WO2015141873A1

WO2015141873A1 - Raman analysis method and device for high-speed quantitative analysis of wide-area sample

Info

Publication number: WO2015141873A1
Application number: PCT/KR2014/002290
Authority: WO
Inventors: 정대홍; 이윤식; 이호영
Original assignee: 서울대학교산학협력단
Priority date: 2014-03-18
Filing date: 2014-03-18
Publication date: 2015-09-24

Abstract

The present invention relates to a Raman analysis method and device for high-speed quantitative analysis of a wide-area sample which enable high-speed quantitative analysis of a plurality of search targets existing in a wide-area sample while reducing the size and cost of equipment. Unlike the conventional spot-unit-based analysis, the present invention has such effects as highly improving the speed of multidimensional multiple marker analysis by reading-out continuous spectrum information on one or more consecutive scanning intervals during one camera operation period, thus reducing the noise created by the camera operation and enhancing the scanning speed.

Description

Raman analysis method and apparatus for high speed quantitative analysis of wide range samples

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.

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.

As 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.

As a spectrometer, it is necessary to sufficiently remove stray light such as Rayleigh scattering, and a method of using two or three double monochromators has been used. Recently, an interference filter having excellent optical characteristics is used. The use of one monochromator has become commonplace.

1 is a configuration diagram of a conventional Raman analysis device, Figure 2 is an illustration of a conventional Raman analysis method.

Referring to FIG. 1, in the conventional Raman analysis apparatus, 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 By adjusting the light source incident through the objective lens 20 to meet the sample 21 on the stage 29 may be finely moved for each spot (Spot).

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.

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. .

In this case, 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.

In the conventional Raman analysis apparatus, 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.

Of course, there are cases where the stage moves instead of mirrors or supports both, but the precision and speed are low, the cost is high, and the size is large, so both economic and performance are undesirable.

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 / academia because of its large size and high cost. However, there is a limit to increase the area of the sample to be measured while reducing the volume and cost so that it can be used for various purposes because of the large volume, high cost, and small 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 case of the Raman analysis apparatus of such a famous manufacturer, scan inspection of a certain area is possible, but the limited area that can be inspected makes it difficult to quickly inspect a sample of a sufficient area, and the low resolution makes it difficult to obtain a target quality and expensive. It is composed of a driving unit, which is high in cost, making it difficult to disseminate it for small industrial or small medical analysis.

9 is a configuration diagram of a Raman analysis apparatus of "JY-Horiba" published in European Patent Publication EP1983332A1.

Referring to FIG. 9, 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.

In the meantime, "Renishaw" discloses a technique for improving scan speed and precision with a so-called "line scan" technique in PCT Publication WO 2009 / 093050A1. In this case, the stage to which the sample is applied moves in the X and Y axes. By taking this configuration, it is possible to measure a larger area of the sample, but this configuration is sensitive to the external environment in that the stage must be operated in precise connection with the optical measuring unit, and the cost for driving the stage is high and bulky. Because of the growing limitations, it is also difficult to disseminate them for use in small industries or medical analysis.

As such, 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.

As an example 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. In addition, 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.

In recent years, nanospectrometry has been used to search for diagnostic reagents and diagnose diseases using multiple markers. Recently, 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. Alternatively, 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.

The most common nano-probe technology recently applied to the diagnosis of such multiple markers is fluorescence-based multi-measurement technology such as Fluorescense-Assisted Cell Sorter (FACS). The wide range of multiple diagnostics limits the number of markers that cannot be used in the field of labeling or searching for millions of candidates.

Despite the structural limitations of the conventional Raman measuring apparatus and the limitations of the multi-label analysis method described above, as the level of medical service increases, the demand for multi-biomarker analysis is increasing. The low cost of distributing the Raman analysis device to the general hospital, the small size of the table top and the rapid measurement, the new technology with high signal sensitivity and accuracy is required.

On the other hand, 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.

In addition, since the conventional Raman analysis apparatus acquires images of every measurement position individually, a configuration to reduce the noise due to noise generated at each image acquisition operation of the camera has to be added. Not efficient at

In particular, in the field of detecting a large amount of different substances at high speed through multi-dimensional labeling technology to diagnose multiple diseases at the same time or searching for a suitable reagent in a candidate group of diagnostic reagents, 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.

As a result, an adequate level of Raman analysis apparatus has not yet been provided for the analysis of medical biomarkers, and current camera imaging methods and analysis methods have not been optimized for biomarker analysis. Implementing is a technically and economically difficult situation.

Thus, there is a need for new Raman analysis devices and methods that can provide adequate volume, cost and performance for such medical analysis and small industries.

[Preceding technical literature]

[Patent Documents]

Korean Unexamined Patent No. 2011-7019504

European Publication No. EP1983332A1

PCT Publication WO 2009 / 093050A1

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 according to an embodiment of the present invention for achieving the above object 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.

On the other hand, as an example, 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 according to another embodiment of the present invention for achieving the above object 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.

In this case, 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 according to an embodiment of the present invention, 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 according to an embodiment of the present invention 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 according to an exemplary embodiment of the present invention 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 according to an embodiment of the present invention 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.

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.

5 is a view illustrating an operation of a flip mirror unit according to an embodiment of the present invention.

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

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

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.

9 is a block diagram of a conventional Raman analysis device.

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

The present invention as described above will be described in detail with reference to the accompanying drawings and embodiments.

Prior to the description, the Raman analysis apparatus for high-speed quantitative analysis of a wide-area sample according to an embodiment of the present invention described below 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.

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.

Referring to FIG. 3, 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. 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.

Although 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.

According to a preferred embodiment, 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. .

That is, high speed driving of one axis is required for the scan operation, but the driving speed of the other axis may be relatively low.

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 (for example, the X 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.

Here, if 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.

However, the adjustment of the other axis (e.g., Y-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.

On the other hand, 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 .

In this case, 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.

In the Raman analysis apparatus, 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. .

Here, 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. .

4 is an exemplary diagram of a Raman analysis method for quantitatively analyzing a plurality of biological markers in a sample according to an embodiment of the present invention. In the Raman analysis apparatus according to an embodiment of the present invention, 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.

As 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.

In particular, in the field of detecting a large amount of different substances at high speed through multi-dimensional labeling technology to diagnose multiple diseases at the same time or searching for a suitable reagent in a candidate group of diagnostic reagents, 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.

In addition, since the measurement of a large area sample of at least one square millimeter (mm) in which blood is applied to multiple biological markers is required, the size of the test area is increased and thus the speed is improved.

In addition, since 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.

In addition, 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.

On the other hand, the Raman analysis apparatus according to an embodiment of the present invention 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.

In addition, 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.

In addition, 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.

Meanwhile, referring again to FIG. 3, 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.

In addition, 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.

Meanwhile, 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. Although it is preferable to include and comprise, different wavelength range can be used according to the conditions of a sample.

In addition, 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.

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. In addition, by applying the scanner structure optimized for this operation method, 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.

In particular, as shown in FIG. 5, 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.

Referring to FIG. 6, when the Raman analysis apparatus scans a sample, one axis moves at a high speed and the other axis moves at a lower speed than the one axis, and scans the sample 23.

In particular, in order to search for candidate candidates for diagnosis and diagnosis of multiple diseases using nano spectroscopy, 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.

In addition, as described above, 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 according to an embodiment of the present invention 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.

In addition, it is possible to quickly measure or search for multiple-dimensional markers as well as Raman spectral signal patterns, as well as multiple signals for complex analysis of fluorescence, graphics, and optical patterns.

In addition, in addition to the scan type of the illustrated method, it is possible to variously change the scan method having a predetermined angle (for example, interlaced scan or sequential scan type of CRT) or a method of dividing the area and sequentially scanning the areas. , All belong to the scope of the present invention.

10 is an exemplary view of a sample image and a 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, as described above, 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.

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

In other words, using the method proposed in the present invention, 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.

On the other hand, since the Raman signal is a very fine signal, since a very long exposure time is required for accurate determination, the

scan controllers

65 and 75 of the Raman analysis apparatus according to an embodiment of the present invention 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.

For example, the Raman analysis apparatus according to an embodiment of the present invention 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. After performing the pre-scan, 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.

If 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.

7 is an exemplary view 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 moving one of the flip mirror units 81 by controlling one axis at a lower speed than the other axis. Of course, this scan pattern may vary.

According to a preferred embodiment, 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.

In addition, for in vivo samples, a near infrared ray laser may be further configured to utilize infrared spectroscopy in the Raman analyzer.

In addition, 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.

More preferably, 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.

Referring to FIG. 8, in the Raman analysis method, 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. (S10), 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 In step S20, 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. To control the incident light provided to the sample 21 through the objective lens 20 to sequentially scan at least a portion of the inspection area (S30) and the spectroscopic unit 50 of the Raman analysis device is By 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).

According to another preferred embodiment, 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.

At this time, 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.

Alternatively, 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.

As described above, 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.

Claims

A Raman analysis device for collectively collecting and analyzing a plurality of types of search target information present in a wide-area sample,

A flip-mirror unit for reflecting the incident light provided from the laser light source and varying a reflection path with respect to one axis at a speed in a preset range;

An actuator unit configured to vary the position of the flip mirror unit in synchronization with an operation of the flip mirror unit at a speed slower than the flip mirror unit variable speed with respect to another axis perpendicular to the variable axis of the flip mirror unit;

A scan controller configured to control the flip mirror unit and the actuator unit to sequentially scan at least a portion of an inspection region from incident light provided to the sample through an objective lens; and

The spectroscopic information on the reflected light is output through the camera by receiving the reflected light in which the Raman signals generated by the plurality of types of search targets are mixed through the camera by the scanning control part while sequentially scanning the inspection area. However, the Raman analysis device for a high-speed quantitative analysis of a wide-area sample comprising a; spectroscope for outputting the continuous spectroscopic information for one or more sequential scan intervals set for each sample during one camera operation period.
The method of claim 1,

The search target is a Raman analysis device for high-speed quantitative analysis of a wide-area sample, characterized in that the industrial manufacturing or medical biomarker.
The method of claim 1, wherein the actuator unit

Raman analysis device for high-speed quantitative analysis of a wide-area sample comprising a linear controllable universal drive structure including a small motor, piezo actuator, ultrasonic actuator.
The method of claim 1, wherein the flip mirror unit

Raman analysis device for high-speed quantitative analysis of a wide-area sample comprising a voice coil motor or a MEMS driver.
The method of claim 1, wherein the scan control unit

And 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 method of claim 1, wherein the spectroscopic portion

Raman analysis device for high-speed quantitative analysis of a wide range sample including a CCD having the highest quantum efficiency between 500 nanometers and 600 nanometers, or the maximum efficiency in another optical domain.
The method of claim 1,

Raman analysis device for high-speed quantitative analysis of the wide-area sample, characterized in that the table-like and integral through the optimization of the device elements.
The method of claim 1, wherein the sample

The Raman analysis device for high-speed quantitative analysis of a wide-area sample, characterized in that the sample area is more than 1 square millimeter while the high efficiency condensing of the confocal optical system does not affect the flip mirror unit and the actuator unit.
In the Raman analysis method for high-speed quantitative analysis of the search target in the sample using the Raman analysis device of any one of claims 1 to 8,

a) analyzing the continuous spectroscopic information in the form of a Raman map including predetermined distribution and coefficient information to derive an analysis result; a Raman analysis method comprising a high-speed quantitative analysis of a sample of a wide area.
The method of claim 9,

And b) performing a multi-quantitative diagnosis for simultaneously analyzing individual cases of multiple diseases based on the information on the Raman map through the analysis and measurement of multi-dimensional multi-label markers when the search target is a biomarker. Raman analysis method for rapid quantitative analysis of samples.