus be optimized without the risk of photobleaching any of the hybridized spots 114 and 118 in the microarrays 111 and 115.
While the invention has been described with reference to particular embodiments, it will be understood that the present invention is by no means limited to the particular constructions and methods herein disclosed and/or shown in the drawings, but also comprises any modifications or equivalents within the scope of the claims.
What is claimed is:
MICROARRAY SCANNING SYSTEM
BACKGROUND OF THE INVENTION
Field Of the Invention s This invention in general relates to optical scanning systems and, in particular, to an apparatus and method for calibration of a microarray scanning system.
Description Of The Prior Art The use of excitation radiation to produce fluorescence in a series of scanned genetic samples is known. U.S. Patent No. 5,689,110 issued to Dietz et al., for ~o example, discloses a calibration method and device for a fluorescence spectrometer which uses fluorescence from a homogenous solid state standard as the source of calibration fluorescence. Fluorescent imagers are used to acquire data in experiments that utilize fluorescent labels, or fluorophores, to identify the state of a sample being tested. In some cases the presence of or lack of fluorophores in the sample determines is the experimental result. In other cases the fluorophore concentration, which is a function of the intensity of the emission radiation received from the sample, is the measurement of interest and the experimental result can be inferred by measuring the intensity of the detected radiation.
An example of a process that uses fluorophores is the microarray which is a set zo of experiments utilizing genetic material such as DNA or RNA, bound to a glass substrate. Reference or 'target' DNA is spotted onto a glass substrate -typically a one-by three-inch glass microscope slide - where it chemically binds to the surface. Each spot, or sample, of DNA constitutes a separate experiment. A sample of 'probe' DNA
or RNA, to which has been added the fluorophore material, is subsequently placed on zs the target spots on the surface of the substrate and is allowed to hybridize with the target DNA. Excess probe DNA that does not bind with target DNA is removed from the surface of the slide in a subsequent washing process.
_2_ The experiments measure the binding affinities between the probe DNA and the target DNA to determine the similarity in molecular structure; complementary molecules have a much greater probability of binding than do unrelated molecules. The fluorophore added to the probe DNA emits a range of radiation energy centered about a wavelength ~, when illuminated by incident excitation radiation of a emission particular, shorter wavelength ~. excitation' The brightness of the emitted radiation, measured by the detection system of a microarray scanning system, is a function of the fluorophore concentration present in the illuminated spot. Because the fluorophore concentration is a function of the binding affinity or likeness of the probe molecule to ~o the target molecule, the brightness of a hybridized spot is an indication of the degree of similarity between the probe DNA and the target DNA present in the hybridized spot.
A typical microarray sample may provide for up to tens of thousands of experiments to be performed simultaneously on the probe DNA, thus producing a detailed characterization of a particular gene under investigation.
is In a microarray scanning system, the area of interest is usually divided into an array of discrete elements referred to as 'pixels.' Each pixel is illuminated independently as it is being addressed by the scanning system. The optical radiation source is typically a single-wavelength laser device focused down to form an excitation radiation spot of the desired size. Emission radiation is emitted by the illuminated Zo fluorophore in an outward, spherical beam. A portion of this emission beam is collected by an optical system and transmitted to a detection apparatus. In addition to the emitted radiation, some of the incident excitation radiation scattered from the surface of the sample is also collected by the optical system. To minimize the amount of excitation radiation reaching the detector assembly, the optical system may be Zs designed using filtering components, such as dichroic and band-pass filters, to provide discrimination between excitation and emission radiation wavelengths.
In order to obtain accurate information from the scanning of a microarray, it is important to know which fluorophore materials have been used in order to use the correct wavelengths in illuminating the spots and to filter the correct wavelengths of the so fluorescent emissions. Furthermore, it is advantageous to excite the fluorophores with a high-intensity excitation beam so as to return the maximum signal to the microarray scanning system detector. However, the intensity of the excitation beam must be kept below the level at which the flurorophore becomes saturated or the sample material may degrade.
Furthermore, analysis of raw data collected by the microarray scanning system must be performed in accordance with protocols that may vary in accordance with experiment parameters. In conventional scanning systems, entry of the scanning and analysis protocols is performed manually. This involves significant operator time and, fiu-ther, is a source of errors in the scanning and analysis procedure.
io The sensitivity of the detection system is a critical parameter in a microarray scanning system. The possible range of fluorescence emission varies enormously between samples and often exceeds the dynamic range of the detection system, causing saturation of signals. The occurrence of saturated signals in a data set makes it impossible to quantify the fluorophore brightness emitted from the hybridized spots is exhibiting saturation.
In a conventional microarray scanning system, sensitivity adjustment of the detection system is an iterative procedure. The user performs a partial scan using a particular channel of the system, views the image, and adjusts the excitation radiation power and/or the gain of the detector system accordingly such that the optimal range of ao sensitivity lies within the dynamic range of the detection system. This process is time consuming for the user and, further, degrades the experimental samples by a process of photobleaching the fluorescently-tagged spots on the substrate.
While the relevant art provides iterative procedures for calibration of microarray scanning systems, there remains a need for improvements that offer advantages and as capabilities not found in presently available methods of calibration, and it is a primary object of this invention to provide such improvements.
SUMMARY OF THE INVENTION
In accordance with the present invention a series of dilution spots is imprinted on a microarray sample which includes an array of genetic material samples containing 30 one or more fluorophores. A microarray scanning system, which includes an excitation W~ 01/35074 CA 02390651 2002-05-08 PCT/US00/41170 radiation source, a detection system, and a computational device, is used to analyze the fluorophores in the genetic material samples. Automatic calibration adjustment of either or both the detection system and the excitation radiation source is achieved by i) irradiating the dilution spots with the source of excitation radiation; ii) detecting s emission radiation produced by the dilution spot fluorophore material in response to the irradiation; iii) deriving a series of brightness readings corresponding to the levels of emission radiation detected at corresponding dilution spots; iv) analyzing the brightness readings with the computational device to obtain a fluorophore brightness characteristic as a function of fluorophore concentration; and v) adjusting the sensitivity of the io detection system and/or the intensity level of the source of excitation radiation in accordance with the fluorophore brightness characteristic.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention description below refers to the accompanying drawings, of which:
is Fig. 1 is a diagrammatical view of a microarray scanning system as used in the analysis of a microarray sample;
Fig. 2 is a diagrammatical view of the sample surface of the microarray sample of Fig. 1;
Fig. 3 is a diagram illustrating a fluorophore brightness as a function of zo fluorophore concentration; and Fig. 4 is a diagram illustrating response of fluorophores at various concentrations to a constant level of incident excitation radiation.
DETAILED DESCRIPTION OF AN ILLUSTRATIVE EMBODIMENT
There is shown in Fig. 1 a diagrammatical representation of a microarray is scanning system 10 as can be used in the analysis of a microarray sample 100. The microarray scanning system 10 includes an illumination head 20, an optical system 30, and a detector assembly 40. The illumination head 20 comprises an excitation radiation source 21 producing source radiation 25 of two or more different wavelengths and a shutter assembly 23 which functions to pass any one of the different wavelengths W~ Ol/350~4 CA 02390651 2002-05-08 PCT/US00/411~0 received from the excitation radiation source 21. In the example shown, the excitation radiation source 21 is producing radiation 25 having wavelength 7~, and wavelength ~,2 . The shutter assembly 23 is blocking radiation of wavelength ~,, and is allowing radiation of wavelength 7~z to pass as a beam of single-wavelength excitation radiation s 27. The excitation radiation source 21 may include, for example, two or more single-wavelength coherent optical radiation sources such as lasers, one or more mufti-wavelength coherent optical radiation sources, or one or more broadband sources.
It should thus be understood that operation of the microarray scanning system 10 is not limited to the use of only two wavelengths and that the illumination head 20 may ~o provide excitation radiation of three or more different wavelengths.
The optical system 30 includes an excitation mirror 33 positioned to redirect the excitation radiation beam 27 onto the microarray sample 100 as an incident excitation beam 27'. An objective lens 31 is disposed between the excitation mirror 33 and the microarray sample 100 in the optical path of the incident excitation radiation beam 27'.
is The objective lens 31 serves to focus the incident excitation beam 27' to a desired spot size on the microarray sample 100.
When the incident excitation radiation 27' illuminates a fluorescent label, or fluorophore, present in the microarray sample 100, there is produced a corresponding emission radiation beam 29 of wavelength 7~ , typically 20 to 40 nm longer emission zo than the wavelength (i.e., ~,~ or 7~z ) of the incident radiation beam 27'.
In the configuration shown, the excitation mirror 33 functions as a geometric beamsplitter where the width of the incident excitation beam 27' is much smaller than the width of the emission radiation beam 29. The relatively small excitation mirror 33 thus reflects the incident excitation beam 27' scattered from the microarray sample 100 back to the Zs illumination head 20 while allowing the greater portion of the emission radiation beam 29 to pass upstream of the objective lens 31.
The detector assembly 40 includes a photomultiplier tube 41 and a variable high-voltage reference 43. In an alternative embodiment, an avalanche photodiode or a solid state optical detection device (e.g., a CCD) can be used in place of the so photomultiplier tube 41. The photomultiplier tube 41 outputs a signal to a variable-gain WU ~1/35~74 CA 02390651 2002-05-08 PCT/US00/41170 amplifier 45.
A band-pass or long-pass filter 37, substantially transmissive to the emission radiation beam 29 and substantially non-transmissive to the excitation radiation beam 27 may be disposed in the optical path of the optical system 30 between the objective s lens 31 and a focusing lens 39. In a preferred embodiment, the focusing lens 39 forms a confocal system with the objective lens 31 and images the emission radiation beam 29 onto the photomultiplier tube 41. The optical system 30 may further include a broadband mirror 35 to provide a folded transmission path for the emission radiation beam 29, and an aperture stop 34 may be provided between the focusing lens 39 and io the photomultiplier tube 41. The aperture stop 34 serves to block that portion of the illuminated microarray sample 100 which is not in focus at the photomultiplier tube 41.
As can be appreciated by one skilled in the relevant art, the microarray scanning system may further include a corresponding band-pass or long-pass filter for each of the other excitation-emission wavelength pairs utilized by the microarray scanning system is 10.
The operation of the microarray scanning system 10 can best be described with reference to Fig. 1 and to Fig. 2 which is a diagrammatical plan view of the microarray sample 100. The microarray sample 100 includes a planar substrate 101, such as a one by three-inch glass microscope slide. A sample surface 103 of the planar substrate 1 O1 Zo may, for example, include a marking 105 and/or an etched or 'frosted' region 107 extending from a boundary 108 to the edge of the planar substrate, either or both produced by the substrate manufacturer. The microarray sample 100 includes at least a first microarray 111 comprising a plurality of first target spots 113 (denoted by open circles), containing genetic target material, disposed on the sample surface 103 and may as further include a second microarray 115 comprising a plurality of second target spots 117. The first target spots 113 and second target spots 117 are typically arrayed in rows and columns as shown. As probe material (not shown) containing a predetermined concentration of fluorophore material is added to successive first target spots 113, hybridized spots 114 (denoted by filled circles) remain after excess probe 3o material is removed. Similarly, hybridized spots 118 result from the addition of probe material to second target spots 117.
_7_ The microarray sample 100 is removably secured to a test platform 55, in Fig.
1, such as by mechanical restraint or by a suction device, as is well-known in the relevant art. A positioning system S 1 imparts translational movement in an X-Y plane to the test platform 55, and thus to the microarray sample 100, by means of a mechanical linkage 53. The microarray scanning system 10 further includes a computational device 60, such as a computer, connected to the positioning system S 1 so as to provide control by the computational device 60 via positioning software 61. When the microarray sample 100 has been secured to the test platform 55, the detector assembly 40 can be used to optimize the focus position of the objective lens 31. This can be io done, for example, by imaging the marking 105 or a user-applied fiducial mark 106 with the optical system 30. The focusing procedure is described in greater detail in the related application, incorporated herein in its entirety by reference.
The computational device 60 also receives the signal output of the variable-gain amplifier 45, which provides positional feedback as the microarray sample 100 is is aligned and scanned via the positioning system 51. The positional feedback obtained by illuminating the test surface 103 with the incident excitation radiation 27' and imaging the illuminated portion of the test surface 103 back to the positioning software 61 via the detector 40 as the microarray sample 100 is moved in the X-Y plane.
The sensitivity of the microarray scanning system can be adjusted for a zo particular microarray sample 100 by using a dilution spots 119 provided on the sample surface 103, in Fig. 2. The dilution spots 119 includes a plurality of dilution spots 119a through 119g each having a different fluorophore concentration. The user quantifies the dilution spots 119 on a spot-by-spot basis to obtain a concentration-to-brightness curve for a particular fluorophore. It should be understood that although seven dilution zs spots are shown, a greater or lesser number can be used.
In a preferred embodiment, the first microarray 11 l, the second microarray 115, and the dilution spots 119 are placed at predetermined positions relative to one another by using as a reference feature any of, for example, the marking 1 O5, the etched region 107 and boundary 108, the user-applied fiducial mark 106, or an edge 109 of the 3o substrate 101. This configuration enables use of automated equipment to image the WO 01/35074 CA 02390651 2002-05-08 pCT/US00/4117U
first microarray 111, the second microarray 115, and the dilution spots 119, and to perform subsequent calibration as described in greater detail below.
The microarray scanning system 10 divides the dilution spots 119 into pixels.
As the fluorophore material in each of the dilution spots 119a through 119g is s illuminated by the incident excitation radiation 27', each pixel is successively acquired by the detector assembly 40 and analyzed for the presence of fluorophore material by the computational device 60. Each analysis measurement results in a data point that represents the relative fluorophore concentration of the measured pixel. The pixel data is then reconstructed to produce a quantified description of the scanned dilution spots io 119. A similar procedure is used to analyze the fluorescent emission from the hybridized spots 114 and 118.
It is known in the relevant art that the brightness characteristics of the hybridized spots 114 are typically nonlinear functions of the fluorophore concentration, as shown in Fig. 3. As exemplified by a concentration-to-brightness curve 121, a is fluorophore may have a more useful response within a relatively narrow concentration range (e.g., from about 10 to 1000 '~uor in the example provided), and an essentially pm flat response outside this concentration range. It is important to be able to measure the concentration-to-brightness curve on a known fluorescent sample for the purpose of quantifying the fluorophore concentration in the corresponding hybridized spot 114.
2o Once the characteristic curve of the corresponding fluorescent imager has been determined, operational parameters of the microarray scanning system 10 can be specified.
By way of example, a comparison of various fluorescent dyes is provided in Fig. 4. In a Cy3 dilution fiducial series 123 containing seven individual dilution spots is having fluorophore concentrations ranging from 0.01 '~uor to 10,000 '~uor , a pm pm brightness of 15,155 was measured at a concentration of 100 '~uor and saturation ~m occurred at a concentration of 1000 ~uor for a constant level of incident excitation ~,m WO 01/35074 CA 02390651 2002-05-08 PCT/[J$00/41170 _9_ radiation. The signal values are average pixel values obtained in a 2 millimeter circle.
For an Alexa532 dilution fiducial series 125, a concentration of 100 '~uor produced a ~m measured brightness of 21,280. For an Alexa594 dilution fiducial series 127 and a concentration of 1000 '~uor , the brightness measurement was 38,363, and for a Cy5 ~m s dilution fiducial series 129, saturation was reached at a concentration of 10,000 ~uor .
~m If the sensitivity of the detector system 40 is set too high, saturated signals are produced, reducing the usefulness of the resulting data set. If, on the other hand, the sensitivity of the detector system 40 is set too low, the full resolution of the microarray scanning system 10 is not used and maximum differentiation in fluorescence levels ~o between the hybridized spots 114 is not obtained. Moreover, if two or more channels of the microarray scanning system 10 are being used, the channels need to be balanced such that the dynamic range of the fluorophore sensitivity of each channel lies within the dynamic range of the microarray scanning system 10.
The computational device 60, in Fig. 1, includes dilution software 63, or other ~s machine-readable code, for obtaining a concentration-to-brightness curve from the dilution spots 119. In a preferred embodiment, the dilution spots 119 are set by protocol, and the position and characteristics of the dilution spots 119 are predetermined. Prior to imaging the hybridized spots 114, the microarray scanning system 10 images the dilution spots 119 while adjusting any combination of: i) the Zo emitted power of the excitation radiation source 21, ii) a high-voltage reference 43 in the photomultiplier tube 41, and iii) the gain of the variable-gain amplifier 45, for all applicable channels. The outputs of the excitation radiation source 21 and the photomultiplier tube 41 can typically be adjusted over a range of at least 100:1. This allows the sensitivity of the microarray scanning system 10 to be adjusted over a range is of 10,000:1 or greater. The sensitivity of the microarray scanning system 10 can thus be optimized without the risk of photobleaching any of the hybridized spots 114 and 118 in the microarrays 111 and 115.
While the invention has been described with reference to particular embodiments, it will be understood that the present invention is by no means limited to the particular constructions and methods herein disclosed and/or shown in the drawings, but also comprises any modifications or equivalents within the scope of the claims.
What is claimed is:
Claims (23)
1. A microarray sample suitable for use in a microarray scanning system, said microarray sample comprising:
a planar substrate comprising a sample surface;
a plurality of target spots disposed on said sample surface, each said target spot comprising a genetic target material; and a plurality of dilution spots disposed on said sample surface, each said dilution spot comprising a predetermined concentration of fluorophore material with the plurality when scanned providing information from which a fluorophore brightness characteristic that is a function of fluorophore concentration can be determined.
a planar substrate comprising a sample surface;
a plurality of target spots disposed on said sample surface, each said target spot comprising a genetic target material; and a plurality of dilution spots disposed on said sample surface, each said dilution spot comprising a predetermined concentration of fluorophore material with the plurality when scanned providing information from which a fluorophore brightness characteristic that is a function of fluorophore concentration can be determined.
2. The microarray sample of claim 1 wherein said planar substrate comprises a microscope slide.
3. The microarray sample of claim 1 wherein said predetermined concentration of said fluorophore material in at least one said dilution spot is different from said predetermined concentration of said fluorophore material in any other said dilution spot.
4. The microarray sample of claim 1 wherein said dilution spots are located at predetermined positions with respect to said plurality of target spots.
5. The microarray sample of claim 1 wherein said dilution spots are located at predetermined positions with respect to a fiducial mark disposed on said sample surface.
6. The microarray sample of claim 1 further comprising a fiducial mark disposed at a predetermined distance from said plurality of dilution spots.
7. The microarray sample of claim 1 further comprising a plurality of second dilution spots disposed on said sample surface, each said second dilution spot comprising a second fluorophore material.
8. The microarray sample of claim 1 further comprising one or more probe spots disposed upon a corresponding one or more of said target spots, said probe spots comprising a genetic probe material and a predetermined concentration of said fluorophore material.
9. A microarray scanning suitable system for conducting experiments on a planar substrate supporting a plurality of target spots containing genetic material and a plurality of dilution spots with each dilution spot having a predetermined concentration of fluorophore material, said microarray scanning system comprising:
irradiating means for irradiating the plurality of dilution spots;
detecting means for detecting emission radiation produced by the dilution spot fluorophore material in response to said irradiation;
means for deriving a plurality of brightness readings, each said brightness reading corresponding to the level of emission radiation detected at a corresponding dilution spot;
means for analyzing said plurality of brightness readings to obtain a fluorophore brightness characteristic as a function of fluorophore concentration; and adjusting means for adjusting the sensitivity of said microarray scanning system in response to said fluorophore brightness characteristic, the irradiating means and the detecting means irradiating the target spots and detecting the corresponding emission radiation after the adjusting means has adjusted the sensitivity of the system.
-12a-
irradiating means for irradiating the plurality of dilution spots;
detecting means for detecting emission radiation produced by the dilution spot fluorophore material in response to said irradiation;
means for deriving a plurality of brightness readings, each said brightness reading corresponding to the level of emission radiation detected at a corresponding dilution spot;
means for analyzing said plurality of brightness readings to obtain a fluorophore brightness characteristic as a function of fluorophore concentration; and adjusting means for adjusting the sensitivity of said microarray scanning system in response to said fluorophore brightness characteristic, the irradiating means and the detecting means irradiating the target spots and detecting the corresponding emission radiation after the adjusting means has adjusted the sensitivity of the system.
-12a-
10. The microarray scanning system of claim 9 wherein said means for irradiating comprises at least one member of the group consisting of a single-wavelength coherent optical source, a multiple-wavelength coherent optical source, and a broadband radiation source.
11. The microarray scanning system of claim 9 wherein said means for detecting comprises an optical system.
12. The microarray scanning system of claim 11 wherein said optical system comprises a confocal system.
13. The microarray scanning system of claim 9 wherein said means for detecting comprises at least one member of the group consisting of a photomultiplier tube, an avalanche photodiode, and a solid state optical detection device.
14. The microarray scanning system of claim 9 wherein said means for deriving comprises a machine-readable code.
15. The microarray scanning system of claim 9 wherein said means for adjusting the sensitivity comprises means for adjusting an output level of said means for irradiating.
16. The microarray scanning system of claim 9 wherein said means for adjusting the sensitivity comprises means for adjusting an output signal of said means for detecting.
17. The microarray scanning system of claim 9 wherein said means for adjusting the sensitivity comprises means for adjusting a reference source in electrical communication with said means for detecting.
18. The microarray scanning system of claim 9 further comprising means for securing the substrate.
19. The microarray scanning system of claim 9 further comprising means for translating the substrate in at least two axes.
20. A method for performing experiments on a planar substrate supporting a plurality of dilution spots and a plurality of target spots containing genetic material, -13a-each dilution spot having a predetermined concentration of fluorophore material, said method comprising the steps of:
irradiating the plurality of dilution spots with a source of excitation radiation;
detecting emission radiation produced by the dilution spot fluorophore material in response to said irradiation;
deriving a plurality of brightness readings, each said brightness reading corresponding to the level of emission radiation detected at a corresponding dilution spot;
analyzing said plurality of brightness readings to obtain a fluorophore brightness characteristic as a function of fluorophore concentration;
adjusting the sensitivity of said microarray scanning system in response to said fluorophore brightness characteristic; and irradiating the plurality of target spots after adjusting the sensitivity of the system.
irradiating the plurality of dilution spots with a source of excitation radiation;
detecting emission radiation produced by the dilution spot fluorophore material in response to said irradiation;
deriving a plurality of brightness readings, each said brightness reading corresponding to the level of emission radiation detected at a corresponding dilution spot;
analyzing said plurality of brightness readings to obtain a fluorophore brightness characteristic as a function of fluorophore concentration;
adjusting the sensitivity of said microarray scanning system in response to said fluorophore brightness characteristic; and irradiating the plurality of target spots after adjusting the sensitivity of the system.
21. The method of claim 20 further comprising the step of applying a probe sample of genetic material to one or more of said test spots so as to produce one or more corresponding hybridized spots, said probe sample comprising a predetermined concentration of the fluorophore.
22. The method of claim 21 further comprising the step of irradiating said one or more hybridized spots following said step of adjusting the sensitivity of said microarray scanning system.
23. The method of claim 20 wherein the plurality of dilution spots are located at predetermined positions with respect to the plurality of target spots.
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| PCT/US2000/041170 WO2001035074A1 (en) | 1999-11-09 | 2000-10-12 | Apparatus and method for calibration of a microarray scanning system |
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- 2000-10-12 AT AT00989657T patent/ATE315222T1/en not_active IP Right Cessation
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| EP1228354B1 (en) | 2006-01-04 |
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| WO2001035074A9 (en) | 2002-08-01 |
| EP1228354A1 (en) | 2002-08-07 |
| DE60025400D1 (en) | 2006-03-30 |
| WO2001035074A1 (en) | 2001-05-17 |
| ATE315222T1 (en) | 2006-02-15 |
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