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WO2015141873A1 - Procédé et dispositif d'analyse raman pour analyse quantitative à vitesse élevée d'échantillon à aire étendue - Google Patents

Procédé et dispositif d'analyse raman pour analyse quantitative à vitesse élevée d'échantillon à aire étendue Download PDF

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Publication number
WO2015141873A1
WO2015141873A1 PCT/KR2014/002290 KR2014002290W WO2015141873A1 WO 2015141873 A1 WO2015141873 A1 WO 2015141873A1 KR 2014002290 W KR2014002290 W KR 2014002290W WO 2015141873 A1 WO2015141873 A1 WO 2015141873A1
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sample
raman
analysis
speed
wide
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English (en)
Korean (ko)
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정대홍
이윤식
이호영
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SNU R&DB Foundation
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SNU R&DB Foundation
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/02—Details
    • G01J3/0202—Mechanical elements; Supports for optical elements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/02—Details
    • G01J3/0205—Optical elements not provided otherwise, e.g. optical manifolds, diffusers, windows
    • G01J3/021—Optical elements not provided otherwise, e.g. optical manifolds, diffusers, windows using plane or convex mirrors, parallel phase plates, or particular reflectors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/02—Details
    • G01J3/0289—Field-of-view determination; Aiming or pointing of a spectrometer; Adjusting alignment; Encoding angular position; Size of measurement area; Position tracking
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/02—Details
    • G01J3/0291—Housings; Spectrometer accessories; Spatial arrangement of elements, e.g. folded path arrangements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/28—Investigating the spectrum
    • G01J3/44—Raman spectrometry; Scattering spectrometry ; Fluorescence spectrometry
    • 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
    • G01N21/65—Raman scattering
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2201/00—Features of devices classified in G01N21/00
    • G01N2201/10—Scanning
    • G01N2201/105—Purely optical scan

Definitions

  • the present invention relates to an apparatus and method for analyzing a sample labeled with a Raman signal, and in particular, capable of high-speed quantitative analysis of a plurality of search targets present in a wide range of samples, while reducing the size and cost of equipment.
  • Raman analysis apparatus and method capable of high-speed quantitative analysis of a plurality of search targets present in a wide range of samples, while reducing the size and cost of equipment.
  • Raman analyzer is a device that analyzes the physical properties of the sample represented by the Raman spectrum using a spectrometer that measures the Raman spectrum, and is composed of a light source and a spectrometer that mainly use a laser to generate the Raman spectrum.
  • lasers to be used lasers having various wavelengths ranging from ultraviolet rays to infrared wavelength ranges are commonly used. In the case of a biological sample, lasers having energy in the visible and near infrared ranges are mainly used. Examples include the 514.5 nm line of argon laser, the 647 nm line of krypton laser, and the 532 nm line, 660 nm line and 785 nm line of solid state lasers such as YAG.
  • FIG. 1 is a configuration diagram of a conventional Raman analysis device
  • Figure 2 is an illustration of a conventional Raman analysis method.
  • the light source starting from the laser light source 10 includes a spatial filter 11, a mirror 12, a mirror, and a dichroic mirror 13. It arrives at the X-axis mirror 14 through the optical path.
  • Conventional Raman analyzers arrange an X-axis mirror (14: X-axis mirror) and a Y-axis mirror (15: Y-axis mirror) moving on the X axis and the Y axis perpendicular thereto, and finely respectively
  • X-axis mirror 14: X-axis mirror
  • Y-axis mirror 15: Y-axis mirror
  • the X-axis mirror 14 and the Y-axis mirror 15 can be finely adjusted through the voice coil motors 16 and 17 (Voice Coil Motor) and the drivers 18 and 19 controlling their driving.
  • voice coil motors 16 and 17 Vehicle Coil Motor
  • the light source incident through the objective lens 20 is scattered by colliding with the sample 21. Most of the light sources are scattered with the same energy as incident light, but some of them are inelastic scattered by exchanging energy with the sample 21 to a unique degree. .
  • the wavelengths of Stokes scattering in which the light source loses energy and the Anti-Stokes scattering in which the light source obtains energy appear in a unique form, and the spectrometer 50 has an edge.
  • An edge filter 28 can be collected (typically Stokes scattering) and displayed as a uniquely shaped spectrum.
  • the spot-specific spectrum image is repeatedly photographed by the camera 40 and analyzed by the computer 30 to determine the characteristics of the sample 21.
  • the above-described configuration is divided into a configuration of the X-axis mirror 14 and the Y-axis mirror 15, and then finely adjusted to each one (usually referred to as galvo mirror), expensive accessories that require a high degree of precision Since it must be mounted in plural, the cost increases, and when the measurement range is widened, there is a problem in that the quality of an area far from the center is degraded by optical distortion, and thus there is a limit in the measurement area. In addition, since the laser light must be reflected to the X-axis mirror 14 and the Y-axis mirror 15 in common, the width of the measurable sample is limited by the reflective physical reflection area limitation. It is a situation that does not correspond to the area required for a wide range of samples, such as a sample or a semiconductor wafer.
  • FIG. 2 schematically illustrates a method of collecting and analyzing information of the Raman analysis apparatus illustrated in FIG. 1, in which the Raman analysis apparatus finely adjusts the X-axis mirror 14 and the Y-axis mirror 15 for each spot 22.
  • the Raman map (36: Raman Map) including the predetermined spot information and the Raman information collected by the spectrometer 50 by the spot 22 to move the optical path 25. Correspond and analyze.
  • the Raman analysis apparatus of FIG. 1 used in the previous example is used for crack inspection of a semiconductor wafer, foreign material inspection, analysis of a fine sample, and the like, and is mainly used for industrial equipment or research / TECH because of its large size and high cost.
  • the Raman analysis apparatus also includes a separate X-axis mirror and Y-axis mirrors 14a and 14b so that the scan area is narrow and the cost of the driving unit for precision driving is increased, so that the small-scale industrial or medical analysis may be used. It is difficult to spread.
  • the conventional Raman analysis apparatus and method for analysis or medical analysis for small industries has limitations in performance, size, and cost, and this problem is particularly noticeable in the use of medical analysis.
  • the diagnosis of a disease using a single biomarker requires a large amount of samples and the diagnosis rate is very slow, which increases the cost of diagnosis and makes a large number of diagnosis difficult.
  • a candidate group of diagnostic reagents in order to search for a candidate group of diagnostic reagents, a plurality of candidates should be examined. However, when a single candidate is tested, a long search time is required to find an optimal candidate.
  • nanospectrometry has been used to search for diagnostic reagents and diagnose diseases using multiple markers.
  • the most popular technology is to obtain independent signals for a plurality of biomarkers using multiple labeled probes and analyze them at once to detect multiple targets simultaneously. Is being reported.
  • the signal read-out method applied to the multi-diagnosis is not a multi-spotting technique in which a single sample is divided into multiple spots and analyzed so that the amount of the sample increases according to a test item.
  • a nanoquantum probe (nanoprobe) technology for producing a probe using silver nanoparticles to increase the sensitivity of the signal to derive quantitative analysis results
  • a multi-dimensional labeling technology can be used to label a large amount of different materials at the same time.
  • FACS Fluorescense-Assisted Cell Sorter
  • the conventional Raman analysis apparatus repeatedly acquires a spot or a line image of a certain size and analyzes the obtained result corresponding to the Raman map, so that it may be efficient in a sample sensitive to location information or Raman information, but the biomarker There are limitations when it is necessary to quickly and precisely quantitate a large number of trace biomarkers present in a wide range of samples for analysis.
  • the Raman throughout the sample rather than the accuracy of the Raman information according to the location of the sample. Because the probabilistic distribution of information and the coefficient information used therein have a much greater impact on the accuracy of analysis, it is important to collect continuous results through high-speed measurements in a uniform measurement environment.
  • An object of the present invention for improving the above-described problem is different from the conventional spot unit analysis, which is generated by the camera operation by reading out the continuous spectral information for one or more sequential scan intervals during one camera operation period. It is to provide a Raman analysis apparatus and method to improve the scanning speed while reducing the noise.
  • Another object of the embodiment of the present invention for improving the above problems is to configure a single flip mirror unit in consideration of the characteristics of the continuous sequential scan method and to move the entire flip mirror unit about the other axis perpendicular to the variable one axis, the accuracy of the analysis It is to provide a Raman analysis apparatus and method to reduce the size and cost of the shaft drive without deterioration.
  • Another object of the present invention for improving the above-mentioned problem is to derive the results of analysis of the continuous spectral information for the sequential scan interval to the Raman map including the preset distribution and coefficient information than the exact position and absolute spectral information of the sample It is to provide a Raman analysis apparatus and method for optimizing speed and quantitative analysis.
  • Another object of the embodiment of the present invention for improving the above-mentioned problem is to minimize the etendue of the light source by moving the stage seating the sample through the auto focusing sensor facing the objective lens and correcting the change in the sample height according to the scan.
  • One Raman analysis apparatus and method are provided.
  • Raman analysis device for high-speed quantitative analysis of a light domain sample is a Raman analysis device for collectively collecting and analyzing a plurality of types of search target information present in the sample, from a laser light source
  • a flip mirror unit for reflecting the incident light provided and varying a reflection path with respect to one axis at a speed in a preset range, and positioning the flip mirror unit with respect to another axis perpendicular to the variable axis of the flip mirror unit.
  • An actuator unit configured to vary and synchronize the operation of the flip mirror unit at a slow speed, and a scan to control the flip mirror unit and the actuator unit so that incident light provided to the sample through the objective lens continuously scans at least a portion of the inspection region sequentially
  • the incident light is inspected by the control unit and the scan control unit
  • One or more sequential scans are provided by receiving a reflected light mixed with Raman signals generated by the plurality of types of search targets generated by sequentially scanning the inverse through a preset optical path, and outputting spectroscopic information about the reflected light through a camera. It includes a spectroscope for outputting continuous spectroscopic information on the interval during one camera operation period.
  • the search object may be an industrial product or a medical biomarker.
  • the actuator unit preferably includes a linear controllable universal drive structure including a small motor, piezo actuator, and ultrasonic actuator.
  • the flip mirror unit preferably includes a voice coil motor or a MEMS driver.
  • the scan control unit may further include a correction unit configured to move the stage on which the sample is seated in a vertical direction with respect to the surface of the objective lens, and to correct a change in the sample height according to the scan.
  • the spectrometer preferably includes a CCD having the highest quantum efficiency between 500 nanometers and 600 nanometers, and may be selected in different wavelength ranges according to the sample purpose.
  • the Raman assay device may be tabletop and integral.
  • the sample may be one square millimeter area or more in which a plurality of biomarkers are mixed with blood.
  • Raman analysis method for high-speed quantitative analysis of a light domain sample is a measurement method of the Raman analysis device for collectively collecting and analyzing a plurality of types of search target information present in the sample, And analyzing the continuous spectral information in the form of a Raman map including preset distribution and coefficient information to derive an analysis result.
  • the Raman analysis method is a step of performing a multi-quantitative diagnosis to simultaneously analyze the possibility of individual diseases for a plurality of diseases based on the information on the Raman map through the multi-dimensional multi-label analysis and measurement when the search target is a biomarker It may further include.
  • Raman analysis apparatus and method for quantitatively analyzing a wide-area sample unlike the conventional spot unit analysis, continuous spectral information for one or more sequential scan interval read-out during one camera operation period As a result, scan speed is improved while reducing noise generated by camera operation, thereby greatly improving the speed of multi-dimensional multi-marker analysis.
  • Raman analysis apparatus and method for quantitatively analyzing a wide-area sample in consideration of the characteristics of the continuous sequential scan method constitutes a single flip mirror unit and the entire flip mirror unit with respect to the other axis perpendicular to the variable one axis By moving, it is possible to reduce the size and cost of the axis drive without sacrificing the accuracy of analysis, thereby increasing the efficiency of the Raman analysis device configuration.
  • the Raman analysis apparatus and method for quantitatively analyzing a wide-area sample analyzes the continuous spectral information of a sequential scan interval with a Raman map including preset distribution and coefficient information to derive an analysis result. Optimized for speed and quantitative analysis rather than accurate location and absolute spectral information, it is very well suited for the interpretation of multiple disease diagnosis or analysis of multiple diagnostic reagents.
  • the Raman analysis apparatus and method for quantitatively analyzing a wide-area sample moves a stage on which a sample is seated through an auto focusing sensor to face an objective lens and corrects a change in sample height according to a scan. By minimizing etendue, Raman image analysis efficiency is further increased.
  • FIG. 1 is a block diagram of a conventional Raman analysis device.
  • Figure 2 is an illustration of a conventional Raman analysis method.
  • Figure 3 is a block diagram of a Raman analysis device for quantitative analysis of a plurality of biological markers in a sample according to an embodiment of the present invention.
  • Figure 4 is an illustration of a Raman analysis method for quantitatively analyzing a plurality of biological markers in a sample according to an embodiment of the present invention.
  • FIG. 5 is a view illustrating an operation of a flip mirror unit according to an embodiment of the present invention.
  • FIG. 6 is an exemplary view of a sample image scan according to an embodiment of the present invention.
  • FIG. 7 is an exemplary diagram of a beam scan pattern according to an embodiment of the present invention.
  • FIG. 8 is a flow chart of a Raman analysis method for quantitatively analyzing a plurality of biological markers in a sample according to an embodiment of the present invention.
  • FIG. 9 is a block diagram of a conventional Raman analysis device.
  • FIG. 10 is an exemplary view of a sample image and spectrum according to an embodiment of the present invention.
  • the Raman analysis apparatus for high-speed quantitative analysis of a wide-area sample targets a crack or a foreign substance present in a sample for a semiconductor wafer or specifies a micro sample such as a nanomaterial.
  • the present invention can be applied to the analysis of industrial products for the detection of substances, the analysis of medical biomarkers, etc.
  • the description of the present invention describes an embodiment for the analysis of biomarkers that are difficult to apply among these target samples and have a large sample size. Among them, a case where a plurality of biological markers present in a sample are used as an analysis target is described as an example. Raman analysis apparatus and method for the analysis of a number of biological markers in the sample can of course be utilized in the analysis of industrial products.
  • FIG. 3 is a block diagram of a Raman analysis device for quantitatively analyzing a plurality of biological markers in a sample according to an embodiment of the present invention.
  • the Raman analysis apparatus is a Raman analysis apparatus for collectively analyzing and analyzing a plurality of kinds of trace biomarker information present in the sample 21.
  • the Raman analysis apparatus reflects incident light provided from the laser light source 10 and is provided with respect to one axis.
  • the flip mirror unit 81 positions the flip mirror unit 81 to change the reflection path at a speed within a preset range, and the flip mirror unit 81 is positioned on the other axis perpendicular to the variable axis of the flip mirror unit 81.
  • the objective lens 20 is controlled by controlling the actuator unit 60, the flip mirror unit 81, and the actuator unit 60, which are synchronized with the operation of the flip mirror unit 81 at a slower speed than the variable speed.
  • Incident light sequentially scans the inspection area by the scan control units 65 and 75 and the scan control unit 65 and 75 so that the incident light provided to the sample 21 sequentially scans at least some regions of the inspection area.
  • Ha While receiving the reflected light mixed with the Raman signal generated by the plurality of biological markers generated through a predetermined optical path and outputs the spectroscopic information on the reflected light through the camera 40, at least one sequential scan interval set for each sample It includes a spectroscope 50 for outputting the continuous spectroscopic information for one camera operation period.
  • the flip mirror unit 81 is illustrated as a quadrangle including the flip mirror 80 for convenience of illustration and identification, the flip mirror 80 and the variable axis and the other axis or the flip mirror 80 are illustrated. And a drive configuration (70, 75) of the variable shaft and a drive configuration (60, 65) of the other shaft, etc. can be configured to be variable and easy to move.
  • the incident light from the laser light source 10 in the Raman analysis apparatus includes a spatial filter 11, one or more mirrors 12 and 14, and a dichroic mirror 13. Arrive at the flip-mirror part 81 via the optical path comprised by.
  • the Raman analysis apparatus moves the optical path in the variable axis direction at a high speed through the drive unit 70 of the variable axis of the flip mirror unit 81 disposed on the optical path, and the other axis direction is moved at a slower speed than this. .
  • the Raman analysis apparatus finely adjusts this operation through the scan controllers 65 and 75 so that the point where the light source incident through the objective lens 20 meets the sample 21 on the stage 29 has a continuous path. Can be configured.
  • Fine adjustment of the variable axis may be performed by a voice coil motor (70) or a MEMS (MEMS) driver such as an actuator and a driver for controlling the driving thereof. 75), high-speed fine adjustment is possible. This may use a commercial Galvo Mirror.
  • MEMS MEMS
  • the drive unit is a MEMS oscillation at a fixed frequency, it is difficult to adjust the absolute position, but it is suitable for the scan drive and can also integrate the mirror, thereby greatly reducing the size and cost.
  • the adjustment of the other axis may use a relatively inexpensive and easy linear control motor such as a small motor 60, a piezo actuator or an ultrasonic actuator, and a driver for controlling the driving thereof. 65) can be adjusted. This lowers costs and reduces control burden.
  • the light source incident through the objective lens 20 is scattered by striking the sample 21, most of which is scattered with the same energy as the incident light, but some are inelastic scattering by exchanging energy with the sample to a unique degree .
  • the wavelengths of Stokes scattering in which the sample 21 loses energy and Anti-Stokes scattering in which the sample obtains energy appear in a unique form according to the physical properties of the sample 21. 50 may collect (typically Stokes scatter) through an edge filter 28 and display it as a uniquely shaped spectrum.
  • continuous spectroscopic information on one or more sequential scan sections set for each sample 21 may be output during one camera 40 operation period and analyzed by the computer 30 to determine characteristics of the sample 21. .
  • the individual operation of the camera 40 refers to a series of processes of camera initialization (image memory clean, automatic exposure adjustment, white balance adjustment, etc.), light collection, and output format processing of the collected image according to the camera shutter operation. .
  • the spectroscope 50 includes Receives the reflected light mixed with the Raman signal by the biomarker through the preset optical path and outputs the spectral information of the reflected light through the camera 40, one or more sequential scan periods 22, 23 set for each sample 21 Continuous spectral information 24 is output during one camera operation period, and the analysis tool 35 analyzes the Raman map 37 including the distribution and coefficient information on the sample to derive the analysis result. do.
  • the conventional Raman analysis apparatus repeatedly acquires an image for each spot as described above, and analyzes it in correspondence with the Raman map including the information for each spot, it may be efficient in a sample sensitive to the location information for each spot and the resulting Raman information. It is not suitable for the Raman analysis method which enables the quantitative analysis of a plurality of trace biomarkers present in the above-mentioned wide range of samples in an early time.
  • the Raman throughout the sample rather than the accuracy of the Raman information according to the location of the sample. This is because the probabilistic distribution of information and the coefficient information used therein have a much greater influence on the precision of analysis.
  • the conventional Raman analysis apparatus measures the spot in units, the precision is lower than that of the continuous scan due to the noise generated during each operation by the spot-specific operation of the camera.
  • the conventional Raman analysis apparatus uses a so-called "line scan" scanning scheme or the above-mentioned multi-spotting scheme, which does not perform a sequential scan for a continuous region as in the present invention, but at least a portion of the region.
  • the discontinuous scanning method is performed on the A, thereby increasing the noise generated during shooting.
  • the Raman analysis apparatus reads continuous spectroscopic information on one or more sequential scan sections (whole area or a predetermined divided area) during one camera operation period differently from conventional spot unit analysis. -out improves the speed and quality of high-speed multi-dimensional multi-marker analysis by reducing scan noise while initializing the camera operation every time and improving scan speed.
  • the Raman analysis apparatus since the Raman analysis apparatus according to an embodiment of the present invention derives an analysis result by analyzing the continuous spectral information on the sequential scan interval with the Raman map 37 including distribution and coefficient information, the exact position of the sample 21. (22) and for the analysis of multiple disease diagnosis or analysis of multiple diagnostic reagents that require quantitative analysis rather than absolute spectroscopic information.
  • the Raman analysis apparatus according to an embodiment of the present invention, the movable range of the flip mirror unit 81 and the actuator unit (for example, the small motor 60, the piezo actuator, the ultrasonic actuator) for moving it to another axis Since they do not affect each other (the mirror part of the existing X and Y configuration is limited in consideration of the optical path of each moving range), it is possible to scan a large area sample so that it is easy to cope with samples larger than 1 square millimeter (mm). Can be.
  • the actuator unit for example, the small motor 60, the piezo actuator, the ultrasonic actuator
  • the actuator unit 60 may be configured as a general-purpose driving unit and a driver having a relatively inexpensive, easy linear control and proper control precision, such as a small motor, a piezo actuator, an ultrasonic actuator, and the like.
  • the scan controllers 65 and 75 move the stage 29 on which the sample 21 is seated in a vertical direction with respect to the surface of the objective lens 20, and correct the sample height change according to the scan ( Not shown) may be further included.
  • the scan controllers 65 and 75 may include an auto focus sensor to move the stage 29 on which the sample is placed to face the objective lens 20 to detect and correct a change in the sample height according to the scan. In this case, the etendue of the light source can be minimized, thereby further increasing the analysis efficiency of the Raman image.
  • the Raman analysis apparatus may be configured to further include not only Raman spectroscopy, but also additional configurations to analyze optical patterns such as fluorescent patterns to further improve the reliability of the results of multidimensional multiple analysis.
  • the spectroscope 50 considers a CCD (Charged Coupled Device) having the highest quantum efficiency (Quantum Efficiency) between 500 nanometers and 600 nanometers compared to other wavelength bands in the visible light region in consideration of the peak value of the Raman spectrum.
  • CCD Charged Device
  • quantum Efficiency Quantum Efficiency
  • different wavelength range can be used according to the conditions of a sample.
  • the Raman analysis apparatus is configured to drive and control the flip mirror 80 and the like compared to the case of using a conventional X, Y axis galvo mirror or adjusting the stage to the X, Y axis (60, 70, 65, 75) is very inexpensive and can be configured in a single configuration so that the size can be reduced and it can be configured to easily scan a large area compared to the case of adjusting the X and Y axes only by the mirror, and thus it is advantageous to miniaturize it into a table or an integrated type.
  • FIG 5 is a view illustrating an operation of the flip mirror unit 81 according to an embodiment of the present invention.
  • the scan portion of the illustrated Raman analysis apparatus quantitatively analyzes the continuous information reception instead of the spectral information reception in units of spots when generating the Raman map through the sample 21 to reduce noise and improve speed.
  • the inspection area can be extended while reducing the cost and size, allowing for high-speed quantitative analysis of a large number of trace biomarkers in a wide range of samples (21). It can be configured inexpensively.
  • the optical path 5 of the light source incident on the flip mirror unit 81 passes through the objective lens 20 through the rotational movement 71 of the variable axis of the flip-mirror 80 and the movement movement 61 of the other axis. 29 is set to continuously scan the entire inspection area sequentially on the sample 21.
  • the Raman analysis apparatus of the present invention configures the flip mirror units 80 and 81 as a single unit to reduce its volume, and the distribution information of the Raman information is more important due to the characteristics of multiple analysis. Even if the Y-axis 61 is slower than the X-axis 71 and the control precision is slightly lower than that of the X-axis 71, the same performance can be maintained for the characteristics and quantitative analysis of the continuous sequential scanning method of the present invention described above.
  • FIG. 6 is an exemplary view of a sample image scan according to an embodiment of the present invention.
  • the number of search targets exceeds the limit of one-dimensional labeling technology.
  • Labeling and searching operations in this multi-dimensional multiple labeling technique may label or search up to millions of candidate substances, so the reduction of the scan speed and noise in the sample image is very important.
  • the distribution or pattern of Raman information on the entire sample is more important in the analysis than the Raman information for each spot due to the characteristics of the multiple analysis.
  • the Raman analysis apparatus enables fast scan and precise pattern analysis of Raman information through a characteristic configuration that outputs continuous spectroscopic information on one or more sequential scan intervals during one camera operation period, thereby enabling multi-dimensional multiplexing.
  • the effectiveness is very high in the field of nano probes, diagnostic reagents and multiple diagnostics for performing marker analysis and measurement.
  • FIG. 10 is an exemplary view of a sample image and a spectrum according to an embodiment of the present invention.
  • the Raman analysis apparatus detects the label 1 in the sample by rapid scan through Raman analysis for multi-dimensional multi-marker analysis and measurement, so as to provide accurate Raman information. Pattern analysis is possible.
  • the Raman analysis apparatus detects the label 1 in the sample by rapid scan through Raman analysis for multi-dimensional multi-marker analysis and measurement, so as to provide accurate Raman information. Pattern analysis is possible.
  • the sample is about 0.5 mm * 0.5 mm sized sample, and the Raman map obtained by the point scan method while irradiating a laser light of 532 nm at 1.5 mW output is the image of Figure 10a.
  • the measurement time can be increased while maximizing the imaging function or minimizing the imaging function while sacrificing the measurement time according to the purpose while maintaining the high-efficiency signal collection capability of the confocal without missing a large-area sample. It can be minimized.
  • the scan controllers 65 and 75 of the Raman analysis apparatus are more preferable. Before performing the sequential scan, control to perform a pre-scan to quickly determine the position of the label (1) in the sample by a fluorescence and elastic scattered light scanning method rather than a Raman method and based on this signal generation unit By rescanning the bay, the S / N ratio can be maximized while reducing measurement time.
  • the Raman analysis apparatus creates a two-dimensional map (2D MAP) in a relatively less time-consuming manner rather than a Raman method to primarily identify only the position of the label 1 in the sample.
  • 2D MAP two-dimensional map
  • the Raman measurement is performed on the position 1 where the presence of the label is confirmed according to the pre-scan, and the other part 2 may be skipped.
  • Examples of such pre-scanning methods include a method of directly checking linear light using a laser, a method of obtaining an image with a confocal microscope, a method of obtaining a fluorescence image by applying fluorescence to a label, or a method of obtaining a 2D image through vision, etc. It is possible to apply various methods which are faster than Raman method.
  • the cover 2 is substantially unmarked or not covered by the pre-scan, it may be recognized as noise even if the actual Raman measurement is applied.
  • the application of the method allows for high-speed measurements without degrading the quality.
  • FIG. 7 is an exemplary view of a beam scan pattern according to an embodiment of the present invention.
  • the Raman analysis apparatus scans an image of a sample while moving one of the flip mirror units 81 by controlling one axis at a lower speed than the other axis.
  • this scan pattern may vary.
  • the Raman analysis apparatus may use a laser having a wavelength range of 500-550 nm optimized for the nano probes as the laser light source 10 when utilized in the analysis using the nano probes.
  • a near infrared ray laser may be further configured to utilize infrared spectroscopy in the Raman analyzer.
  • the Raman analysis apparatus may be configured to modularize each configuration shown in FIG. 3 so as not to require alignment between the modules, or to easily upgrade.
  • the Raman analysis device may be configured such that the edge filter 28 is suitable for Stokes scattering, or the laser light source 10 can be configured to replace the laser easily.
  • FIG. 8 is a flow chart of a Raman analysis method for quantitatively analyzing a plurality of biological markers in a sample according to an embodiment of the present invention.
  • the flip-mirror unit 81 of the Raman analysis apparatus reflects incident light provided from the laser light source 10 and varies the reflection path on one axis at a speed in a predetermined range.
  • the actuator unit 60 of the Raman analysis device is variable to the flip mirror unit 81 to position the flip mirror unit 81 on the other axis perpendicular to the variable axis of the flip mirror unit 81
  • the control unit 65 and 75 of the Raman analysis apparatus are configured to synchronize the flip mirror unit 81 with the operation of the flip mirror unit 81 at a slower speed than that of the flip mirror unit 81 and the actuator unit 60.
  • the scan control unit 65,75 While the incident light is sequentially scanned the Raman signal generated by the plurality of types of biomarkers generated by scanning the inspection area receives the reflected light through a predetermined optical path and outputs the spectroscopic information on the reflected light through the camera 40, And outputting continuous spectroscopic information on one or more sequential scan sections set for each sample during one camera operation period (S40).
  • the Raman analysis method is a Raman analysis method of a Raman analysis device for collectively collecting and analyzing a plurality of types of trace biomarker information present in a sample, wherein the Raman analysis method includes the Raman analysis including distribution and coefficient information. Analyzing in the form of a map to derive the analysis results.
  • the step of deriving the analysis result may be to search for a candidate group of diagnostic reagents based on the multi-dimensional labeling technology and derive the analysis result.
  • the Raman analysis method may further include performing a multi-quantitative diagnosis of simultaneously analyzing individual cases of multiple diseases through multi-dimensional multiple marker analysis and measurement based on the information on the Raman map.
  • the Raman analysis apparatus and method for analyzing a plurality of biomarkers present in a sample as an object of analysis are described as a target for searching for cracks or foreign substances present in a sample for a semiconductor wafer or nanomaterials. It can be used in the analysis of various industrial products, such as to search for a specific material of a fine sample, such as, etc., and is not limited to the apparatus and method for the analysis of the biomarker as in the embodiment, and the present invention Various modifications can be made by those skilled in the art without departing from the gist of the invention.

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Abstract

La présente invention concerne un procédé et un dispositif d'analyse Raman pour l'analyse quantitative à vitesse élevée d'un échantillon à aire étendue qui permettent l'analyse quantitative à vitesse élevée d'une pluralité de cibles de recherche existant dans un échantillon à aire étendue tout en réduisant la taille et le coût de l'équipement. Contrairement à l'analyse à base d'unité ponctuelle conventionnelle, la présente invention a des effets tels qu'une forte amélioration de la vitesse d'analyse de marqueur multiple multidimensionnel par lecture d'informations de spectre continu sur un ou plusieurs intervalles de balayage consécutifs pendant une période de fonctionnement de caméra, de manière à réduire le bruit créé par le fonctionnement de la caméra et l'augmentation de la vitesse de balayage.
PCT/KR2014/002290 2014-03-18 2014-03-18 Procédé et dispositif d'analyse raman pour analyse quantitative à vitesse élevée d'échantillon à aire étendue Ceased WO2015141873A1 (fr)

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CN117405649A (zh) * 2023-12-12 2024-01-16 中国科学院长春光学精密机械与物理研究所 细胞拉曼流式光谱成像分析系统及分析方法
CN117405649B (zh) * 2023-12-12 2024-03-22 中国科学院长春光学精密机械与物理研究所 细胞拉曼流式光谱成像分析系统及分析方法

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