Optical measuring device, optical measuring method

Abstract

To provide a technology that can suppress a distortion of image information accompanying a temperature change or chronological change of a scan mirror, without adding an optical system such as an angular sensor and the like.SOLUTION: A light measurement device is configured to: scan a light spot of laser light with which a sample is irradiated, by a scan mirror in a first direction and in a second direction opposite the first direction; thereby generate first image information and second image information on the sample respectively; and determine an amount of correction for correcting an action delay of the scan mirror on the basis of the first image information and second image information.SELECTED DRAWING: Figure 8

Description

Technical Field

[0001] The present invention relates to a technique for measuring the size of particles in a liquid sample using light.

Background Art

[0002] In recent years, the target of pharmaceutical development is shifting from small molecule drugs to biopharmaceuticals. Since biopharmaceuticals are macromolecules, they tend to aggregate, and aggregation may cause toxicity. For example, the U.S. Food and Drug Administration and others are trying to strengthen the regulation of the concentration of aggregates. Therefore, there is a need for a technique to quantitatively measure the size distribution of aggregates with a desired density in the submicron region of 0.1 to 1 μm. Protein aggregates float in a solvent, and their positions change with time due to Brownian motion. Hereinafter, in the present invention, a technique for measuring the size and density of protein aggregates and standard particles such as polystyrene beads will be described. These test substances are collectively described as "particles".

[0003] Patent Document 1 describes a technique for detecting particles using optical measurement. The document discloses a "light measurement method for condensing light to generate a light spot and measuring a test object having a size of approximately three times or less the size of the light spot, which includes a signal acquisition step of irradiating the test object while at least moving the focal position of the light in the optical axis direction to detect reflected light reflected from the test object, a step of acquiring correspondence relationship data describing the correspondence relationship between the intensity of the reflected light and the size of the test object, and a size calculation step of acquiring the size of the test object by querying the correspondence relationship data using the intensity of the reflected light." (Claim 1). The technique described in the document can realize high-resolution measurement without pretreatment by interfering the reflected light and the reference light to enhance the signal.

[0004] Patent Document 2 discloses a technique for rapidly scanning a light spot by driving scanning mirrors in the X-axis and Y-axis directions, which are perpendicular to the optical axis, in order to avoid being affected by the motion of particles undergoing Brownian motion in a liquid, based on the technology of Patent Document 1.

[0005] Patent Document 3 addresses the problem of "suppressing errors associated with changes in the scanning conditions of the laser beam" and describes a technology that "the X-axis scanning means 22 and the Y-axis scanning means 23 drive the X-scanning mirror 18 and the Y-scanning mirror 19 according to a drive signal based on predetermined scanning conditions to scan the laser beam, and the angle sensors 22b and 23b output a position signal corresponding to the irradiation position of the laser beam on the sample 13. Then, with the irradiation of the laser beam to the sample suppressed (including stopped) by the light transmittance variable means 16 that adjusts the irradiation of the laser beam to the sample 13 to an intensity lower than that during observation, a drive signal is supplied to the X-axis scanning means 22 and the Y-axis scanning means 23 to pre-drive the X-scanning mirror 18 and the Y-scanning mirror 19, the position signal is measured at a predetermined time, and the deviation time from the position signal delay time predicted in advance relative to the drive signal is calculated. The present invention can be applied, for example, to a laser scanning microscope." (See abstract). [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Japanese Patent Publication No. 2017-102032 [Patent Document 2] WO2020 / 144754 issue [Patent Document 3] Japanese Patent Publication No. 2011-154312 [Overview of the project] [Problems that the invention aims to solve]

[0007] When measuring the size and density of protein aggregates using the techniques described in Patent Documents 1 and 2, resonant galvanometer mirrors and non-resonant galvanometer mirrors are suitable as scanning mirrors in the X and Y axes. However, since these galvanometer mirrors are mechanical parts, their spring constant and resonant frequency change due to temperature changes and changes over time, which in turn changes the amount of operating delay. These changes distort the image information generated from the detection signal stream obtained by scanning the light spot, so the measurement accuracy may decrease due to the effects of temperature changes and changes over time. To suppress this effect, it is conceivable to apply the technique using an angle sensor described in Patent Document 3.

[0008] The techniques described in Patent Documents 1 and 2 involve irradiating a liquid particle to be measured with laser light, generating three-dimensional image information from the reflected light, and measuring the size information of the target particle. The reflected light converted into image information has the same wavelength as the light source and is extremely weak in intensity. Therefore, if the technique using the angle sensor described in Patent Document 3 is combined with Patent Documents 1 and 2, and an angle sensor for laser light deflected by a galvanometer mirror and the associated optical system are added, it becomes difficult to avoid a decrease in measurement accuracy due to the influence of stray light. Consequently, it is considered inappropriate to use the technique of Patent Document 3 to suppress the effects of temperature and changes over time in Patent Documents 1 and 2.

[0009] This invention has been made in view of the above problems, and aims to provide a technology that can suppress distortion of image information due to temperature changes and changes over time in a scanning mirror without adding optical systems such as angle sensors. [Means for solving the problem]

[0010] The optical measuring device according to the present invention generates first image information and second image information of a sample by scanning the light spot of a laser beam irradiated onto the sample in a first direction and a second direction opposite to that direction using a scanning mirror, and determines a correction amount to correct the operating delay of the scanning mirror based on the first image information and the second image information. [Effects of the Invention]

[0011] According to the present invention, distortion of image information due to temperature changes and changes over time in a galvanometer mirror can be suppressed without adding optical systems such as angle sensors. Other problems, configurations, and effects will be clarified by the following description of embodiments. [Brief explanation of the drawing]

[0012] [Figure 1] This is a schematic diagram showing the relationship between focus shift, detection signal, and particle size in the technology described in Patent Document 1. [Figure 2] This is a simulation result showing the relationship between particle diameter and signal intensity. [Figure 3A] This is a schematic diagram showing a container holding a solvent containing the particles to be measured, an optical system, and a signal processing system. [Figure 3B] This shows the configuration when observing a test sample in which area marks are formed with metal on the back surface of a transparent flat plate 202, instead of sample 204. [Figure 4A] This is a magnified view of the image information of the area markings on the test sample. [Figure 4B] This is a magnified view of the image information of the area markings on the test sample. [Figure 5A] This is a reproduction of Figure 1 from Patent Document 3. [Figure 5B] This is a reproduction of Figure 2 from Patent Document 3. [Figure 6A] This diagram schematically shows the time variation of the drive signal or monitor signal of a scanning mirror. [Figure 6B]It is a diagram schematically showing the time change of a drive signal or a monitor signal of a scanning mirror suitable in the present invention. [Figure 7A] It is a diagram showing a detailed configuration of a sample container suitable in the present invention and a frame and a base plate provided in the apparatus. [Figure 7B] It is a schematic diagram showing the relationship between the sample container 703 and the base plate 701. [Figure 8] It is a schematic diagram showing the configuration of a processing system that generates image information from a detection signal in an embodiment. [Figure 9] It is a schematic diagram showing data conversion of a processing system that generates image information from a detection signal. [Figure 10A] It is XY image information of the base plate 701 obtained based on the processing system of the embodiment. [Figure 10B] It is XY image information of the base plate 701 obtained based on the processing system of the embodiment. [Figure 10C] It is XY image information of the base plate 701 obtained based on the processing system of the embodiment. [Figure 11A] It is XY image information of the base plate 701 obtained based on the processing system of the embodiment. [Figure 11B] It is XY image information of the base plate 701 obtained based on the processing system of the embodiment. [Figure 11C] It is XY image information of the base plate 701 obtained based on the processing system of the embodiment. [Figure 12A] It is XY image information of the base plate 701 obtained based on the processing system of the embodiment. [Figure 12B] It is XY image information of the base plate 701 obtained based on the processing system of the embodiment. [Figure 12C] It is XY image information of the base plate 701 obtained based on the processing system of the embodiment. [Figure 13A] It is an experimental result showing the relationship between the correction amount of the operation delay of the scanning mirror in the X-axis direction and the correlation coefficient between ImageX+ and ImageX-. [Figure 13B] This is another experimental result showing the relationship between the correction amount for the operating delay of the scanning mirror in the X-axis direction and the correlation coefficient between ImageX+ and ImageX-. [Figure 14] This experimental result shows the relationship between the correction amount for the operating delay of the scanning mirror in the Y-axis direction and the correlation coefficient between ImageY+ and ImageY-. [Figure 15A] This experimental result shows the relationship between the correction amount for the operating delay of the scanning mirror in the X-axis direction and the correlation coefficient between ImageX+ and ImageX-. [Figure 15B] This experimental result shows the relationship between the correction amount for the operating delay of the scanning mirror in the Y-axis direction and the correlation coefficient between ImageY+ and ImageY-. [Figure 16] This is a flowchart showing the measurement process according to the embodiment. [Figure 17] This is another flowchart showing the measurement process according to the embodiment. [Figure 18] This image information was obtained when the correction amount for the operating delay of the scanning mirror was updated at the edge of the sample container. [Figure 19] This is a schematic diagram showing the configuration of an optical measurement device for measuring particle size distribution. [Figure 20] This schematic diagram shows the time evolution of laser light power and the position of the light spot according to the flow chart in Figure 16. [Figure 21] This flowchart explains the procedure for determining the correction amount using the sample in Figure 18. [Modes for carrying out the invention]

[0013] <Embodiment 1: Problems of the Conventional Technology> Figure 1 is a schematic diagram showing the relationship between focus shift, detection signal, and particle size in the technology described in Patent Document 1. The upper left of Figure 1 geometrically shows the optical system when there is a focus shift for relatively small particles. The geometric spot size at the z position of the particle (coordinate along the optical axis) is geometrically determined by the numerical aperture of the objective lens and the amount of focus shift. The detection signal is attenuated according to the ratio of the projected area of ​​the particle within the area of ​​the geometric beam. This ratio of projected area corresponds to the reaction cross-section of light reflection. The upper right of Figure 1 is a schematic diagram showing the case when there is a focus shift for relatively large particles. Compared to the upper left of Figure 1, the particle size is larger, so the reaction cross-section is larger and the amount of reflected light is also larger. Therefore, it is possible to detect the difference in particle size as a difference in the amount of reflected light (difference in the amount of detection signal). The bottom of Figure 1 shows experimental results showing the relationship between the amount of detection signal at the focal position and the particle size.

[0014] Figure 2 shows the simulation results illustrating the relationship between particle diameter and signal intensity. The simulation method used was the "wave-dynamic ray tracing method" described in Patent Document 1, with a light source wavelength of 785 nm and an objective lens numerical aperture of 0.45. As can be seen in the figure, the magnitude of the detection signal changes depending on the particle size. By pre-storing this correspondence data, it is possible to measure the particle size from the magnitude of the detection signal. The detected reflected light changes according to the refractive index as well as the particle size. In the techniques described in Patent Documents 1 and 2, a detection signal can be obtained by converting the electric field amplitude of the reflected light into a voltage using light interference.

[0015] Patent Document 3 discloses a technique for appropriately correcting changes in the amount of operational delay due to temperature changes and changes over time in a laser scanning microscope, using an angle sensor that detects the angle of laser light deflected by a galvanometer mirror. The technique in Patent Document 3 is primarily intended to generate image information from the fluorescence of organic matter excited by laser light. Therefore, the light that forms the basis of the image information is fluorescence with a wavelength different from that of the light source. Since this is wavelength-separated by a dichroic mirror or the like, even if an angle sensor that detects the amount of deflection of the laser light from the light source by a scanning mirror is added, it will not be affected by stray light generated by the addition of reflective surfaces such as optical path branching mirrors or the end face of the sensor chip.

[0016] Patent documents 1 and 2 suggest using the angle sensor technology described in Patent Document 3 to compensate for the effects of temperature changes and changes over time. However, as mentioned above, the signals converted into image information in Patent Documents 1 and 2 are extremely weak, so if the optical system is changed as in Patent Document 3, the effects of stray light are unavoidable. Therefore, the present invention provides a technology that can suppress the effects of temperature changes and changes over time without using such a sensor.

[0017] In the following description of the present invention, the coordinate system will be unified to one in which the optical axis direction is the Z-axis, and the X and Y axes are located in a plane perpendicular to it. Unless otherwise specified, the light source will be a semiconductor laser with a wavelength of 795 nm, the objective lens will have a numerical aperture of 0.45, the image information range will be 300 μm in both the X and Y directions, the number of pixels will be 400 pixels in both the X and Y directions, the A / D converter will have a sampling frequency of 20 MHz, and the image information acquisition speed will be 10 fps. Furthermore, the description will proceed assuming that a fast-operating resonant galvanometer mirror is used for scanning in the X-axis direction, and a standard galvanometer mirror is used for scanning in the Y-axis direction.

[0018] <Embodiment 1: Optical Measurement Method> Figure 3A is a schematic diagram showing a container holding a solvent containing the particles to be measured, an optical system, and a signal processing system. Here, the size and density of suspended particles are measured by positioning the focal point of a laser beam in the solvent. The laser beam emitted from the light source 100 is focused inside the sample 204 by passing through the transparent plate 202 at the bottom of the container 200 via the objective lens 108. The scanning optical system 107 (scanning mirror) is controlled by the drive circuit 160. The laser beam reflected from the sample 204 interferes with the laser beam reflected by the reference light mirror 106 and is converted into an electrical signal by the detection optical system 112 to become the detection signal. The time-series changes of the detection signal are stored as a digital information sequence in the A / D converter 161. The CPU (Central Processing Unit) 162 (arithmetic unit) generates image information from the time-series data of the detection signal stored in the A / D converter and performs the measurement processing of the size and density of particles in the sample 204. This measurement principle can be based, for example, on the technology described in Patent Documents 1 and 2.

[0019] Figure 3B shows the configuration when observing a test sample in which a region mark is formed with metal on the back surface of a transparent plate 202 instead of sample 204. This can be used in experiments to easily understand the temperature effect of the scanning mirror.

[0020] Figures 4A and 4B are enlarged views of the image information of the area marks on the test sample. Figure 4B shows the same image information when the temperature of the scanning mirror changes by 1°C from that in Figure 4A. Comparing Figures 4A and 4B, it is clear that the change in the operating delay amount of the scanning mirror due to temperature changes has a very large impact on the image information.

[0021] Figure 5A is a reproduction of Figure 1 from Patent Document 3. In the figure, the X-axis and Y-axis scanning mirrors are 22 and 23, and 22b and 23b are sensors that detect the scanning angle of the laser beam by each scanning mirror. By acquiring the signals from these sensors, the control device can suppress distortion of image information due to temperature changes and changes over time of the scanning mirrors.

[0022] Figure 5B is a reproduction of Figure 2 from Patent Document 3. This figure shows the relationship between the drive signal and the position signal of the scanning mirror. The difference between the two during the image acquisition interval is the operating delay amount of the scanning mirror.

[0023] Figure 6A schematically shows the time variation of the drive signal or monitor signal of a scanning mirror. For ease of understanding, it is assumed here that the scanning mirror operates in a sawtooth pattern. The monitor signal is a signal used to electrically monitor the position, velocity, or acceleration of the scanning mirror by a circuit implemented inside the scanning mirror. While it does not generate stray light due to optical influences, it does not accurately represent the scanning angle or the position of the light spot, but rather is an electrical signal that serves as an indicator of the state of the scanning mirror as electrically acquired. In the figure, the image acquisition section of Patent Document 3 is in the section where the drive signal or monitor signal is rising to the right. At this time, the effect of the operating delay amount due to temperature changes or changes over time of the scanning mirror results in a shift in the X-axis or Y-axis direction of the generated image information. In devices such as laser scanning microscopes, where the position of the observed object projected onto the image information is important, it is important to suppress this shift in image information.

[0024] Figure 6B schematically shows the time variation of the drive signal or monitor signal of a scanning mirror preferred in the present invention. In the technologies described in Patent Documents 1 and 2, in order to avoid the influence of Brownian motion of particles suspended in the sample, it is desirable to acquire image information at twice the frame rate by providing image acquisition sections not only in the section where the drive signal or monitor signal is rising to the right, but also in the section where it is falling to the right, as shown in the figure. The experimental data shown in Figures 4A and 4B were generated by utilizing both the forward and return paths of a reciprocating scanning mirror to generate image information, as shown in Figure 6B. Since the shift of image information in the rightward and leftward directions of the paper overlaps for each pixel, this appears as a large distortion in Figure 4B.

[0025] Figure 7A shows a detailed configuration of a sample container and a frame and base plate provided within the apparatus, which are suitable for the present invention. In the figure, 701 is the base plate, 702 is the frame, and 703 is the sample container. The sample container 703 is positioned and set on the base plate 701 along the frame 702. At this time, the transparent plate (described later) formed on the bottom surface of the sample container 703 and the base plate 701 are in contact. It is desirable to use a metal material with high thermal conductivity for the base plate 701. Here, a graphite-based aluminum composite material with high thermal conductivity and excellent processing accuracy was used. With this structure, the sample container 703 can be disposed of after each measurement, avoiding the need for container cleaning and the influence of adhering substances on the measurement, thereby improving user convenience. In this embodiment, considering user handling, the case where the sample container 703 is a set of 4 wells is shown, but it is also possible to create containers with 32 wells as a set, and the number of sets can be appropriately selected considering the cost and yield of the containers.

[0026] Figure 7B is a schematic diagram showing the relationship between the sample container 703 and the base plate 701. In the figure, 701 is the base plate, 703 is the sample container, 704 is the transparent plate, 705 is the sample, 706 is the seal to prevent evaporation of the sample, 710 is the void to improve the shape accuracy of the container by injection molding, and 711 is the heat flow from the sample to the base plate.

[0027] This structure allows the transparent plate 704 formed on the bottom of the container to be in direct contact with the base plate 701. The base plate 701 is provided with a hole corresponding to the transparent plate 704, and its diameter is larger than the diameter of the well containing the sample. In this invention, if there is a difference between the temperature of the sample and the temperature of the measuring device, particle flow due to convection occurs, reducing the measurement accuracy. In this embodiment, by using a glass material with high thermal conductivity as the transparent plate 704, and further by making the transparent plate in contact with a metal base plate 701 with high thermal conductivity, the temperature of the sample can be made the same as the temperature inside the device in a short time, shortening the waiting time for temperature stabilization and improving user convenience. The waiting time for temperature stabilization that the inventors experimentally confirmed was about 10 minutes. In addition, in this embodiment, 96 samples can be measured continuously in a single operation. The time required to complete the measurement of 96 samples under the condition of repeating the above standard measurement 10 times for each sample was about 4.8 hours.

[0028] In this invention, when measuring the operating delay of the scanning mirror, the laser beam 712 is focused on the back surface of the base plate 701 (the side not in contact with the sample container 703), and machining marks are used as image information.

[0029] Figure 8 is a schematic diagram showing the configuration of the processing system for generating image information from the detection signal in this embodiment. This processing system can be implemented by the CPU 162. Here, it is assumed that the time change of the detection signal is stored as time-series data in the internal memory of the A / D converter 161. The time-series data describes the detection signal I, the detection signal Q which is 90 degrees out of phase with respect to I, the drive signal or monitor signal X of the X-axis scanning mirror, and the drive signal or monitor signal Y of the Y-axis scanning mirror. Normally, the entire time-series data of the detection signal is used to generate the measurement image information. When updating the operating delay amount of the scanning mirror, as shown in the figure, the scanning speed is determined and two sets of image information consisting of ImageX+, ImageX-, ImageY+, and ImageY- are generated depending on whether the speed is positive or negative. ImageX+ and ImageY+ are generated from the upward-sloping section shown in Figure 6B, and ImageX- and ImageY- are generated from the downward-sloping section. In the case of the ImageX+ and ImageX- pair, the effect of the scanning mirror's operating delay on the drive signal manifests as a shift in pixel position that is the same magnitude but opposite in orientation in both ImageX+ and ImageX-. This is used to determine the conditions under which ImageX+ and ImageX- best match by varying the amount of correction for the operating delay. For example, the optimal value for the scanning mirror's operating delay correction is determined as the condition that maximizes the correlation coefficient between the two. The same process is performed for ImageY+ and ImageY- in the Y-axis direction.

[0030]

number

[0031] Figure 9 is a schematic diagram showing the data conversion of the processing system that generates image information from the detection signal. The time-series data of the detection signal held by the A / D converter 161 is expressed as I(t), Q(t), X(t), and Y(t) as a function of time t. I(t) and Q(t) are subjected to predetermined gain correction and offset correction as needed to be converted into intensity data A(t) to be included in the image information. In this invention using a homodyne detection optical system, A(t) is expressed by the following equation 2: A(t) = I(t) 2 +Q(t) 2 ...(Equation 2).

[0032] The drive signal or monitor signal X(t) of the scanning mirror in the X-axis direction is converted to the X-axis pixel index X'(t) of the image information based on the motion delay correction parameter. Similarly, the drive signal or monitor signal Y(t) of the scanning mirror in the Y-axis direction is converted to the X-axis pixel index Y'(t) of the image information based on the motion delay correction parameter. Using these, the intensity data A(t) at time t is subjected to predetermined processing such as averaging as intensity data of pixels described by pixel positions i=X'(t) and j=Y'(t). Through these processes, the time-series data of the detection signal is converted to image information. This processing is common to all data used in normal measurements, as well as to ImageX+, ImageX-, ImageY+, and ImageY- used to determine the motion delay correction amount of the scanning mirror in the present invention.

[0033] Figures 10A, 10B, and 10C show the XY image information of the base plate 701 obtained based on the processing system of this embodiment. Figure 10A shows ImageX+, Figure 10B shows ImageX-, and Figure 10C shows the image information for measurement. Figure 10C appears blurred compared to Figures 10A and 10B because the correction amount for the operating delay of the X-axis scanning mirror is smaller than the optimal value. A closer comparison reveals that in Figure 10C, Figures 10A and 10B are shifted in the X direction (horizontal direction of the paper).

[0034] Figures 11A, 11B, and 11C show the XY image information of the base plate 701 obtained based on the processing system of this embodiment. Figure 11A shows ImageX+, Figure 11B shows ImageX-, and Figure 11C shows the image information for measurement. Figure 11C appears to have the same clarity as Figures 11A and 11B because the correction amount for the operating delay of the X-axis scanning mirror is set to the optimal value.

[0035] Figures 12A, 12B, and 12C show the XY image information of the base plate 701 obtained based on the processing system of this embodiment. Figure 12A shows ImageX+, Figure 12B shows ImageX-, and Figure 12C shows the image information for measurement. Figure 12C appears blurred compared to Figures 12A and 12B because the correction amount for the operating delay of the X-axis scanning mirror is greater than the optimal value. A closer comparison reveals that in Figure 12C, Figures 12A and 12B are shifted in the X direction (horizontal direction of the paper), and the shift direction is opposite to that of Figures 10A and 10B.

[0036] Figure 13A shows experimental results illustrating the relationship between the correction amount for the X-axis scanning mirror's operating delay and the correlation coefficient between ImageX+ and ImageX-. The condition that maximizes the correlation coefficient shown in the figure represents the optimal operating delay correction amount. Figure 13A was obtained when the internal temperature of the device was 7°C.

[0037] Figure 13B shows another experimental result illustrating the relationship between the correction amount for the operating delay of the scanning mirror in the X-axis direction and the correlation coefficient between ImageX+ and ImageX-. Figure 13B was obtained when the internal temperature of the apparatus was 28°C. Similarly, the condition that maximizes the correlation coefficient is the optimal operating delay correction amount. As is clear from comparing with Figure 13A, it is shown that this value differs from that at 7°C depending on the internal temperature of the apparatus. If the optimal operating delay correction amount is used at 7°C, it can be seen that the correlation coefficient between ImageX+ and ImageX- drops to almost zero. Therefore, according to this embodiment, by updating the operating delay correction amount before measuring the size and density of particles suspended in the sample, distortion of image information can be prevented and accurate measurements can be performed.

[0038] Figure 14 shows experimental results illustrating the relationship between the correction amount for the operating delay of the scanning mirror in the Y-axis direction and the correlation coefficient between ImageY+ and ImageY-. Similar to the X-axis direction, the condition that maximizes the correlation coefficient represents the optimal correction amount for the operating delay. Figure 14 was obtained when the internal temperature of the device was 28°C.

[0039] Figure 15A shows experimental results illustrating the relationship between the correction amount for the operating delay of the scanning mirror in the X-axis direction and the correlation coefficient between ImageX+ and ImageX-. Here, for the purpose of reducing processing time, the results are shown when the detection range of the correlation coefficient is limited to the central 200x200 pixels for NX=NY=400 pixels. One period corresponds to 5000 sampling points. The horizontal axis (sampling point delay) is the index number representing the amount of shift of the pixel reference point in the time-series data of the detection signal accumulated by sampling at 20MHz. A correlation coefficient of 0.9 or higher was obtained at its maximum, indicating that good correction of the operating delay is possible even under conditions where the detection range of the correlation coefficient is reduced to shorten processing time.

[0040] Figure 15B shows experimental results illustrating the relationship between the correction amount for the operating delay of the Y-axis scanning mirror and the correlation coefficient between ImageY+ and ImageY-. Similar to Figure 15A, to reduce processing time, the results are shown when the detection range of the correlation coefficient is limited to the central 200×200 pixels for NX=NY=400 pixels. One period corresponds to 4 million sampling points. The reason why the half-width of the correlation coefficient is wider and has a larger margin compared to that of the X-axis shown in Figure 15A is that the operating speed of the Y-axis scanning mirror is 1 / 400, which corresponds to the number of pixels, compared to the X-axis scanning mirror.

[0041] Figure 16 is a flowchart showing the measurement flow according to this embodiment. In step S1601, the focal position is moved in the XY axis direction to align with the base plate 701. In steps S1602 and S1603, the sample folder including the base plate is moved in the Z axis direction while the magnitude of the detection signal is measured, and the focal position Z0 at which it is maximized is determined. In step S1604, time-series data of the measurement signal is acquired. In S1605 and S1606, ImageX+ and ImageX- are generated while changing the correction amount of the operating delay amount of the scanning mirror in the X axis direction, and the condition that maximizes the correlation coefficient between the two is determined as the optimal correction amount in the X axis direction. In S1607 and S1608, ImageY+ and ImageY- are generated while changing the correction amount of the operating delay amount of the scanning mirror in the Y axis direction, and the condition that maximizes the correlation coefficient between the two is determined as the optimal correction amount in the Y axis direction. Using the correction amounts of the operating delay of the scanning mirrors in the X axis direction and Y axis direction that have been updated as described above, the size and density of particles suspended in the sample are measured in step S1609.

[0042] Figure 17 is an alternative flowchart showing the measurement process according to this embodiment. The update of the correction amount for the scanning mirror's operating delay according to this embodiment does not necessarily have to be performed before measuring all samples, as the primary cause is temperature change. This flowchart is a modified version of Figure 16 from this perspective.

[0043] In step S1701, the measurement conditions for measuring one sample are initialized. In step S1702, the measurement conditions of the optical system and electrical system, including the optical path length of the reference optical mirror, are adjusted. In step S1703, it is determined whether or not it is necessary to update the operating delay amount of the scanning mirror, and only if it is determined to be necessary, the correction amount for the operating delay amount of the scanning mirror is updated in step S1704. The update process is the same as in Figure 16. The determination of whether or not an update process is necessary can be made, for example, as follows: (a) necessary for the first measurement after the device is started up, (b) necessary for the first measurement in a series of continuous measurements of a series of samples, (c) necessary if a certain amount of time has passed since the last update, (d) necessary if the current device temperature has changed by a certain amount or more from the device temperature at the time of the last update, (e) not necessary otherwise. If it is determined that an update process is not necessary, the latest correction amount that is retained is used as is. In step S1705, the size and density of particles suspended in the sample are measured.

[0044] Figure 18 shows image information obtained when the correction amount for the operating delay of the scanning mirror is updated at the edge of the sample container. The image information used to update the correction amount for the operating delay of the scanning mirror by the optical measurement method of this embodiment is not limited to time-series data of the detection signal acquired on the back surface of the base plate. It is also possible to use time-series data of the detection signal acquired at a different location. In the figure, the left side is the area containing the sample, and the right side is the wall surface of the sample container. If the image information acquired in this way is not uniform in the plane, time-series data of the detection signal acquired at any position within the position range observable by the device can be used.

[0045] <Embodiment 1: Summary> The optical measurement method according to this embodiment aligns the focal position of the laser beam with the base plate 701 (or the edge of the sample container), scans the optical spot in the XY direction, and divides the time-series data of the detection signal into cases where the scanning speed of the optical spot is positive and negative, generating a set of image information from each detection signal stream. The image information for the positive scanning speed case and the image information for the negative scanning speed case are compared to search for the condition in which the two best match, and the correction amount for the operating delay of the scanning mirrors in the XY direction is determined based on that condition. This makes it possible to achieve stable measurement that is not affected by temperature changes or changes over time without placing additional devices such as angle sensors.

[0046] <Embodiment 2: Optical Measuring Device> Figure 19 is a schematic diagram showing the configuration of the optical measuring device for measuring particle size distribution according to the present invention. Laser light emitted from a light source 100, whose emission state is controlled by a laser driver 101 that controls high-frequency superposition and emission power, is converted into parallel light by a collimating lens 102, and after the polarization direction is adjusted by a λ / 2 plate 103 whose optical axis is set at approximately 22.5 degrees with respect to the horizontal direction, it is separated into signal light and reference light by a polarizing beam splitter 104.

[0047] The reference light is converted to a circularly polarized state by the λ / 4 plate 105, reflected by the reference light mirror 106, and then reflected by the λ / 4 plate 105 with a polarization state rotated 90 degrees from the forward path, and then reflected by the polarizing beam splitter 104. The signal light is deflected in the direction of propagation by the scanning optical system 107 (composite deflection mirrors in the X and Y directions), converted to a circularly polarized state by the action of the built-in λ / 4 plate, and focused in the sample 204 by the objective lens 108. The sample 204, the sample container 203 enclosing it with a transparent flat plate 202 formed on its bottom surface, and the base plate 201 are positioned in the Z-axis direction by a drive mechanism 109 that moves in the Z-axis direction. As a result, the focal position of the signal light is scanned along the Z-axis direction. The signal light component reflected from particles suspended within sample 204 is deflected by the scanning optical system 107 in the same direction as the forward path, and, due to the action of the built-in λ / 4 plate, becomes polarized in a state rotated 90 degrees from the forward path, before passing through the polarizing beam splitter 104. Here, the sample container 203 holds the sample 204 and guides the signal light into the sample via the transparent plate 202. The base plate 201 contacts the transparent plate 202 to mechanically hold the sample container 203 and also stabilizes the sample temperature.

[0048] The signal light and the reference light are combined by a polarizing beam splitter 104 and guided to the detection optical system 112, where they are split into transmitted light and reflected light by a half-beam splitter 113 via a pinhole 111.

[0049] The reflected light passes through a λ / 4 plate 114 with its optical axis set at approximately 45 degrees to the horizontal, then is focused by a focusing lens 115 and split into two by a polarizing beam splitter 116. Each split is then photoelectrically converted by photodetectors 150 and 151, and differentially amplified by a current differential amplifier 152 to produce a detection signal Q123.

[0050] The transmitted light passes through a λ / 2 plate 118 whose optical axis is set at approximately 22.5 degrees with respect to the horizontal direction, and is then focused by a focusing lens 119 and split into two by a polarizing beam splitter 120. Each of these is then photoelectrically converted by photodetectors 153 and 154, and differentially amplified by a current differential amplifier 155 to produce a detection signal I122.

[0051] The detection optical system 112 shown here constitutes a homodyne phase diversity method, and the detection signals 122 and 123 are the real and imaginary parts of the complex electric field obtained by intensifying the reflected light from the target particle with reference light.

[0052] The A / D converter 161 acquires and stores the detection signal I122, detection signal Q122, and the drive signals or monitor signals X and Y124 of the X-axis and Y-axis scanning mirrors built into the scanning optical system 107 as time-series data. The intensity signal S required for particle size measurement is calculated from the detection signals I122 and Q122 using (Equation 2). The control signals or monitor signals X and Y of the X-axis scanning mirror are converted into pixel position signals X' and Y' based on the correction amount of the latest scanning mirror's operating delay. The image information required for measurement is generated from the time-series data of the intensity signal S and the pixel position signals X' and Y'. The signal processing unit 124 generates the image information for measurement shown here, controls the operation of the device components including the light source, and determines the correction amount of the operating delay for at least one of the X-axis scanning mirrors and the Y-axis scanning mirrors through the processing systems shown in Figures 8 and 9, and performs correction amount update processing based on the flows in Figures 16 and 17. The display unit 170 displays the calculation results from the signal processing unit 124 and presents them to the user.

[0053] Using this device, continuous automatic measurement of an array of multiple sample containers shown in Figure 7A can be easily achieved by adding a sample movement function in the XY direction as well as the Z direction to the drive mechanism 109.

[0054] <Embodiment 3> Embodiment 3 of the present invention describes the detailed operation or modified form of each part when determining the correction amount. The configuration of the optical measuring device, the operating procedure, and other matters are the same as in the embodiments described above.

[0055] Figure 20 is a schematic diagram showing the time evolution of laser beam power and optical spot position according to the flow in Figure 16. In the figure, the measurement section follows the calibration section of the scanning mirror delay. Regarding the optical spot position, after moving from the initial value to a predetermined (X,Y) position, the focal position is determined by scanning in the Z direction, and the signal is measured at this focal position, and the delay correction amount is updated. Regarding the laser beam power, the delay correction amount is updated at a power P0 suitable for measuring the effect signal from a predetermined base plate. For measuring the size and density of particles suspended in the sample, the measurement process is performed at a different predetermined power P1. Since the magnitude of the reflected light obtained from the base plate is larger than the magnitude of the reflected light from the particles suspended in the sample, setting P0 ≤ P1 makes it possible to effectively utilize the dynamic range and SNR of the A / D converter and circuit system.

[0056] Figure 21 is a flowchart illustrating the procedure for determining the correction amount using the sample shown in Figure 18. The edge portion of the sample container shown in Figure 18 has different image information brightness in the left-right direction, making it suitable for updating the delay correction amount of the scanning mirror in the X direction. On the other hand, to update the delay amount of the scanning mirror in the Y direction, it is preferable to perform the update process at a position (X,Y) where the image information brightness differs in the up-down direction. Therefore, Figure 21 shows the processing flow when updating the delay amount using image information appropriate for each direction.

[0057] In step S2101, the optical spot is moved to a position (X1, Y1) suitable for updating the delay correction amount in the X-axis direction. In step S2102, the detection signal is acquired while moving the optical spot in the Z-axis direction, and the position of the optical spot is determined to be at the focal position Z1 as the condition in which the magnitude of the signal is maximized. In step S2103, time-series data of the measurement signal is acquired. In S2104, ImageX+ and ImageX- are generated while changing the correction amount of the operating delay amount of the scanning mirror in the X-axis direction, and the condition in which the correlation coefficient between the two is maximized is determined as the optimal correction amount in the X-axis direction. In step S2105, the optical spot is moved to a position (X2, Y2) suitable for updating the delay correction amount in the Y-axis direction. In step S2106, the detection signal is acquired while moving the optical spot in the Z-axis direction, and the position of the optical spot is determined to be at the focal position Z2 as the condition in which the magnitude of the signal is maximized. In step S2107, time-series data of the measurement signal is acquired. In step S2108, ImageY+ and ImageY- are generated while varying the correction amount for the operating delay of the scanning mirror in the Y-axis direction, and the condition that maximizes the correlation coefficient between the two is determined as the optimal correction amount in the Y-axis direction. Using the updated correction amounts for the operating delay of the scanning mirrors in the X-axis and Y-axis directions, the size and density distribution of particles suspended in the sample are measured in step S2109.

[0058] <Regarding variations of the present invention> The present invention is not limited to the embodiments described above, and includes various modifications. For example, the embodiments described above are described in detail to make the present invention easier to understand, and are not necessarily limited to those having all the configurations described. Furthermore, it is possible to replace parts of the configuration of one embodiment with the configuration of another embodiment, and it is also possible to add configurations from other embodiments to the configuration of one embodiment. In addition, it is possible to add, delete, or replace parts of the configuration of each embodiment with other configurations.

[0059] In the embodiments described above, the present invention was applied to the optical measurement methods for particle size and density described in Patent Documents 1 and 2. However, the method for determining and updating the correction amount for the operating delay of the scanning mirror shown here can also be applied to laser scanning microscopes such as those described in Patent Document 3. The technology described in Patent Document 3 determines the correction amount for the operating delay of the scanning mirror using an angle sensor that detects the scanning angle of the laser beam by the scanning mirror. However, it cannot compensate for the effects of changes in shape or voltage drift due to temperature changes or changes over time, such as fluctuations in the power supply voltage of the microscope tube that holds the angle sensor or the circuit that drives the sensor. On the other hand, the technology disclosed in the present invention directly optimizes the image information, which is the ultimate goal of measurement, so it is possible to generate stable image information without being affected by the changes or drifts described above. In this case, if image information of an optically nearly uniform object, such as the surface of a silicon substrate, is used, the divided image information does not change much with respect to pixel shift, so it is not possible to accurately determine the correction amount for the operating delay of the scanning mirror. In such cases, it is desirable to determine the correction amount at a location where the image information is not uniform in the plane, such as the edge of a slide.

[0060] The above embodiments describe a method for simultaneously determining and updating the correction amount for the operating delay for both the X-axis and Y-axis scanning mirrors. However, the technology of the present invention can also be applied to only one of the X-axis or Y-axis. In that case, it should be noted that a reduction in processing time can be obtained.

[0061] In the embodiments described above, for example, the condition that maximizes the correlation coefficient between ImageX+ and ImageX- was used to determine the optimal correction amount for the operating delay of the scanning mirror in the X-axis direction. This means finding the condition that maximizes the degree of agreement between the two. Therefore, in addition to the condition for maximizing the correlation coefficient, the condition for minimizing the RMS error between the two, the condition for minimizing the sum of the absolute values ​​of the differences, etc., can also be used to determine the optimal correction amount for the operating delay. It is mathematically guaranteed that the same result can be obtained within the CPU's calculation error range even if indicators other than the correlation coefficient are used. For example, depending on the objective of simplifying processing or shortening the device development period, such as reducing processing time or reusing existing FPGAs, the indicators and conditions for obtaining the optimal correction amount can be appropriately selected based on the present invention.

[0062] In the embodiments described above, the objective lens 108 and the drive mechanism 109 serve as illumination units that focus and irradiate the sample with signal light, and the detection optical system 112 serves as a detection unit that detects interference signals generated by the interference between signal light and interference light. [Explanation of symbols]

[0063] 100: Light source 108: Objective lens 109: Drive mechanism 112: Detection Optics 161: A / D converter 162:CPU (arithmetic unit) 201: Base Plate 202: Transparent flat plate 203: Sample container

Claims

1. A light measuring device that measures a sample by irradiating it with light, Irradiation unit that irradiates the sample with laser light, A mechanism for scanning the light spot of the laser beam in a plane perpendicular to the optical axis of the laser beam by using a scanning mirror to move the light spot of the laser beam back and forth in a first direction and a second direction opposite to the first direction. A detector that detects the amount of light optically obtained by the aforementioned scanning as a detection signal. A calculator that processes the aforementioned detection signal, Equipped with, The arithmetic unit generates first image information of the sample from the detection signal when the light spot is moved in the first direction, The arithmetic unit generates second image information of the sample from the detection signal when the light spot is moved in the second direction. The arithmetic unit determines a correction amount to correct the operating delay of the scanning mirror based on the first image information and the second image information. The calculator determines a correction value as the correction amount that optimally corrects the delay amount between the drive signal of the scanning mirror and the position signal of the light spot. The calculator determines the correction amount by searching for the correction value that maximizes the degree of agreement between the first image information and the second image information while changing the candidate values ​​of the correction value. An optical measuring device characterized by the following features.

2. The mechanism scans the light spot along a first in-plane direction within the plane, The arithmetic unit generates first image information and second image information obtained by scanning the light spot in the first in-plane direction, The arithmetic unit determines the correction amount for correcting the operating delay of the scanning mirror in the first in-plane direction. The optical measuring device according to claim 1, characterized in that it is a light measuring device.

3. The aforementioned arithmetic unit, A step of scanning the light spot in a third direction and a fourth direction opposite to the third direction in a second in-plane direction that is perpendicular to the first in-plane direction within the plane, A step of generating third image information of the sample from the detection signal when the light spot is moved in the third direction, A step of generating a fourth image information of the sample from the detection signal when the light spot is moved in the fourth direction, We will implement the following: The arithmetic unit determines, in addition to the correction amount in the first in-plane direction, a correction amount for correcting the operating delay of the scanning mirror in the second in-plane direction. The optical measuring device according to claim 2, characterized in that it is a light measuring device.

4. When determining the correction amount, the illumination unit sets the focus of the light spot at a position where the image in a plane perpendicular to the optical axis is not uniform. The optical measuring device according to claim 1, characterized in that it is a light measuring device.

5. When determining the correction amount, the irradiation unit sets the focus of the light spot on the surface of the base plate, which is located on the bottom surface of the container containing the sample, that is not in contact with the container. The optical measuring device according to claim 1, characterized in that it is a light measuring device.

6. The aforementioned arithmetic unit, The first measurement after the aforementioned optical measuring device has been started up, The first measurement when measuring multiple samples consecutively, After a predetermined amount of time has elapsed since the last update of the aforementioned correction amount, When the temperature of the optical measuring device changes by more than a threshold value relative to the temperature at the time the correction amount was last updated, In at least one of the above, the correction amount is determined. The optical measuring device according to claim 1, characterized in that it is a light measuring device.

7. The aforementioned sample is a liquid sample containing particles, The optical measuring device further includes a branching unit that splits the laser light into signal light and reference light, The irradiation unit focuses the signal light onto the sample and irradiates it. The optical measuring device further includes an interference optical system that obtains an interference signal by interfering the signal light reflected from the particle with the reference light, The calculator generates image information of the sample from the interference signal and uses that image information to measure at least one of the particle size or the particle density distribution. The optical measuring device according to claim 1, characterized in that it is a light measuring device.

8. When determining the correction amount, the irradiation unit sets the focus of the light spot on a member with a higher light reflectance than the sample. When determining the correction amount, the irradiation unit emits the laser light at the first power, When measuring the sample, the irradiation unit emits the laser light with a second power greater than the first power. The optical measuring device according to claim 1, characterized in that it is a light measuring device.

9. The illumination unit sets the focus of the light spot at a first position where the brightness value of the sample image is not uniform in the first plane direction, The arithmetic unit generates first image information and second image information, respectively, obtained by scanning the light spot in the first in-plane direction with the first position as the initial position. The illumination unit sets the focus of the light spot at a second position where the brightness value of the sample image is not uniform in the second plane direction, The arithmetic unit generates the third image information and the fourth image information, respectively, obtained by scanning the light spot in the second plane direction with the second position as the initial position. The optical measuring device according to claim 3.

10. A photometric measurement method that measures a sample by irradiating it with light, The step of irradiating the sample with laser light emitted from a light source, A step of scanning the light spot of the laser beam in a plane perpendicular to the optical axis of the laser beam by moving the light spot back and forth in a first direction and a second direction opposite to the first direction using a scanning mirror. A step of detecting the amount of light optically obtained by the scanning as a detection signal, A step of generating first image information of the sample from the detection signal when the light spot is moved in the first direction, A step of generating second image information of the sample from the detection signal when the light spot is moved in the second direction, A step of determining a correction amount for correcting the operating delay of the scanning mirror based on the first image information and the second image information. It has, In the step of determining the correction amount, a correction value that optimally corrects the delay between the drive signal of the scanning mirror and the position signal of the light spot is determined as the correction amount. In the step of determining the correction amount, the correction amount is determined by searching for the correction value that maximizes the degree of agreement between the first image information and the second image information while changing the candidate values ​​of the correction value. A method for measuring light characterized by the following features.

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