JP6977995B2 - Microscopic Raman spectroscopic measurement device, microscopic Raman spectroscopic measurement method and program - Google Patents

Description

The present invention relates to Raman spectroscopic measurement, and particularly relates to an apparatus and method for microscopic Raman spectroscopic measurement using a microscope as an optical system.

Using Raman spectroscopy, detailed knowledge about the molecular structure and molecular environment of a sample can be obtained in a minimally invasive manner without pretreatment of the sample. For this reason, new non-biopsy medical diagnostic methods utilizing Raman spectroscopy are being actively developed in Japan and overseas. In particular, microscopic Raman spectroscopy using a microscope as an optical system can extract information on minute regions in a sample, so that precise measurement and diagnosis with high resolution can be performed.

In order to accurately diagnose the pathological condition of a disease based on the Raman spectrum by microscopic Raman spectroscopy, for example, a large number of Raman spectra showing the pathological condition of the disease and a healthy biopsy sample are measured and data is accumulated. There is a need to. For that purpose, it is necessary to quickly perform a large number of Raman spectrum measurements under favorable conditions.

When performing micro-Raman spectroscopic measurements, first fix the biopsy sample to a light-transmitting fixing member (for example, sandwiched between plate-shaped quartz glass (slide glass and cover glass)), and then place the sample on the stage of the microscope. Place it on the surface. Laser light is radiated there through the optical system of the microscope, and the Raman scattered light scattered from the sample is focused (a part of the optical system is shared for irradiation and focusing). In the case of an inverted arrangement in which the objective lens facing the stage is arranged below the sample, the objective lens, the slide glass, the biopsy sample, and the cover glass are arranged in order from the bottom along the optical axis of the laser beam. The typical thickness of a biopsy sample is, for example, about 1 mm.

In order to perform micro-Raman spectroscopy under favorable conditions in the above arrangement, it is necessary to make adjustments for focusing to align the focal point of the objective lens on the optical axis with the position of the biopsy sample (for example, within the range of 1 mm thickness). be. When the objective lens is in focus on the fixed member instead of the sample, the Raman spectrum of the fixed member becomes dominant over the sample, and the purpose of measurement cannot be achieved. Therefore, conventionally, the distance in the optical axis direction between the objective lens and the stage (hereinafter referred to as the facing distance) is manually changed little by little (the stage is fixed and the lens barrel to which the objective lens is attached is moved up and down little by little). Or, fix the lens barrel and move the stage up and down little by little), read the change in the Raman spectrum measured each time, and determine the facing distance that satisfies the focusing condition (hereinafter referred to as the focusing distance). Adjustments have been made to do so.

However, since doctors and medical technicians who measure biopsy samples are not specialists in spectroscopic measurements in most cases, there is a limit to how quickly and efficiently the above adjustment work can be performed (for example, it takes 10 minutes or more). There is.). In particular, if an attempt is made to perform each micro-Raman spectroscopic measurement while replacing a large number of biopsy samples having non-uniform thickness, the workload may become excessive.

In microscopic Raman spectroscopy, a technique for reducing the influence of background light generated from a transparent body for holding a sample such as glass is known (see, for example, Patent Document 1). According to Patent Document 1, it is described that the transparent body for holding a sample is made of CaF2 single crystal, synthetic quartz, or fluoride glass. The technique of Patent Document 1 reduces the influence of background light by selecting a material that can reduce the level of fluorescence emitted from the transparent material, but enables the laser light to be quickly focused on the sample. A different approach is needed to solve this problem.

Japanese Unexamined Patent Publication No. 2012-237646

The problem to be solved by the present invention is to quickly focus the laser beam on the sample placed on the stage fixed to the light-transmitting fixing member, which is efficient even for non-experts of spectroscopic measurement. It is to enable microscopic Raman spectroscopy of a large number of samples.

In order to solve the above-mentioned problems, the micro-Raman spectroscopic measuring device according to the present invention is a micro-Raman spectroscopic measuring device capable of detecting and analyzing Raman scattered light scattered from a sample irradiated with laser light. A stage that has a light source and a sample mounting surface and can be mounted on the sample mounting surface by fixing the sample to a light-transmitting fixing member, and facing the stage and opposite to the stage. An objective lens arranged so as to focus light incident from the side toward the stage, a mechanical drive unit capable of changing the facing distance between the objective lens and the stage, and Raman scattered light. The spectroscopic unit capable of measuring the spectrum and the laser light emitted from the laser light source are directed to the stage via the objective lens, and the Raman scattered light incident through the objective lens is directed to the spectroscopic unit. The optical discrimination unit that can be used and the mechanical drive unit are controlled to control the laser light source each time the facing distance is changed to emit laser light, and the spectrum data measured by the spectroscopic unit is acquired. From the value of at least one of the intensity of the Raman scattered light peculiar to the fixing member and the intensity of the Raman scattered light peculiar to the sample extracted from the data of the spectrum to the sample of the laser beam. It is characterized by including a control unit capable of determining the value of the facing distance when the focusing condition for which the focus is estimated is estimated as the focusing distance.

Further, in the method of microscopic Raman spectroscopic measurement according to the present invention, a laser light source, a stage, and an objective lens arranged so as to face the stage and focus light incident on the opposite side of the stage to the direction of the stage. In a method of micro-Raman spectroscopic measurement performed using a micro-Raman spectroscopic measuring device including a mechanical drive unit, an optical discrimination unit, and a spectroscopic unit capable of changing the facing distance between the objective lens and the stage. The sample is fixed to a light-transmitting fixing member and placed on the sample mounting surface of the stage, the initial value of the facing distance is set by the mechanical drive unit, and the laser beam is emitted from the laser light source for a specified time. When the laser beam passes through the optical discrimination portion and the objective lens and is fixed to the fixing member and irradiates the sample placed on the sample mounting surface, the fixing member and the sample are exposed to light. From the spectrum of light including Raman scattered light scattered from at least one of them and measured in the spectroscopic section through the objective lens and the optical discrimination section, the intensity of Raman scattered light peculiar to the fixing member and the sample can be obtained. The intensity of the Raman scattered light peculiar to the fixing member is extracted, and the intensity of the Raman scattered light peculiar to the fixing member and the intensity of the Raman scattered light peculiar to the sample are combined from at least one value of 1 to the sample. It is determined whether or not the focusing condition for which the focus is estimated is satisfied, and until it is determined that the focusing condition is satisfied, each time the value of the facing distance is changed by the mechanical drive unit, the laser beam of the laser beam is used. The focusing distance is the value of the facing distance when it is determined that the focusing condition is satisfied by repeating the emission and extraction of the intensity of the Raman scattered light peculiar to the fixing member and the intensity of the Raman scattered light peculiar to the sample. It is characterized by being confirmed as.

According to the present invention, a large number of samples can be efficiently focused even by a non-expert in spectroscopic measurement by quickly focusing a laser beam on a sample placed on a stage fixed to a light-transmitting fixing member. Microscopic Raman spectroscopy can be performed.

FIG. 1 is a block diagram of a microscopic Raman spectroscopic measuring device according to an embodiment of the present invention. FIG. 2 is a flowchart illustrating a method of microscopic Raman spectroscopic measurement according to an embodiment of the present invention. FIG. 3 is a conceptual diagram illustrating the setting of the facing distance in the embodiment of the present invention. FIG. 4 is a flowchart illustrating a part of the flow of FIG. 2 in detail. FIG. 5 is actual measurement data of a Raman spectrum obtained by executing a part of the flow of FIG. FIG. 6 is a diagram showing an experimental example of establishing the focusing condition in the embodiment of the present invention using a chicken piece as a sample. FIG. 7 is a diagram showing an experimental example of establishing the focusing condition in the embodiment of the present invention using a silicon piece as a sample.

Hereinafter, embodiments of the present invention will be described with reference to the drawings. The examples described below do not limit the technical scope of the invention.

FIG. 1 is a block diagram of a micro-Raman spectroscopic measuring device 100 according to an embodiment of the present invention. A sample 503 sandwiched and fixed by a light-transmitting fixing member composed of a cover glass 501 and a slide glass 502 can be placed on the sample mounting surface 101S of the stage 101 included in the micro-Raman spectroscopic measuring device 100. Since the micro-Raman spectroscopic measurement device 100 is arranged upside down, an optical path between the optical system described later of the microscopic Raman spectroscopic measurement device 100 and the sample 503 is secured by opening the center of the stage 101 or the like. The cover glass 501, the slide glass 502, and the sample 503 are not included in the configuration of the micro Raman spectroscopic measuring device 100.

The optical system of the micro-Raman spectroscopic measuring device 100 includes a laser light source 102, an optical discrimination unit 103, an objective lens 104, a mirror 105, and a mirror 106. The optical discrimination unit 103 is, for example, a dichroic mirror that reflects light having the same wavelength as the laser light emitted from the laser light source 102 and transmits light having other wavelengths. In the configuration shown in FIG. 1, the optical discrimination unit 103 can reflect the laser light emitted from the laser light source 102 and arriving at the optical discrimination unit 103 via the mirror 105 in the direction of the mirror 106. Further, the light discrimination unit 103 transmits light (including Raman scattered light having a wavelength different from that of the laser light) generated from the sample 503 or the like irradiated with the laser light and reflected by the mirror 106 via the objective lens 104. Then, it can be directed to the spectroscopic unit 108, which will be described later.

The objective lens 104 converges the luminous flux of the laser beam arriving from the mirror 106 toward the focal point, and irradiates the sample 503 that is expected to be placed at the focal point. Further, the light including the Raman scattered light generated from the sample 503 or the like is received, and the light flux thereof is directed to the light discrimination unit 103 in the direction opposite to the traveling direction of the laser light.

The configuration of the optical system of the micro-Raman spectroscopic measuring device 100 is not limited to that shown in FIG. For example, the optical discrimination unit 103 selectively directs light having the same wavelength as the laser light to the sample irradiation through the objective lens 104, and directs light having a wavelength different from the laser light coming from the objective lens 104 to the spectroscopic unit 108. Anything can be used as long as it can be used. Each configuration of the optical system does not have to have the positional relationship as shown in FIG. 1, and is not limited to the inverted arrangement in the first place. Optical elements such as mirrors such as the mirror 105 and the mirror 106, various lenses, and edge filters may be appropriately selected and arranged according to the arrangement of each configuration of the optical system.

The micro-Raman spectroscopic measuring device 100 includes a spectroscopic unit 108 and a mechanical driving unit 109. The spectroscopic unit 108 has a known configuration including a spectroscope that decomposes incident light into components for each wavelength and a detector including an element such as a CCD. The mechanical drive unit 109 is configured to include, for example, a piezoelectric actuator (piezo element or the like) or an optical actuator (optomicrometer or the like), and can change the facing distance between the stage 101 and the objective lens 104. .. For example, in the case where the lens barrel including the objective lens 104 is fixed, the mechanical drive unit 109 can move the stage 101 in the vertical direction in FIG.

The mechanical drive unit 109 can further change the relative position of the stage 101 with respect to the objective lens 104 in parallel with the sample mounting surface 101S. For example, in the case where the lens barrel including the objective lens 104 is fixed, the mechanical drive unit 109 can move the stage 101 in the left-right direction in FIG. 1 and in the direction perpendicular to the paper surface. Such a configuration is known and commercialized as a scanning microscope stage.

The micro-Raman spectroscopic measuring device 100 includes a control unit 110. The control unit 110 implements, for example, a program for causing a personal computer (PC) to execute the method of microscopic Raman spectroscopy according to the embodiment. The control unit 110 is connected to the laser light source 102, the spectroscopic unit 108, and the mechanical drive unit 109 by wire or wirelessly (including the case where they are connected via a network such as the Internet). These connections are represented in FIG. 1 by dashed double-headed arrows.

The control unit 110 can control the mechanical drive unit 109 to change the facing distance between the stage 101 and the objective lens 104. The control unit 110 can control the mechanical drive unit 109 and further change the relative position of the stage 101 with respect to the objective lens 104 in parallel with the sample mounting surface 101S. The control unit 110 can control the laser light source 102 to emit laser light for a specified time. The control unit 110 can control the spectroscopic unit 108 to acquire the spectral data measured by the spectroscopic unit 108.

The method according to the example of the microscopic Raman spectroscopic measurement performed using the microscopic Raman spectroscopic measurement apparatus 100 will be described below. FIG. 2 is a flowchart illustrating a method according to an embodiment. First (START), the sample 503 is sandwiched between fixing members (cover glass 501 and slide glass 502) and fixed, and then placed on the sample mounting surface 101S of the stage 101 (step S20). Subsequently, the control unit 110 instructs the mechanical drive unit 109 to set the facing distance to an initial value (for example, the value of the facing distance when the stage 101 is set to the lowest position in the movable range) (step S21). .. At this time, the control unit 110 also instructs the mechanical drive unit 109 to set the initial value of the relative position (hereinafter referred to as a two-dimensional position) of the stage 101 with respect to the objective lens 104 in the plane parallel to the sample mounting surface 101S. Set.

The control unit 110 designates a time for continuously measuring the Raman spectrum, and emits a laser beam from the laser light source 102 for the designated time (step S22). The specified time can be arbitrarily selected within the range of the conditions of the device according to the purpose of measurement (usually, the time in seconds is selected, but when it is necessary to wait for the attenuation (bleach) of the fluorescence intensity, etc., it is in minutes. You may choose the time of.).

The laser light emitted from the laser light source 102 passes through the optical system of the micro-Raman spectroscopic measuring apparatus 100 described with reference to FIG. 1 and heads in the direction of irradiating the sample 503 from the objective lens 104. Light including Raman scattered light or background light generated from a fixing member composed of a sample 503 or a cover glass 501 and a slide glass 502 irradiated with laser light is collected by an objective lens 104 and passed through an optical discrimination unit 103 for spectroscopy. Reach unit 108.

The spectroscopic unit 108 disperses the light including the Raman scattered light or the background light described above, and obtains the data of the spectrum plotted for the Raman shift in wave number as a unit. The control unit 110 is fixed from the data of the spectrum on the assumption that the Raman shift value peculiar to the fixing member (cover glass 501 or slide glass 502) and the Raman shift value peculiar to the sample 503 are known. The intensity of the Raman scattered light peculiar to the member (cover glass 501 or the slide glass 502) and the intensity of the Raman scattered light peculiar to the sample 503 can be extracted (step S23).

Since the target of the microscopic Raman spectroscopy measurement is the intensity of the Raman scattered light peculiar to the sample 503, the facing distance is set so that the intensity of the Raman scattered light peculiar to the fixing member (cover glass 501 or slide glass 502) is not dominant. It is desirable to do. FIG. 3 is a conceptual diagram illustrating the setting of the facing distance. The horizontal axis represents the facing distance, and the vertical axis represents the intensity of Raman scattered light (both are arbitrary units). The intensities of Raman scattered light of the fixing member and the sample are represented by solid line curves and broken line curves, respectively.

In FIG. 3, when the facing distance is monotonically increased by a certain unit length (in the example of FIG. 1, the position of the objective lens 104 is fixed and the height of the stage 101 is gradually increased), the laser beam is initially fixed. Focusing on the member (cover glass 501 in the example of FIG. 1), the intensity of the Raman scattered light of the fixing member (solid line curve) is the intensity of the Raman scattered light of the sample (sample 503 in the example of FIG. 1) (broken curve). It exceeds the peak and reaches the peak. After that, as the facing distance is increased, the focal point of the laser beam gradually shifts to the sample 503, so that the broken line curve exceeds the solid line curve and reaches a peak.

It is assumed that the threshold value "TH1" is given to the solid line curve and the threshold value "TH2" is given to the broken line curve. Further, the facing distance is monotonically increased as a condition of the facing distance (hereinafter referred to as a focusing condition) in which the intensity of the Raman scattered light peculiar to the fixing member is not dominant and the focusing of the laser light to the sample 503 is estimated. It is assumed that the solid line curve exceeds the threshold value “TH1” and then falls below the threshold value “TH1” when the direction is changed to, and the broken line curve exceeds the threshold value “TH2”.

In FIG. 3, when the facing distance is monotonically increased from a small value on the left side of the horizontal axis to reach the value “D1”, the intensity of Raman scattered light of the fixing member exceeds the threshold value “TH1”. After that, the intensity of the Raman scattered light of the fixing member reaches a peak at the facing distance "D2", and when the facing distance is further increased, it falls below the threshold value "TH1" at "D3". The above focusing condition is not satisfied until the facing distance reaches “D3” (“NO” in step S24). If the above focusing condition is not satisfied, the control unit 110 controls the mechanical drive unit 109 to increase the facing distance by one unit length (step S25). After that, the flow is returned to step S22, and steps S22 to S24 are repeatedly executed until the facing distance reaches “D3”.

After that, the control unit 110 executes step S24 in a state where the facing distance reaches “D3”. At the facing distance "D3", the solid line curve exceeds the threshold value "TH1" and then falls below the threshold value "TH1", and the broken line curve exceeds the threshold value "TH2". Focusing condition is satisfied (“YES” in step S24). If the focusing condition is satisfied, the intensity of the Raman scattered light of the sample 503 is determined in a state where the intensity of the Raman scattered light peculiar to the fixing member is not dominant and the focusing of the laser light to the sample 503 is estimated. Can be measured. Therefore, the control unit 110 determines the facing distance “D3” as the focusing distance for the subsequent micro-Raman spectroscopic measurement (step S26).

Next, the control unit 110 controls the mechanical drive unit 109 to repeatedly move the two-dimensional position of the stage 101 in parallel with the sample mounting surface 101S, and each time the control unit 110 moves, the same process as in steps S22 and S23 of FIG. Is executed (step S27). FIG. 4 is a flowchart illustrating step S27 in detail.

It is assumed that the position as (N + 1) is predetermined as the two-dimensional position. These two-dimensional positions are represented by the symbols "P0", "P1", ..., "PN" and are collectively referred to as "Pi". "P0" in "Pi" as shown in (N + 1) is the initial value of the two-dimensional position set at the same time as step S21 in FIG. Referring to FIG. 4, the value of “Pi” at the start time is “P0” (step S40).

The control unit 110 designates a time for continuously measuring the Raman spectrum, and emits a laser beam from the laser light source 102 for the designated time (step S41). This step is the same as step S22 in FIG. Next, as in step S23 in FIG. 2, the control unit 110 obtains the intensity of the Raman scattered light peculiar to the fixing member (cover glass 501 or slide glass 502) and the sample 503 from the spectral data obtained by the spectroscopic unit 108. The intensity of the inherent Raman scattered light can be extracted (step S42). This step is the same as step S23 in FIG. However, in step S42, the latter can be extracted in a state where the focus of the laser beam on the sample 503 is estimated at all the two-dimensional positions.

If the two-dimensional position Pi has not reached the end point PN (“NO” in step S43), the control unit 110 updates the Pi to the next two-dimensional position (step S44) and returns the flow to step S41. If the two-dimensional position Pi has reached the end point PN (“YES” in step S43), the control unit 110 ends the flow. At this point, the flow shown in FIG. 2 is also completed, and the Raman scattered light peculiar to the fixing member (cover glass 501 or slide glass 502) is not dominant and the focus of the laser light on the sample 503 is estimated. , The micro-Raman spectroscopic measurement for the two-dimensional position according to (N + 1) of the sample 503 is completed.

In the above description, the focusing condition is such that the solid line curve exceeds the threshold value “TH1” and then falls below the threshold value “TH1” when the facing distance is changed in the direction of monotonous increase as shown in FIG. Moreover, it is assumed that the broken line curve exceeds the threshold value “TH2”. However, the change in the facing distance may be in the direction of monotonically decreasing (in the example of FIG. 1, the case where the laser beam first focuses on the slide glass 502 and then gradually shifts the focus to the sample 503. ).

Further, depending on the measurement conditions, it may be difficult to obtain a clear broken line curve due to strong background light or the like. In such a case, the focusing condition may be only that the solid line curve exceeds the threshold value “TH1” and then falls below the threshold value “TH1”. After determining the focusing distance, the sample may not be automatically scanned in two dimensions, but the two-dimensional position of the sample may be artificially selected and microscopic Raman spectroscopy may be performed.

Hereinafter, an example of actual measurement data according to the above-described embodiment will be described with reference to FIGS. 5 to 8. FIG. 5 is actual measurement data of the Raman spectrum when a chicken piece of a biopsy sample is sandwiched between fixing members made of quartz glass and steps S22 to S24 of FIG. 2 are repeatedly executed (the bottom curve faces each other). Corresponds to the initial value of the distance, and the top curve corresponds to the value of the opposite distance that satisfies the focusing condition.).

The horizontal axis of FIG. 5 is Raman shift (unit is cm ^-1 ), and the vertical axis is light intensity (arbitrary unit). The solid arrow in the figure indicates the direction in which the facing distance is monotonically increased. Although the quartz glass Raman spectrum (420 cm ^-1) and chicken Raman spectrum (1448cm ^-1) are both observed, in this actual measurement based only on changes in the Raman spectra of quartz glass in view of the influence of background light The focusing condition was determined.

FIG. 6 is a diagram showing an experimental example of where it was determined that the focusing condition was satisfied when the facing distance was monotonically increased from the initial value using the chicken piece sandwiched between the quartz glasses as a sample. The micro-Raman spectroscope used in the experiment has the same inverted arrangement as in FIG. 1, and the horizontal axis in FIG. 6 represents the height of the stage (unit: μm) when the height of a certain reference is 0 (the horizontal axis is the horizontal axis). The direction from left to right is the direction of monotonous increase in the facing distance.)

The change in the intensity (normalized value of the maximum value within the measured range) of ^{the Raman spectrum (1448 cm -1} ) of chicken when the facing distance is changed in 25 μm steps (maximum 36 points) is shown in the dark color of FIG. It is represented by the curve of. The change in the intensity (normalized value of the maximum value within the measured range) of ^{the Raman spectrum (420 cm -1} ) of the quartz glass with the same change in the facing distance is represented by the light-colored curve in FIG. It should be noted that both the light and shade curves show the values normalized by their respective maximum values, and do not show the comparison of the absolute values of the two.

In the experimental example of FIG. 6, a laser beam having an output of 40 mW was irradiated for 1 second (time specified in step S22 of FIG. 2) for measurement. The facing distance corresponding to the stage height indicated by the arrow line of "focusing condition OK" shown in FIG. 6 is a value determined as the focusing distance. This focusing distance appears in the phase where the Raman spectral intensity of the quartz glass is declining after passing the maximum value, and is evaluated to be close to the target value.

In experiments using chicken pieces as a sample, the focusing distance could not be determined properly due to the influence of background light such as fluorescence, so a piece of silicon, which is expected to have a smaller effect, was sandwiched between quartz glass fixing members. A similar experiment was performed in. The results are shown in FIG. The horizontal axis of FIG. 7 is the same as the horizontal axis of FIG. 6, and the vertical axis is the intensity (arbitrary unit) of the Raman spectrum.

^{The change in the intensity of the Raman spectrum (520 cm -1} ) of silicon with the change in the facing distance is represented by the dark curve in FIG. 7. ^{The change in the intensity of the Raman spectrum (420 cm -1} ) of quartz glass due to the change in the facing distance is represented by the light-colored curve in FIG. It should be noted that both the light and shade curves show values that are appropriately standardized, and do not show a comparison of the absolute values of the two. The facing distance corresponding to the stage height indicated by the arrow line of "focusing condition OK" shown in FIG. 7 is a value determined as the focusing distance. This focusing distance appears in the phase where the Raman spectral intensity of the quartz glass is declining after passing the maximum value, and is evaluated to be close to the target value.

According to the embodiment of the present invention, the facing distance (distance between the sample and the objective lens) capable of measuring the Raman spectrum derived from the sample in a state where the Raman spectrum derived from the fixed member is not dominant is automatically determined. This allows non-experts in spectroscopic measurements to typically complete tasks that can take up to 10 minutes in tens of seconds (waiting for a decay (bleach) of the fluorescence intensity for a strongly fluorescent sample. Even if it is necessary, it is about 4 minutes including the bleaching time.)

Even when two-dimensional scanning of about 100 points of the sample is performed after the focusing distance is determined, the automatic measurement is completed in about 5 minutes in total including the time until the focusing distance is determined. Therefore, even a non-expert in spectroscopic measurement can efficiently perform microscopic Raman spectroscopic measurement. The configuration of the micro-Raman spectroscopic measurement apparatus and the method of microscopic Raman spectroscopic measurement according to the above-described embodiment can be appropriately changed within the scope of the gist of the invention according to the conditions such as the sample, the instrument, and the measurement environment. ..

100 Microscopic Raman spectroscopic measuring device 101 Stage 101S Sample mounting surface 102 Laser light source 103 Optical discrimination unit 104 Objective lens 105, 106 Mirror 108 Spectroscopic unit 109 Mechanical drive unit 110 Control unit 501 Cover glass 502 Slide glass 503 Sample

Claims (11)

In a microscopic Raman spectroscopic measuring device capable of detecting and analyzing Raman scattered light scattered from a sample irradiated with laser light.
Laser light source and
A stage having a sample mounting surface and capable of fixing the sample to a light-transmitting fixing member and mounting the sample on the sample mounting surface.
An objective lens that faces the stage and is arranged so that light incident from the opposite side of the stage is focused in the direction of the stage.
A mechanical drive unit capable of changing the facing distance between the objective lens and the stage,
A spectroscopic unit capable of measuring the spectrum of Raman scattered light,
An optical discrimination unit capable of directing the laser light emitted from the laser light source toward the stage via the objective lens and directing the Raman scattered light incident through the objective lens toward the spectroscopic unit.
Each time the mechanical drive unit is controlled to change the facing distance, the laser light source is controlled to emit laser light, and the spectrum data measured by the spectroscopic unit can be acquired, and the spectrum can be obtained. Focusing of the laser beam on the sample is estimated from at least one of the intensity of the Raman scattered light peculiar to the fixing member and the intensity of the Raman scattered light peculiar to the sample extracted from the above data. A microscopic Raman spectroscopic measuring apparatus including a control unit capable of determining the value of the facing distance when the focusing condition is satisfied as the focusing distance. The control unit can control the mechanical drive unit to change the facing distance in the direction of monotonically increasing or monotonically decreasing, and each time the facing distance is changed in the direction of monotonically increasing or monotonically decreasing. The spectroscopy according to claim 1, wherein the focusing condition is that the intensity of the Raman scattered light peculiar to the extracted fixing member exceeds the threshold value given in advance and then falls below the threshold value. Raman spectroscopy measuring device. The control unit can control the mechanical drive unit to change the facing distance in the direction of monotonically increasing or decreasing, and each time the facing distance is changed in the direction of monotonically increasing or monotonically decreasing. The intensity of the Raman scattered light peculiar to the fixing member and the intensity of the Raman scattered light peculiar to the sample are extracted, and the intensity of the Raman scattered light peculiar to the fixing member exceeds the first threshold value given in advance. Claim 1 is characterized in that the focusing condition is that the Raman scattered light intensity inherent in the sample exceeds the second threshold value given in advance while the Raman scattered light is subsequently lowered below the first threshold value. The micro-Raman scattering measuring device according to the above. The micro-Raman spectroscopic measurement device according to any one of claims 1 to 3, wherein the mechanical drive unit includes a piezoelectric actuator or an optical actuator. The mechanical drive unit can change the relative position of the stage with respect to the objective lens in parallel with the sample mounting surface.
After determining the focusing distance, the control unit controls the mechanical drive unit to control the laser light source each time the stage changes its relative position with respect to the objective lens in parallel with the sample mounting surface. The microscopic Raman spectroscopic measuring apparatus according to any one of claims 1 to 3, wherein the laser beam is emitted and the spectrum data measured by the spectroscopic unit can be acquired. The laser light source, the stage, the objective lens arranged so as to face the stage and focus the light incident from the opposite side to the stage in the direction of the stage, the objective lens, and the facing distance between the stages are changed. In a method of micro-Raman spectroscopic measurement performed using a micro-Raman spectroscopic measuring device including a mechanical drive unit, an optical discrimination unit, and a spectroscopic unit capable of performing the same.
The sample is fixed to a light-transmitting fixing member and placed on the sample mounting surface of the stage.
The initial value of the facing distance is set by the mechanical drive unit, and the initial value is set.
The laser beam is emitted from the laser light source for a specified time.
When the laser light passes through the optical discrimination portion and the objective lens and irradiates the sample mounted on the sample mounting surface fixed to the fixing member, at least one of the fixing member and the sample is irradiated. From the spectrum of the light including the Raman scattered light scattered from the objective lens and the light measured in the spectroscopic part through the optical discrimination part, the intensity of the Raman scattered light peculiar to the fixing member and the Raman scattering peculiar to the sample. Extract the light intensity,
Whether the focusing condition for which the laser light is estimated to be focused on the sample is satisfied from at least one of the intensity of the Raman scattered light peculiar to the fixing member and the intensity of the Raman scattered light peculiar to the sample. Judge whether or not,
Until it is determined that the focusing condition is satisfied, each time the value of the facing distance is changed by the mechanical driving unit, the emission of the laser beam, the intensity of the Raman scattered light peculiar to the fixing member, and the sample are measured. Repeated extraction of the intensity of the unique Raman scattered light,
A method for microscopic Raman spectroscopy, wherein the value of the facing distance when it is determined that the focusing condition is satisfied is determined as the focusing distance. The laser light source, the stage, the objective lens arranged so as to focus the light facing the stage and incident from the opposite side to the stage in the direction of the stage, the objective lens, and the facing distance between the stages are changed. In a method of micro-Raman spectroscopic measurement performed using a micro-Raman spectroscopic measuring device including a mechanical drive unit, an optical discrimination unit, and a spectroscopic unit capable of performing the same.
The step of fixing the sample to the light-transmitting fixing member and placing it on the sample mounting surface of the stage,
The first step of setting the initial value of the facing distance by the mechanical drive unit, and
A second step of emitting laser light from the laser light source for a specified time,
When the laser light passes through the optical discrimination portion and the objective lens and irradiates the sample mounted on the sample mounting surface fixed to the fixing member, at least one of the fixing member and the sample is irradiated. From the spectrum of the light including the Raman scattered light scattered from the objective lens and the light measured in the spectroscopic part through the optical discrimination part, the intensity of the Raman scattered light peculiar to the fixing member and the Raman scattering peculiar to the sample. The third step of extracting the light intensity and
The focusing condition is satisfied in which the focusing of the laser beam to the sample is estimated from at least one of the intensity of the Raman scattered light peculiar to the fixing member and the intensity of the Raman scattered light peculiar to the sample. The fourth step to determine whether or not it is
If it is determined in the fourth step that the focusing condition is not satisfied, the value of the facing distance is updated and set, and then the second step to the fourth step is repeatedly executed. 5 steps and
A method for microscopic Raman spectroscopy, which comprises a sixth step of determining the value of the facing distance when it is determined that the focusing condition is satisfied in the fourth step as the focusing distance. In the fifth step, the value of the facing distance is updated in the direction of monotonically increasing or monotonically decreasing.
The microscopic Raman according to claim 7, wherein the focusing condition is that the intensity of the Raman scattered light peculiar to the fixing member exceeds the threshold value given in advance and then falls below the threshold value. Method of spectroscopic measurement. In the fifth step, the value of the facing distance is updated in the direction of monotonically increasing or monotonically decreasing.
The focusing condition is such that the intensity of the Raman scattered light inherent in the fixing member exceeds the first threshold value given in advance in the fifth step and then falls below the first threshold value, and the focus condition is described. The method for microscopic Raman spectroscopic measurement according to claim 7, wherein the intensity of Raman scattered light peculiar to the sample exceeds a second threshold value given in advance. When the mechanical drive unit can change the relative position of the stage with respect to the objective lens in parallel with the sample mounting surface.
After the sixth step, a seventh step of repeating the second step and the third step each time the relative position of the stage with respect to the objective lens is changed parallel to the sample mounting surface is performed.
The method for microscopic Raman spectroscopy according to any one of claims 7 to 9, further comprising. The laser light source, the stage, the objective lens arranged so as to focus the light facing the stage and incident from the opposite side to the stage in the direction of the stage, the objective lens, and the facing distance between the stages are changed. In a program that causes a computer to control a microscopic Raman spectroscopic measuring device having a mechanical drive unit, an optical discrimination unit, and a spectroscopic unit capable of performing the same.
The first step of setting the initial value of the facing distance by the mechanical drive unit, and
A second step of emitting laser light from the laser light source for a specified time,
When the laser beam is fixed to a light-transmitting fixing member through the light discrimination portion and the objective lens and irradiates the sample placed on the sample mounting surface of the stage, the fixing member and the sample From the spectrum of light including Raman scattered light scattered from at least one of them and measured in the spectroscopic section through the objective lens and the optical discrimination section, the intensity of Raman scattered light peculiar to the fixing member and the sample can be obtained. The third step to extract the intensity of the inherent Raman scattered light,
The focusing condition is satisfied in which the focusing of the laser beam to the sample is estimated from at least one of the intensity of the Raman scattered light peculiar to the fixing member and the intensity of the Raman scattered light peculiar to the sample. The fourth step to determine whether or not it is
If the focusing condition is not satisfied in the fourth step, the value of the facing distance is updated and set, and then the second step to the fourth step is repeated to execute the fifth step. ,
A program comprising causing the computer to perform a sixth step of determining a value of an facing distance when it is determined in the fourth step that the focusing condition is satisfied as a focusing distance.

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