KR101446210B1 - Fast and quantitative raman analysis method and apparatus thereof for large-area multiple bio-targets - Google Patents

Fast and quantitative raman analysis method and apparatus thereof for large-area multiple bio-targets Download PDF

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KR101446210B1
KR101446210B1 KR1020130016619A KR20130016619A KR101446210B1 KR 101446210 B1 KR101446210 B1 KR 101446210B1 KR 1020130016619 A KR1020130016619 A KR 1020130016619A KR 20130016619 A KR20130016619 A KR 20130016619A KR 101446210 B1 KR101446210 B1 KR 101446210B1
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sample
raman
analysis
unit
analyzing
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KR20140103000A (en
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정대홍
이윤식
이호영
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서울대학교산학협력단
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using infra-red, visible or ultra-violet light
    • G01N21/62Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65Raman scattering
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B81MICROSTRUCTURAL TECHNOLOGY
    • B81BMICROSTRUCTURAL DEVICES OR SYSTEMS, e.g. MICROMECHANICAL DEVICES
    • B81B7/00Microstructural systems; Auxiliary parts of microstructural devices or systems
    • B81B7/02Microstructural systems; Auxiliary parts of microstructural devices or systems containing distinct electrical or optical devices of particular relevance for their function, e.g. microelectro-mechanical systems [MEMS]
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01JMEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRA-RED, VISIBLE OR ULTRA-VIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/28Investigating the spectrum
    • G01J3/44Raman spectrometry; Scattering spectrometry ; Fluorescence spectrometry
    • G01J3/4412Scattering spectrometry
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using infra-red, visible or ultra-violet light
    • G01N21/62Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64Fluorescence; Phosphorescence
    • G01N21/6428Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"

Abstract

The present invention relates to a Raman analysis apparatus and method for quantitatively analyzing a plurality of biomarkers in a sample, and more particularly, to a method and apparatus for analyzing a plurality of biological markers in a sample, The scan speed is improved while reducing the noise generated by the camera operation, thereby greatly improving the speed of the multidimensional multi-marker analysis. In addition, a single flip mirror unit is constructed considering the characteristics of the continuous sequential scanning method, By moving the whole flip mirror part relative to the other axis perpendicular to the one axis, the size and cost of the corresponding axis driving part can be reduced without decreasing the accuracy of the analysis, thereby increasing the efficiency of the Raman analyzer and improving the efficiency of the continuous spectroscopy Map information to a Raman map containing predetermined distribution and coefficient information And it is optimized for velocity and quantitative analysis rather than exact position and absolute spectral information of the sample by deriving the analysis result. Therefore, it is very suitable for analysis of multiple disease diagnosis or analysis of multiple diagnosis reagents. The stage is moved to face the objective lens and the change in the sample height due to the scan is corrected to minimize the etendue of the light source, thereby further improving the analysis efficiency of the Raman image.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method and apparatus for rapid quantitative analysis of a plurality of biological markers in a wide-

The present invention relates to an apparatus and a method for analyzing a biological sample labeled with a Raman signal, and more particularly, to a method for quantitatively analyzing a plurality of biological markers in a sample, And more particularly, to a Raman analysis apparatus and method.

The Raman spectrometer is a device for analyzing the physical properties of a sample represented by Raman spectra using a spectrometer measuring Raman spectra. The apparatus is based on a light source and a spectrometer, which mainly use a laser to generate Raman spectra.

As the used laser, various wavelengths of laser ranging from ultraviolet to infrared wavelength are commonly used, and in the case of a biological sample, a laser having energy mainly in visible light and near infrared is used. Examples include the 514.5 nm line of argon laser, the 647 nm line of krypton laser, the 532 nm line, the 660 nm line and the 785 nm line of solid state lasers such as YAG.

As a spectrometer, it is necessary to sufficiently remove mysterious light such as Rayleigh scattering, and a method using two or three double monochromators has been used. In recent years, an interference filter having excellent optical characteristics has been used It has become common to use one monochromator.

FIG. 1 is a configuration diagram of a conventional Raman analyzer, and FIG. 2 is an illustration of a conventional Raman analysis method.

1, in the conventional Raman analyzer, a light source starting from a laser light source 10 is composed of a spatial filter 11, a mirror 12, and a dichroic mirror 13 The X-axis mirror 14 (X-axis mirror).

In the conventional Raman analysis apparatus, an X-axis mirror (14: X-axis mirror) and a Y-axis mirror (15: Y-axis mirror) moving in the X-axis and the Y-axis perpendicular thereto are arranged on the optical path, The path through which the light source is incident through the objective lens 20 and meets the sample 21 on the stage 29 can be moved finely by spot.

The X-axis mirror 14 and the Y-axis mirror 15 can be finely adjusted through voice coil motors 16 and 17 and drivers 18 and 19 for controlling the driving thereof.

The light source incident through the objective lens 20 collides with the sample 21 to be scattered. In most cases, the light is scattered with the same energy as the incident light, but some of the light is inherently scattered by the energy of the sample 21 .

Stokes scattering in which the light source loses energy and anti-Stokes scattering in which the light source obtains energy are shown in a unique form according to the physical properties of the sample 21, and the spectrometer 50 has an edge It can be collected (generally by Stokes scattering) through a filter (28: Edge Filter) and displayed as a spectrum of a unique form.

In the conventional Raman analysis apparatus, the spectral image for each spot is repeatedly photographed by the camera 40 and then analyzed by the computer 30 to grasp the characteristics of the sample 21.

The above-described configuration is advantageous in that a configuration in which the X-axis mirror 14 and the Y-axis mirror 15 are separated from each other and then finely adjusted (usually referred to as a galvo mirror) There is a problem in that the quality of the area away from the center is deteriorated due to optical distortion when the measurement range is widened. Since the laser beam should be commonly reflected to the X-axis mirror 14 and the Y-axis mirror 15, the range of the sample that can be measured by the limit of the reflective physical reflection range is limited. Generally, It does not correspond to the area required for the sample.

Of course, there are cases where the stage is moved instead of the mirror, or both of them are supported, but both the precision and the speed are low, the cost is high, and the size is large.

FIG. 2 schematically shows a method of collecting and analyzing information of the Raman analyzer shown in FIG. 1, wherein the Raman analyzer fine-tunes the X-axis mirror 14 and the Y-axis mirror 15 for each spot 22 The analysis tool 35 moves the Raman information acquired by the spectrometer 50 to the spots 22 by moving the optical path 25 and displays the Raman map 36 Respectively.

The Raman analyzer shown in FIG. 1 is used for crack inspection of semiconductor wafers, foreign matter inspection, and analysis of fine samples. Since the scale is large and the cost is high, it is mainly used for industrial equipment and research / academic use. However, it is bulky, expensive and difficult to be used for multi-biomarker analysis because of the limited area of the sample to be measured.

Currently, "JY-Horiba" (Horiba Jobin Yvon) and "Renishaw" are the most widely known manufacturers of Raman analyzers. In the Raman analyzer of the above-mentioned famous manufacturer, it is possible to perform scan inspection for a certain area, but it is difficult to inspect the sample in a sufficient area for analyzing many biomarkers because the inspection area is limited, It is not easy to obtain, and since it is constituted by an expensive driving part, it is difficult to spread it for biomarker analysis because of high cost.

9 is a block diagram of a Raman analyzer of "JY-Horiba " disclosed in European Laid-Open Publication No. EP 1983332A1.

9, since the X-axis mirror and the Y-axis mirrors 14a and 14b are separated from each other, the scan range is narrow and the cost of the driving unit for precise driving is increased, it's difficult.

On the other hand, in the case of "Renishaw", a technique of improving the scanning speed and accuracy by a so-called "line scan" through PCT publication WO 2009 / 093050A1 is disclosed. However, , And the stage to which the sample is applied moves in the X and Y axes. By taking such a configuration, it is possible to measure a sample of a wider area, but such a structure is sensitive to the external environment in that the stage must operate precisely in conjunction with the optical measuring unit, has a high cost for driving the stage, It is difficult to disseminate it for the purpose of biomarker analysis.

On the other hand, conventional diagnosis of diseases using a single biomarker requires a large amount of samples and the diagnostic rate is very slow, which increases diagnosis cost and makes diagnosis of a large number difficult. Further, in order to search for the candidate group of the diagnostic reagent, a large number of candidate substances must be inspected. In the case of the single substance test, a long search time is required to search the candidate substance.

Recently, nano-spectroscopy has been used to search for diagnostic reagents using multiple markers and to diagnose diseases. Recently, the most popular technique is to acquire independent signals for multiple biomarkers using multiple probe probes, and to simultaneously detect multiple targets by analyzing them at a time. Are reported.

The read-out method of the signal applied to the multiple diagnosis is not a multi spotting technique in which a single sample is divided into a plurality of spots to increase the amount of the sample according to the inspection item, Or nanoprobes that increase the sensitivity of signals by fabricating probes using silver nanoparticles. It is possible to simultaneously label large amounts of different materials through multi-dimensional marking technology.

The most common nanoprobe technique recently applied to this multi-marker diagnosis is fluorescence-based multi-measurement technique such as Fluorescence-Assisted Cell Sorter (FACS). Since this method is based on fluorescence, Due to its wide breadth, the number of multi-diagnostic markers is limited and can not be used in the field of labeling or searching for millions of candidate substances.

Despite the limitations of the conventional Raman measurement apparatus and the limitations of the multi-marker analysis method, as the level of medical service is increased, the demand for the analysis of multiple biomarkers is increasing. Therefore, A new technology with high sensitivity and high accuracy of signals is required, which enables a low-cost Raman analyzer capable of analyzing to be supplied to general hospitals and a small size and quick measurement of the table top.

On the other hand, the conventional Raman analyzer analyzes repeatedly obtained spot images or line images of a predetermined size in correspondence with the Raman map, and thus can be effective in the samples sensitive to the positional information and the Raman information. However, There are limitations in the case of quantitatively analyzing a large number of trace biomarkers present in a wide range of samples for analysis quickly and precisely.

In addition, since the conventional Raman analyzing apparatus acquires images of each measurement position individually, it is necessary to add a structure for reducing the noise due to noise generated during the image acquisition operation of the camera, so that the accuracy is low and the scan speed is slow, .

In particular, in the field of simultaneous diagnosis of multiple diseases by searching a large amount of different materials at high speed through multi-dimensional marking technology or searching for a suitable reagent in a candidate group of diagnostic reagents, Raman information in the entire sample Since the probabilistic distribution of information and the coefficient information used in it have a much greater impact on the accuracy of the analysis, it is important to obtain continuous results through high-speed measurements in a uniform measurement environment.

As a result, it has not yet been possible to provide an appropriate level of Raman analyzer for medical biomarker analysis, and since the present camera shooting method and analysis method are not optimized for biomarker analysis, This is a technically and economically difficult situation.

Korea Patent Publication No. 2011-7019504 European Publication No. EP1983332A1 PCT Publication No. WO 2009 / 093050A1

An object of the present invention to overcome the above-mentioned problems is to provide a method and apparatus for detecting consecutive spectral information for at least one sequential scan period by reading out during one camera operation period, And to provide a Raman analysis apparatus and method for improving scan speed while reducing noise.

Another object of the present invention to solve the above problems is to provide a single flip mirror unit in consideration of the characteristics of a continuous sequential scanning method and move the entire flip mirror unit relative to the other axis perpendicular to the variable single axis, The present invention provides a Raman analysis apparatus and method for reducing the size and cost of a corresponding axis driving unit without deteriorating the accuracy of the Raman analysis.

It is still another object of the present invention to improve the above-described problems by providing an analysis result of Raman map including consecutive spectral information for a sequential scan section with predetermined distribution and coefficient information, And to provide a Raman analyzer and method that are optimized for speed and quantitative analysis.

Another object of the present invention is to minimize the etendue of the light source by correcting the change in the sample height according to the scan by moving the stage facing the objective lens with the objective lens by the automatic focus adjustment sensor And to provide a Raman analysis apparatus and method.

According to an aspect of the present invention, there is provided a Raman analyzer for collecting and analyzing a plurality of kinds of trace bio-marker information in a sample, A flip mirror part for reflecting the incident light provided from the light source and varying a reflection path for one axis at a predetermined range of speeds, a position of the flip mirror part relative to the other axis perpendicular to the variable axis of the flip mirror part, And controlling 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 part of the inspection area in a sequential manner And a scan control unit And the reflected light of the Raman signals generated by the plurality of kinds of biomarkers generated while sequentially scanning the inspection region is input through a predetermined optical path and the spectroscopic information of the reflected light is output through the camera, And a spectroscope unit for outputting the continuous spectroscopic information for the sequential scan period during one camera operation period.

Preferably, the actuator unit includes a linearly controllable universal driver structure including a miniature motor, a piezo actuator, and an ultrasonic actuator.

The flip mirror unit may include a voice coil motor and a MEMS driving unit.

The scan control unit may further include a correction unit that moves the stage for placing the sample on the opposite surface of the objective lens in the vertical direction and corrects a change in the sample height according to the scan.

The spectroscopic unit preferably includes a CCD having a highest quantum efficiency between 500 nm and 600 nm, and can be selected in different wavelength ranges according to the purpose of the sample.

The Raman analyzer may be tabletop and integral.

On the other hand, as an example, the sample may be more than 1 square millimeter area in which plural kinds of biomarkers are mixed with blood.

According to another aspect of the present invention, there is provided a method for quantitatively analyzing a plurality of biomarkers in a sample, comprising the steps of: collecting and analyzing a plurality of kinds of trace bio- And analyzing the continuous spectral information in the form of a Raman map including predetermined distribution and coefficient information to derive an analysis result.

Here, the Raman analysis method may further include a step of performing multiple quantitative diagnosis simultaneously analyzing the possibility of individual onset of multiple diseases based on the information on the Raman map through multidimensional multiple marker analysis and measurement.

The Raman analysis apparatus and method for quantitatively analyzing a plurality of biomarkers in a sample according to an embodiment of the present invention differs from the conventional analysis of spot units in that continuous spectral information for one or more sequential scan intervals is read -out to improve the scanning speed while reducing the noise generated by the camera operation, thereby greatly improving the speed of multidimensional multi-marker analysis.

A Raman analysis apparatus and method for quantifying a plurality of biomarkers in a sample according to an embodiment of the present invention comprises a single flip mirror unit in consideration of the characteristics of a continuous sequential scanning method, It is possible to reduce the size and cost of the shaft driving unit without decreasing the precision of the analysis by moving the whole, thereby improving the efficiency of the Raman analysis apparatus.

The Raman analysis apparatus and method for quantitatively analyzing a plurality of biomarkers in a sample according to an embodiment of the present invention includes analyzing continuous spectral information for a sequential scan section into a Raman map including predetermined distribution and coefficient information, It is optimized for speed and quantitative analysis rather than accurate position and absolute spectral information of the sample, so that it is very suitable for analysis of multiple disease diagnosis or analysis of multiple diagnosis reagent.

The Raman analysis apparatus and method for quantitatively analyzing a plurality of biomarkers in a sample according to an embodiment of the present invention include a stage for placing a sample through an auto-focus adjustment sensor, facing the objective lens, And the etendue of the light source is minimized to further enhance the analysis efficiency of the Raman image.

1 is a block diagram of a conventional Raman analyzer.
2 is an exemplary diagram of a conventional Raman analysis method;
FIG. 3 is a view showing a configuration of a Raman analyzer for quantitatively analyzing a plurality of biomarkers in a sample according to an embodiment of the present invention. FIG.
4 is a diagram illustrating an example of a Raman analysis method for quantitatively analyzing a plurality of biomarkers in a sample according to an embodiment of the present invention.
5 is a view illustrating an operation example 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 illustration of a beam scan pattern according to an embodiment of the present invention;
8 is a flowchart of a Raman analysis method for quantitatively analyzing a plurality of biomarkers in a sample according to an embodiment of the present invention.
9 is a configuration diagram of a conventional Raman analyzer.
10 is an exemplary view of a sample image and spectrum according to an embodiment of the present invention;

BRIEF DESCRIPTION OF THE DRAWINGS The above and other objects, features and advantages of the invention will become more apparent from the following detailed description of the present invention when taken in conjunction with the accompanying drawings.

3 is a block diagram of a Raman analyzer for quantitatively analyzing a plurality of biomarkers in a sample according to an embodiment of the present invention.

3, the Raman analyzer is a Raman analyzer collecting and analyzing a plurality of kinds of trace biomarker marker information existing in the sample 21, and reflects the incident light provided from the laser light source 10, A flip mirror section 81 for changing the reflection path at a predetermined range of speeds and a flip mirror section 81 for positioning the flip mirror section 81 with respect to the other axis perpendicular to the variable axis of the flip mirror section 81 An actuator section 60 for controlling the flip mirror section 81 and the actuator section 60 so as to change the speed of the objective lens 20 in synchronism with the operation of the flip mirror section 81 at a speed lower than the variable speed, Scan control units 65 and 75 for continuously scanning at least a part of the inspection area with incident light provided to the sample 21 and a scan control unit 65 and 75 for sequentially scanning the inspection area Ha And outputs the spectroscopic information of the reflected light through the camera 40. The spectroscopic information of the reflected light is output through the at least one sequential scan interval set for each sample, And outputs the continuous spectroscopic information for one camera operation period.

Although the flip mirror unit 81 is shown as a square including the flip mirror 80 for the convenience of illustration and identification, the flip mirror 80 and the variable shaft and the other shaft or the flip mirror 80 are not shown. And the variable axis driving structures 70 and 75 and the other axis driving structures 60 and 65, so that the variable axis and the moving axis can be configured to be convenient.

In a preferred embodiment of the present invention, incident light originating from the laser light source 10 in the Raman analyzer is incident on a spatial filter 11, one or more mirrors 12 and 14, and a dichroic mirror 13, And arrives at the flip mirror portion 81 via the optical path constituted by the light beam.

The Raman analyzer moves the optical path in the variable axis direction at a high speed through the driving unit 70 of the variable axis of the flip mirror unit 81 disposed on the optical path and moves the other axis direction at a slower speed .

In other words, high-speed driving of one axis is required for the scanning operation, but the driving speed of the other axis may be relatively slow.

The Raman analysis apparatus fine-adjusts such an operation through the scan control units 65 and 75 so that a 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.

The fine adjustment of the variable axis (for example, the X axis) may be performed by using a voice coil motor (70) such as an actuator, a MEMS (Micro Electro Mechanical Systems) 75) can be used for high-speed fine adjustment. It can use the commercial Galvo Mirror.

In this case, when the driving part is a MEMS, since it vibrates at a fixed frequency, it is difficult to adjust the absolute position, but it is suitable for the scan driving and the mirror can be integrated, and the size and cost can be greatly reduced.

However, adjustment of the other axis (e.g., the Y axis) can be performed using a relatively inexpensive and linearly controllable motor such as a small motor 60, a piezo actuator or an ultrasonic actuator, and a driver 65). This can lower costs and reduce control overhead.

On the other hand, a light source incident through the objective lens 20 collides with the sample 21 to scatter, and most of the light is scattered with energy such as incident light, but some of the light is inherently scattered with energy to the sample .

At this time, the Stokes scattering in which the sample 21 loses energy according to the physical property of the sample 21 and the anti-Stokes scattering spectrum in which the sample obtains energy appear in a unique form, (Generally, Stokes scattering) through the edge filter 28 (Edge Filter) to display it in a spectrum of a unique form.

In the Raman analyzer, the continuous spectral information for one or more sequential scan intervals set for each sample 21 is output during one operation period of the camera 40 and analyzed by the computer 30 to determine the characteristics of the sample 21 .

Here, the individual operation of the camera 40 means a series of processes of camera initialization (memory cleaning of the image pickup unit memory, automatic exposure adjustment, white balance adjustment, etc.), and light collection according to the camera shutter operation and output format processing of the collected image .

FIG. 4 is a diagram illustrating a Raman analysis method for quantitatively analyzing a plurality of biomarkers in a sample according to an embodiment of the present invention. In the Raman analysis apparatus according to an embodiment of the present invention, And the spectral information of the reflected light is output through the camera 40. The at least one sequential scan interval 22 or 23 set for each sample 21 ) Is output during one camera operation period, and the analysis tool 35 analyzes the Raman map (37: Raman map) including the distribution and the coefficient information on the sample to derive the analysis result do.

The conventional Raman analyzer analyzes the spot image by repeatedly acquiring the image for each spot as described above and analyzes it in correspondence with the Raman map including the spot-specific information, so that it can be efficient in the sample sensitive to the spot information and the Raman information, It is not suitable for the Raman analysis method that enables quantitative analysis of a large number of trace biomarkers existing in the above-mentioned wide sample within an early time.

In particular, in the field of simultaneous diagnosis of multiple diseases by searching a large amount of different materials at high speed through multi-dimensional marking technology or searching for a suitable reagent in a candidate group of diagnostic reagents, Raman information in the entire sample The probabilistic distribution of information and the coefficient information used for it have a much greater impact on the accuracy of the analysis.

In addition, since it is necessary to measure at least one square millimeter (mm) large area sample to which blood is applied to multiple biomarkers, it is required to enlarge the inspection area and to improve the speed.

In addition, since the conventional Raman analyzer measures the spot as a unit, the precision of the conventional Raman analyzer is lower than that of the continuous scan due to the noise generated in each operation by the operation of each spot of the camera.

In addition, the conventional Raman analyzer uses a scanning method called "line scan" or uses the above-described multispotting method. However, these methods do not perform sequential scanning for a continuous area as in the present invention, The noise generated during the photographing becomes large.

On the other hand, the Raman analysis apparatus according to the embodiment of the present invention differs from the conventional analysis of spots in that continuous spectral information on one or more sequential scan periods (entire region or predetermined region, etc.) -out, it improves the speed and quality of the fast multidimensional multi-marker analysis because it improves the scanning speed by decreasing the noise generated at the initialization by the camera operation every once.

In addition, since the Raman analysis apparatus according to the embodiment of the present invention analyzes the continuous spectral information of the sequential scan section by the Raman map 37 including the distribution and coefficient information to obtain the analysis result, (22) and analysis of multiple disease diagnoses requiring quantitative analysis, rather than spectral information of absolute location, or for the analysis of multiple diagnostic reagents.

In addition, the Raman analysis apparatus according to the embodiment of the present invention may include a movable range of the flip mirror unit 81 and an operation of the actuator unit (for example, a small motor 60, a piezo actuator, and an ultrasonic actuator) (The mirror part of the existing X and Y structure is limited in consideration of the optical path), and it is possible to scan a large area sample, so that it is possible to easily cope with a sample of 1 square millimeter (mm) or more .

Referring to FIG. 3 again, the actuator unit 60 may be constructed of a general-purpose driving unit and a driver, which are relatively inexpensive and linearly controllable, and have appropriate control precision, such as a small-sized motor, a piezo actuator, and an ultrasonic actuator.

The scan control units 65 and 75 include a correction unit for moving the stage 29 on which the sample 21 is placed in the vertical direction with respect to the facing surface of the objective lens 20, (Not shown).

The scan control units 65 and 75 include an auto-focus adjusting sensor, and move the stage 29 to seat the sample against the objective lens 20 to detect a change in the sample height according to the scan, In this case, the etendue of the light source can be minimized and the analysis efficiency of the Raman image can be further increased.

In addition, the Raman analyzer may be configured to further include not only Raman spectroscopy but also additional components so as to analyze the optical pattern such as a fluorescent pattern, thereby further improving the reliability of the result of the multidimensional multiple analysis.

In consideration of the peak value of the Raman spectrum, the spectroscopic unit 50 may include a CCD (Charged Coupled Device) having a maximum quantum efficiency between 500 nanometers and 600 nanometers, as compared with other wavelength bands in the visible light region. But it is possible to use a different wavelength region depending on the conditions of the sample.

In addition, the Raman analyzer has a flip mirror 80 and a configuration (60, 70, 65, and 65) for driving and controlling the flip mirror 80 as compared with a conventional method using the X, 75) is very inexpensive and can be reduced in size by a single structure, and can be configured to easily scan a large area as compared with a case where only the mirror and the X and Y axes are adjusted.

5 is an operational example of the flip mirror unit 81 according to an embodiment of the present invention.

The scan part of the illustrated Raman analyzer is configured to quantitatively analyze continuous information by receiving continuous information instead of receiving spectral information in the spot (spot) unit when generating the Raman map through the scan of the sample 21, thereby reducing the noise and improving the speed And a scanner structure optimized for this operation method is applied to expand the inspection area while reducing the cost and size so that high quantitative analysis can be performed on a large number of trace biomarkers existing in the wide sample 21, And can be configured at low cost.

The light path 5 of the light source incident on the flip mirror unit 81 passes through the objective lens 20 through the rotary motion 71 of the variable axis of the flip mirror 80 and the movement 61 of the other axis, 29 on the sample 21 in a continuous scanning manner.

In particular, as shown in FIG. 5, the Raman analyzer according to the present invention includes a single flip mirror unit 80 and 81 to reduce the volume thereof, and the distribution information of Raman information is relatively more important The same performance can be maintained for the characteristics and quantitative analysis of the continuous sequential scanning method according to the present invention even if the Y axis 61 is slower and the control accuracy is somewhat lower than the X axis 71. [

6 is an illustration of a sample image scan according to an embodiment of the present invention.

Referring to FIG. 6, when the sample is scanned, the Raman analyzer moves one axis at high speed and the other axis at low speed relative to the one axis, thereby scanning (23) the sample.

In particular, multidimensional multi-labeling technology should be utilized because the number of objects to be searched exceeds the limit of one-dimensional marking technology in order to search for candidates of diagnostic reagents using nano spectroscopy and to diagnose multiple diseases.

Since labeling and searching operations in this multi-dimensional multi-labeling technique can mark or search up to millions of candidates, it is very important to reduce the scan speed and the noise in the sample image.

Also, as described above, the distribution or pattern of the Raman information on the entire sample is more important in the analysis than the Raman information per spot because of the characteristics of the multiple analysis.

The Raman analysis apparatus according to an embodiment of the present invention can perform fast scanning and pattern analysis of precise Raman information through a characteristic configuration in which continuous spectral information for one or more sequential scan periods is output during one camera operation period, It is very effective in the fields of nanoprobes, diagnostic reagents and multiple diagnostics that perform marker analysis and measurement.

In addition, it is possible to measure or search for a multi-channel signal for complex analysis of fluorescence, graphic, and optical patterns as well as Raman spectroscopic signal patterns for multi-dimensional multi-markers.

In addition to the above-described scanning method, a scanning method having a predetermined angle (for example, an interlaced scanning or a sequential scanning method of a CRT TV) or a method of dividing an area and sequentially scanning the areas may be used , All of which are within the scope of the present invention.

10 is an illustration of a sample image and spectrum according to an embodiment of the present invention.

As a preferred embodiment, the Raman analysis apparatus according to an embodiment of the present invention detects a label 1 in a sample by a quick scan through Raman analysis for multi-dimensional multi-marker analysis and measurement, as described above, Pattern analysis is possible.

As a preferred embodiment, the Raman analysis apparatus according to an embodiment of the present invention detects a label 1 in a sample by a quick scan through Raman analysis for multi-dimensional multi-marker analysis and measurement, as described above, Pattern analysis is possible.

This sample is a sample of 0.5 mm * 0.5 mm in size, and the Raman map obtained by the point scan method is obtained while irradiating a laser beam of 532 nm with an output of 1.5 mW.

In this case, it took about 21 minutes to measure a large area sample. However, it took only 30 seconds to 2 minutes to remove the image processing function and to measure a large area sample without missing place, and the measurement result was obtained as shown in FIG. 10B.

That is, by using the method proposed in the present invention, it is possible to maximize the imaging function while minimizing the imaging function while maintaining the high-efficiency signal collecting ability of the confocal point without missing the large-sized sample, Can be minimized.

Since the Raman signal is a very fine signal, a considerably long exposure time is required for accurate determination. Therefore, as a more preferable embodiment, the scan control units 65 and 75 of the Raman analysis apparatus according to the embodiment of the present invention Scan to perform a pre-scan for quickly checking the position of the label 1 in the sample using the fluorescence and elastic scattering light scanning method which is not a Raman method before the sequential scanning, It is possible to maximize the S / N ratio while shortening the measurement time.

For example, the Raman analysis apparatus according to an exemplary embodiment of the present invention creates a two-dimensional map (2D map) in a manner that takes relatively little time, rather than a Raman scheme, in order to primarily check only the position of the label 1 in the sample. , The Raman measurement is performed on the position (1) where the presence of the mark is confirmed according to the pre-scan, and the other part (2) is skipped.

As an example of such a pre-scan method, there is a method of directly confirming linear light using a laser, a method of obtaining an image by a confocal microscope, a method of obtaining a fluorescent image by applying fluorescence to a label, or a method of obtaining a 2D image through vision It is possible to apply various methods that are faster than the Raman method.

The Raman analysis apparatus according to an embodiment of the present invention can detect the presence of the above-mentioned pre-scan (2) because it can be recognized as a noise even if an actual Raman measurement is applied, The high-speed measurement can be performed without deteriorating the quality.

7 is an illustration of a beam scan pattern according to an embodiment of the present invention.

As described above, the Raman analysis apparatus scans an image of a sample while controlling one axis at a slower speed than the other axis to move the flip mirror unit 81. Of course, these scan patterns may be different.

As a preferred embodiment, when the Raman analyzer is used for analysis using a nano-probe, a laser having a wavelength range of 500-550 nm optimized for the nano-probe may be used as the laser light source 10.

For the in vivo sample, a near infrared ray laser may be further configured to utilize the infrared spectroscopy in the Raman analyzer.

In addition, the Raman analyzer may be configured such that each configuration shown in FIG. 3 is modularized, for example, so that alignment between the modules is unnecessary, or that upgrading is easy.

More preferably, the Raman analyzer may be configured so that the edge filter 28 is suitable for Stokes scattering, or the laser light source 10 can be replaced to facilitate laser exchange.

FIG. 8 is a flowchart of a Raman analysis method for quantitatively analyzing a plurality of biomarkers in a sample according to an embodiment of the present invention.

Referring to FIG. 8, the Raman analysis method includes the steps of reflecting the incident light provided from the laser light source 10 by the flip mirror unit 81 of the Raman analyzer and varying the reflection path for the uniaxial speed to a predetermined range The actuator unit 60 of the Raman analyzer moves the position of the flip mirror unit 81 to the variable position of the flip mirror unit 81 based on the other axis perpendicular to the variable axis of the flip mirror unit 81, (S20) of synchronizing the operation of the flip mirror unit 81 with the operation of the flip mirror unit 81 at a speed slower than the speed of the flip mirror unit 81. The scan control units 65 and 75 of the Raman analysis apparatus are connected to the flip mirror unit 81 and the actuator unit 60, (S30) of continuously scanning at least a part of the inspection region of the incident light provided to the sample (21) through the objective lens (20) continuously, and a step (S30) of the Raman analysis device By the scan control units 65 and 75 A light source for receiving reflected light obtained by mixing the Raman signals generated by the plurality of kinds of biomarkers generated while incident light is scanned in the inspection region sequentially through a predetermined optical path and outputting spectral information on the reflected light through the camera, And outputting continuous spectral information for at least one sequential scan interval set for each sample during a single camera operation period (S40).

In another preferred embodiment, the Raman analysis method is a Raman analysis method of a Raman analysis apparatus collectively analyzing a plurality of kinds of trace biomarker marker information present in a sample, wherein the continuous spectral information is distributed to Raman And analyzing the result in the form of a map to derive an analysis result.

At this time, the step of deriving the analysis result may be to search the candidate group of the diagnostic reagent based on the multidimensional labeling technique and to derive the analysis result.

Alternatively, the Raman analysis method may further include a step of performing multiple quantitative diagnoses simultaneously analyzing the possibility of individual onset of multiple diseases through multidimensional multi-marker analysis and measurement based on the information on the Raman map.

The foregoing embodiments and advantages are merely exemplary and are not to be construed as limiting the present invention. However, the present invention is not limited to the above-described embodiments, and various changes and modifications may be made by those skilled in the art without departing from the scope of the present invention. .

5: optical path 10: laser light source
11: Spatial Filter 12: Mirror
13: Dichroic Mirror 14: X-axis Mirror 14:
15: Y-axis mirror
16,17: Voice coil motor (Voice Coil Motor)
18, 19: driver 20: objective lens
21: Sample
22: Spot 23: Continuous scan path
24: Continuous spectroscopic information 28: Edge filter
29: stage 30: computer
35: Analysis Tool
36: Raman Map containing spot-specific information
36: Raman Map containing distribution and coefficient information
40: camera 50: spectroscope (spectrometer)
60: actuator unit 65, 75: scan control unit
70: Voice coil motor 80: Flip mirror
81: Flip mirror part

Claims (9)

  1. A Raman analyzer for collectively analyzing a plurality of kinds of trace biomarker markers present in a sample,
    A flip mirror part for reflecting incident light provided from a laser light source and varying a reflection path for one axis at a predetermined range of speed;
    An actuator unit for changing the position of the flip mirror unit in synchronization with the operation of the flip mirror unit at a speed lower than the variable speed of the flip mirror unit with respect to the other axis perpendicular to the variable axis of the flip mirror unit;
    A scan control unit for controlling the flip mirror unit and the actuator unit so that the incident light provided to the sample through the objective lens sequentially and continuously scans at least a part of the inspection area;
    The scan control unit receives the reflected light mixed with the Raman signals generated by the plurality of kinds of biomarkers generated while sequentially scanning the inspection region by the scan control unit through the predetermined optical path and outputs the spectral information about the reflected light through the camera And a spectroscopic unit for outputting consecutive spectroscopic information for at least one sequential scan interval set for each sample during one camera operation period. The apparatus for analyzing a plurality of bio-markers in a sample at a high speed, Analysis device.
  2. 2. The apparatus according to claim 1, wherein the actuator section
    Characterized in that it comprises a linear motor, a linear motor, a linear motor, a linear motor, a linear motor, a linear motor, a linear motor,
  3. The apparatus of claim 1, wherein the flip mirror section
    Wherein the biological analyzer comprises a voice coil motor and a MEMS driving unit.
  4. The apparatus of claim 1, wherein the scan control unit
    Further comprising a correcting unit which moves the stage for placing the sample on the opposite surface of the objective lens in the up and down direction and corrects a change in the sample height according to the scan, .
  5. The apparatus of claim 1, wherein the spectroscope
    A Raman analyzer for quantitatively analyzing a plurality of biomarkers in a sample including a CCD having the highest quantum efficiency between 500 nanometers and 600 nanometers.
  6. The method according to claim 1,
    Wherein the flip mirror unit, the actuator unit, and the scan control unit are a tabletop or an integral unit having a single structure.
  7. The method according to claim 1, wherein the sample
    Wherein the sample area is at least 1 square millimeter.
  8. A method for analyzing a plurality of biological markers in a sample using the Raman analyzer according to any one of claims 1 to 7 at a high-speed quantitative analysis,
    a) analyzing the continuous spectral information in the form of a Raman map including predetermined distribution and coefficient information to derive an analysis result; and quantitatively analyzing a plurality of biomarkers in the sample.
  9. 9. The method of claim 8,
    And b) performing multiple quantitative diagnosis simultaneously analyzing the possibility of individual occurrence of multiple diseases based on the information on the Raman map through multidimensional multi-marker analysis and measurement. Analysis method.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102060280B1 (en) 2019-07-19 2019-12-27 주식회사 라온즈 Complex point-of care test device and network analysis system comprising the complex point-of care test device

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101843541B1 (en) * 2017-11-22 2018-03-30 대한민국 Multifunctional Raman inspection system for research of cultural property

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008116432A (en) 2006-07-06 2008-05-22 Ricoh Co Ltd Raman spectrometric measuring instrument, and raman spectrometry using same
KR20100002742A (en) * 2008-06-30 2010-01-07 서강대학교산학협력단 Method for imaging raman spectroscopy of biologic tissues and cells and an apparatus using the same
JP2010101757A (en) 2008-10-23 2010-05-06 Itt Manufacturing Enterprises Inc System and method for remote unmanned raman spectroscopy
JP2011149822A (en) 2010-01-21 2011-08-04 Sony Corp Optical measuring device and optical measuring method

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008116432A (en) 2006-07-06 2008-05-22 Ricoh Co Ltd Raman spectrometric measuring instrument, and raman spectrometry using same
KR20100002742A (en) * 2008-06-30 2010-01-07 서강대학교산학협력단 Method for imaging raman spectroscopy of biologic tissues and cells and an apparatus using the same
JP2010101757A (en) 2008-10-23 2010-05-06 Itt Manufacturing Enterprises Inc System and method for remote unmanned raman spectroscopy
JP2011149822A (en) 2010-01-21 2011-08-04 Sony Corp Optical measuring device and optical measuring method

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102060280B1 (en) 2019-07-19 2019-12-27 주식회사 라온즈 Complex point-of care test device and network analysis system comprising the complex point-of care test device

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