JP2023123194A - Analysis device, and analysis method - Google Patents

Abstract

To provide an analysis device and analysis method that enable simultaneous and instantaneous measurement of DLS and SLS for the same object.SOLUTION: There are provided an analysis method and an analysis device comprising: a light irradiation unit 140 that forms a focus within a sample 154 on the basis of light generated by a light source 110; a parallel scattering light conversion unit 170 that receives small-angle scattering light 160 from an analysis minute region 156 having the focus formed within the sample 154, and generates parallel small-angle scattering light 168; a scattering light detection unit 200 that measures the small-angle scattering light 160 from the sample 154 on the basis of the parallel small-angle scattering light 168; and an analysis unit 250 that measures the sample 154 on the basis of a measurement result of the scattering light detection unit 200, having a storage device 290. In the analysis method, the scattering light detection unit 200 measures the small-angle scattering light 160, and the analysis unit 250 analyzes both a dynamic scattering light component and static scattering light component on the basis of the measurement result.SELECTED DRAWING: Figure 1

Description

The present invention relates to an analysis apparatus and an analysis method for analyzing a sample based on scattered light obtained by irradiating the sample with light.

Conventionally, the size and shape of fine particles in a sample liquid, the size and structure of their aggregates, especially the fine particles and aggregates of nanometer to sub-micrometer size, have been the subject of analytical evaluation. ing. For example, particles such as nano- to sub-micron metals, plastics, and inorganic substances that are dispersed in a sample liquid and have relatively little interaction with solvent molecules, and the states of their aggregates are analyzed and evaluated. In addition, for example, the state of molecules strongly interacting with solvent molecules and dissolved, such as low and medium molecules such as peptides and nucleic acids, and biopolymers such as proteins, and their aggregates in a sample solution are analyzed and evaluated. is subject to

Two light scattering measurements are commonly used in the above analytical evaluations. One of the light scattering measurement methods is dynamic light scattering (DLS), which detects light scattering from an object to be analyzed and evaluated at a predetermined scattering angle and measures the temporal variation (fluctuation) of light scattering. Scattering) measurement method. DLS measurements primarily provide information about the motion of the scatterers to be analyzed.

Another light scattering measurement method is a static light scattering (SLS) measurement method that measures the dependence of the scattering angle on the average intensity of light scattering from an analyte. SLS measurements primarily provide information about the structure of the scatterer to be analyzed.

Sample analyzers using the above DLS measurement method or SLS measurement method are described in Patent Document 1, Patent Document 2, and Patent Document 3 described below.

Japanese Patent No. 5883233 Japanese Patent No. 6757964 Japanese translation of PCT publication No. 2019-536997

T. Wakamatsu, T.; Onoda, M.; Makoto, "Time-resolved forward-light-scattering monitoring of protein-lysozyme aggregation in precrystalline solutions", Japanese Journal of Applied Physics, (2 018), vol. 57, 058003-1 to 058003-3.

The state of the measurement target, which is a sample, changes with the passage of time. Sample aging is unavoidable. It is very important to obtain a large amount of information on the same measuring object and in the same elapsed time state. For example, if DLS and SLS measurements can be performed simultaneously on the same measurement target, very valuable information regarding the measurement target can be obtained. Although this is just one example, valuable information can be obtained for analyzing and evaluating knowledge about the motion and structure of the analysis target such as particles and aggregates.

A search of known literature was conducted, but no known literature was found that describes or suggests the importance of simultaneously measuring DLS and SLS for the same measurement object. Also, no known document suggesting a configuration for realizing this was found.

SUMMARY OF THE INVENTION An object of the present invention is to provide an analyzer and an analysis method capable of performing both DLS measurement and SLS measurement on the same object under the same elapsed time. .

[First Invention]
The first invention for solving the above problems is
a light source for generating light used for measuring the sample;
a light irradiation unit that irradiates the sample with irradiation light that forms a focal point in the sample based on the light generated by the light source;
a parallel scattered light conversion unit that receives forward or backward small-angle scattered light generated in the analysis microregion, which is the region where the focal point of the irradiation light is formed, and generates parallel small-angle scattered light;
a scattered light detector for measuring the state of the small-angle scattered light from the sample based on the parallel small-angle scattered light;
an analysis unit equipped with a storage device that analyzes the sample based on the measurement result of the scattered light detection unit;
The analysis unit repeatedly stores the measurement result of the scattered light detection unit in the storage device,
Based on the measurement result of the scattered light detection unit stored in the storage device, the analysis unit detects the dynamic scattered light component, the static scattered light component, or the dynamic scattered light component and the static scattered light. The analysis apparatus is characterized in that it analyzes both of the components and analyzes the analysis microregion of the sample.

[Second Invention]
A second invention for solving the above problems is the analysis device of the first invention,
The scattered light detection unit includes a photodetector,
generating a microscope image on the photodetector, which is a magnified image of the state of the sample present in the analysis microregion;
The analysis device is characterized in that the analysis unit repeatedly takes in the microscope image as the measurement result of the scattered light detection unit and stores it in the storage device.

[Third Invention]
A third invention for solving the above problems is the analyzer of the second invention,
The analysis unit stores moving images of the microscope images in the storage device by repeatedly capturing the microscope images from the scattered light detection unit,
The analysis unit is an analysis apparatus characterized by analyzing the dynamic scattered light component and the static scattered light component based on the stored moving image.

[Actions and effects of the first to third inventions]
Next, the effects of the first to third inventions will be described. In the configurations of the first to third aspects of the invention, the analysis unit has a configuration in which the measurement results of the scattered light detection unit are repeatedly stored in the storage device, for example, a predetermined number of times or for a predetermined period of time. are doing. Therefore, it is possible to instantaneously obtain the sample measurement results necessary for the analysis of dynamic scattered light and static scattered light. In many cases, the state of the sample changes with the passage of time, but in the first to third inventions, the measurement results of the sample necessary for the analysis of dynamic scattered light and static scattered light can be viewed as being obtained simultaneously and instantaneously. That is, it is possible to analyze and evaluate the same measurement target region by the dynamic scattering measurement method and the static scattering measurement method in the same state over time.

Moreover, in the conventional technique, the analyzable area in the sample is large, and analysis narrowed down to a minute area is impossible. In the first invention, a light irradiating section for irradiating the sample with irradiation light forming a focal point in the sample is provided, and the area to be analyzed can be narrowed down to a very small analysis minute area.

For example, in the technique described in Patent Document 1, a measurement analysis device that acquires time-averaged autocorrelation function-relaxation data of the correlation function, that is, a DLS measurement analysis device, scattering by a rotating mechanism (scattering angle adjustment mechanism) DLS measurements are performed for each scattering angle by changing the angle. See, for example, paragraph [0045] of Patent Document 1.

Further, in Patent Document 1, as another embodiment of a scanning microscopic light scattering measurement analysis apparatus, only scattered light at a selected scattering angle is detected by using an objective lens and a cover member for scattered light from a sample. For the DLS measurement method with selective detection (for example, as described in paragraph [0103] of Patent Document 1, etc.), a scattering angle selection filter was also placed between the objective lens and the imaging lens, and was similarly selected. A DLS measurement method that selectively detects only the scattered light at the scattering angle is disclosed (eg, as described in paragraph [0115] of US Pat.

However, there is no disclosure of a means for measuring scattered light by continuously changing the scattering angle using the cover member and the scattering angle selection filter (herein referred to as a scattering angle selection device for convenience). Using the scattering angle selection device, the scattering angle can be instantaneously and continuously changed (for example, in the scattering angle range of 1° to 15°, the angular resolution is about 0.1° or less, and the instantaneous measurement), SLS measurement for measuring the intensity spatial pattern of scattered light (scattered light intensity distribution with respect to the scattering angle) is technically difficult. Therefore, with the technique of Patent Document 1, it is difficult to measure DLS and SLS at the same time.

Further, Patent Document 1 discloses DLS measurement at multiple points on a sample, for example, DLS measurement for a minute volume while changing measurement positions at intervals of several μm by a scanning mechanism. See, for example, paragraph [0007] of Patent Document 1. However, as described above, it is difficult to instantaneously measure SLS from a sample on a microscope scale by using the scattering angle selection device, and furthermore, it is difficult to simultaneously and instantaneously measure DLS and SLS on a microscope scale. there were.

In addition, in Patent Document 2, in the measurement of small-angle forward scattered light or small-backward scattered light from a protein crystallization solution, the scattered light is taken in a predetermined scattering angle range, converted into parallel scattered light, and the scattered light The intensity distribution (distribution function for the scattering angle) was measured in real time at once. See, for example, paragraph [0070] of Patent Document 2.

Patent Literature 2 discloses a real-time SLS measurement method and a method for analyzing the SLS measurement data. See, for example, paragraph [0107] of Patent Document 2. However, neither a real-time DLS measurement method nor an analysis method thereof is disclosed or suggested, and a simultaneous measurement method of DLS and SLS and an analysis method thereof are not described. Furthermore, in Patent Document 2, the incident light incident on the solution cell is parallel light (collimated light). See, for example, paragraph [0056] of Patent Document 2. The size of the incident light beam is, for example, about 0.7 mm in diameter. See, for example, paragraph [0133] of Patent Document 2. For this reason, it is difficult to carry out simultaneous DLS and SLS measurements on a microscopic scale in a microscopic region of, for example, several tens to hundreds of μm 2 or less, and to locally analyze and evaluate particles and aggregates. Therefore, in Patent Document 2, DLS and SLS are measured simultaneously and instantaneously, the diffusion coefficient and size of the particles and aggregates to be analyzed, the structure of the aggregates, etc. are analyzed, and the structure of the solution, etc. is analyzed on a microscope scale. It is difficult to analyze the microstructure.

Furthermore, the technique described in US Pat. No. 6,200,000 generates interfering beams in a sample by means of an optical element including an axicon lens to form a substantially unilluminated dark region, thereby capturing scattered light in the dark region in the far field. The technique is positioned to detect and perform DLS and/or SLS measurements at low scattering angles of 0° to 10° from the illumination axis. The scattered light detector has a plurality of detectors arranged in the range of 0° to 10°. See, for example, paragraph [0015] of Patent Document 3. For example, the detector or detectors are configured to detect scattered light in the range of 1° or 2° or 3° or 4° or 5°. See, for example, paragraph [0016] of Patent Document 3.

That is, it is difficult to instantaneously measure a continuous SLS spatial pattern to detect the scattered light at discrete scattering angles using multiple detectors. Here, it is described that an optical fiber is coupled to the detector for the case of measuring scattered light at an arbitrary angle between 0° and 10°. See, for example, paragraph [0016] of Patent Document 3. However, there is no disclosure of a specific method. Scattered light captured by an optical fiber with respect to various angles is detected by a detector and converted into an SLS signal, that is, a scattered light intensity function (scattering angle dependence of scattered light intensity). It is technically difficult to obtain. Therefore, in Patent Document 2, it is difficult to perform DLS and SLS simultaneously and instantaneously.

[Action and effect of the second invention and the third invention]
In addition to the above description, in the second and third inventions, forward or backward small-angle scattered light is received from the analysis minute region in which the focal point of the irradiation light is formed in the sample, and parallel scattered light is received. A configuration is provided in which the scattered light detection section detects parallel small-angle scattered light formed by the conversion section. In the second invention, as described above, the photodetector generates a microscopic image, which is an image magnified on a microscopic scale of the state of the sample existing in the microanalysis area. In addition, by continuously and repeatedly taking in the microscope images, it has become possible to measure the momentary changes in the microscope images as moving images. A DLS analysis can be performed by analyzing the time-varying changes for a particular region. Also, SLS analysis can be performed by capturing and analyzing the microscopic image in a region extending radially from the optical axis. That is, it is possible to capture the process of changes in the sample over time that occur in the same region as moving images of microscope images, which are images magnified on a microscope scale. Further, from this moving image, analysis results of both DLS analysis and SLS analysis can be obtained at a specific point in time in the course of the sample's change over time. Moreover, analysis can be performed using the measurement results in a state of being enlarged on a microscope scale. In the prior art, such analysis could not be performed.

[Fourth invention]
A fourth invention for solving the above problems is the analysis device according to one of the first to third inventions,
A scattering angle filter and a scattered light collecting section are provided between the parallel scattered light conversion section and the scattered light detection section, and the scattering angle filter detects the parallel small angle scattered light from the parallel scattered light conversion section. Arc-shaped specific-angle scattered light having a specific angle and forming an arc shape on a plane perpendicular to the optical axis of the parallel small-angle scattered light is detected through the scattered-light collecting unit. incident on the photodetector of the part,
The analyzer detects the microscopic image, which is an enlarged display of the state of the sample in the analysis minute area, by the photodetector when the parallel small-angle scattered light is incident on the photodetector. is.

[Fifth invention]
A fifth invention for solving the above problems is the analyzer of the fourth invention,
The analysis unit repeatedly acquires and stores the microscopic image, which is an enlarged display of the state of the sample in the microanalysis area, detected by the photodetector, thereby reducing the quality of the sample in the microanalysis area. The analysis device is characterized in that the moving image of the microscope image in which the state is enlarged and displayed is stored in the storage device.

[Sixth invention]
A sixth invention for solving the above problems is the analyzer of one of the fourth invention or the fifth invention,
The scattering angle filter is made of a material that blocks light, and has an arcuate opening with a radius corresponding to the specific angle with respect to the optical axis of the parallel small-angle scattered light. Characterized as an analytical device.

[Effects of the fourth to sixth inventions]
As will be described below with reference to the embodiments shown in FIGS. 10 to 14, small-angle scattered light 160 is generated by the sample existing in the analysis minute region 156 in the sample 154 based on the irradiation light 148 emitted from the light irradiation unit 140. made. This small-angle scattered light 160 is converted into parallel small-angle scattered light 168 by the parallel scattered light converter 170 . When the parallel small-angle scattered light 168 is incident on the scattering angle filter 180 , the arc-shaped specific angle scattered light 186 having a specific scattering angle and an arc shape on a plane perpendicular to the optical axis 146 passes through the scattering angle filter 180 . It passes through and enters the scattered light collector 190 . This arc-shaped specific-angle scattered light 186 is incident on the photodetector 202 via the scattered light collector 190, so that the image representing the state of the sample present in the analysis microregion 156 is enlarged into a microscopic image. , are detected by photodetector 202 . This microscope image can be captured and stored in the analysis unit 250 as the detection result of the photodetector 202 .

Although publicly known documents have been investigated, no known example disclosing or suggesting a technology capable of displaying an enlarged image representing the state of the sample existing in the analysis minute area 156, that is, a microscopic image could be found. Note that Patent Document 1 describes a scattering angle selection filter 19 . However, the aperture of this scattering angle selection filter 19 is a point and does not spread in an arc shape. Therefore, the microscope image cannot be produced.

[Seventh Invention]
A seventh invention for solving the above problems is the analyzer according to one of the first to sixth inventions,
The analyzer is characterized in that the incident light incident on the light irradiation unit is a light beam having a circular cross-sectional shape, and the diameter of the light beam of the incident light is 1.6 mm or less.

[Eighth Invention]
An eighth invention for solving the above problems is the analysis device of one of the first to seventh inventions,
The light irradiation section includes a condenser lens for condensing light and a lens adjustment mechanism for moving the condenser lens along an optical axis of the incident light incident on the light irradiation section. ,
The lens adjustment mechanism is characterized in that the distance that the condenser lens can be moved along the optical axis is ±10 mm or less, and the minimum length that can be moved and adjusted is 50 μm or less.

[Ninth Invention]
A ninth invention for solving the above problems is the analysis device of one of the first to eighth inventions,
An analysis apparatus characterized in that a diameter of a minimum beam spot formed inside the sample by irradiating the sample with the irradiation light from the light irradiation unit is in the range of 10 μm or more and 100 μm or less. is.

[Tenth invention]
A tenth invention for solving the above problems is the analyzer of one invention among the first invention to the ninth invention,
having a sample section,
The sample unit includes a sample cell for holding the sample and a sample position adjustment mechanism for adjusting the position of the sample cell,
The analysis unit outputs an adjustment image for adjusting the position of the sample cell by the sample position adjustment mechanism based on the measurement result of the scattered light detection unit,
The analysis unit repeatedly stores the measurement result of the scattered light detection unit in the storage device based on a measurement instruction for performing the analysis,
The analysis unit is an analysis apparatus, characterized in that the analysis is performed based on the measurement results of the scattered light detection unit repeatedly stored in the storage device.

[Action and effect of the tenth invention]
The effects of the tenth invention will be described. In the first invention, by forming the focus of the irradiation light on the region desired to be analyzed within the sample, there is an effect that the state of the region desired to be analyzed can be accurately measured. However, in order to make use of this effect, it is necessary to adjust the position of the sample cell, for example, in a plane perpendicular to the optical axis, using a sample position adjustment mechanism, so that the focus of the irradiation light is formed on the region to be analyzed. becomes. In the present invention, an adjustment image is displayed so that this work can be performed easily and accurately. This adjustment image is drawn, for example, based on the small-angle scattered light generated from the sample at the focal position of the current irradiation light. By adjusting the position of the sample cell using the sample position adjustment mechanism while viewing this image, the position of the sample cell can be adjusted more accurately and more smoothly.

[Eleventh Invention]
An eleventh invention for solving the above problems is the analysis apparatus according to one of the first to tenth inventions, wherein the maximum incidence of the irradiation light with respect to the optical axis from the light irradiation unit onto the sample is The analytical device characterized in that the angle θi ranges from 0.5 degrees to 20 degrees.

[Twelfth Invention]
A twelfth invention for solving the above problems is the analyzer of one invention among the first invention to the eleventh invention,
a first step in which the analysis unit acquires the measurement results of the repeated scattered light detection unit based on set sampling conditions;
a second step of extracting the measurement result of the dynamic scattered light analysis region and the measurement result of the static scattered light analysis region from the measurement results of the scattered light detection unit that are repeatedly captured;
performing calculations for dynamic scattered light analysis based on the measurement results of the dynamic scattered light analysis area and calculations for static scattered light analysis based on the measurement results of the static scattered light analysis area; 3 steps and
An analysis method characterized by having

[Thirteenth invention]
A thirteenth invention for solving the above problems is the analysis method of the twelfth invention,
After the first step of acquiring repeated measurement results from the scattered light detection unit based on the set sampling conditions, the analysis unit suspends execution of the first step for a set imaging interval,
The analysis unit restarts execution of the first step after the end of the set imaging interval,
According to the set number of times of execution of the first step or the set total imaging time, which is the time during which the execution of the first step and the subsequent stop of execution are continued repeatedly, the first An analysis method characterized by repeating an operation of executing a step and stopping the execution of the first step during the imaging interval.

[14th Invention]
A fourteenth invention for solving the above problems is the analysis method according to one of the twelfth or thirteenth inventions,
The analysis unit performs an operation in an adjustment mode and an operation in a measurement analysis mode based on the instruction,
In the adjustment mode operation, the third step of performing the calculation for the dynamic scattered light analysis and the calculation for the static scattered light analysis based on the measurement data from the scattered light detection unit is stopped. Characterized by an analysis method.

According to the present invention, it is possible to obtain an analysis apparatus and an analysis method capable of performing both DLS measurement and SLS measurement with respect to the same measurement object at the same elapsed time.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is an explanatory diagram for explaining an embodiment of an analyzer for analyzing minute analysis regions of a sample, to which the present invention is applied; 1. It is explanatory drawing explaining the structure and operation|movement regarding a light irradiation part, a sample part, and a parallel scattering light conversion part in the structure of FIG. FIG. 2 is an explanatory diagram for explaining the relationship between irradiation light and generation of small-angle scattered light in the minute analysis region of the sample shown in FIG. 1; It is explanatory drawing of the microscope image measured in the Example to which this invention was applied. FIG. 4 is an explanatory diagram for explaining an analysis method based on a microscope image of small-angle scattered light; FIG. 4 is an explanatory diagram for explaining a detection surface in an example of a photodetector in a scattered light detection section; FIG. 5 is an explanatory diagram for explaining the relationship between a detection result detected by a scattered light detection unit and a storage state; FIG. 4 is an explanatory diagram for explaining the relationship between a microscope image based on small-angle scattered light detected by a scattered light detection unit and a DLS analysis area R1 and an SLS analysis area R2; FIG. 10 is an explanatory diagram for explaining the relationship between the SLS analysis region R2 and the detection result in the detection area of the scattered light detection unit; FIG. 10 is an explanatory diagram for explaining another embodiment to which the present invention is applied; 11A and 11B are explanatory diagrams for explaining the operation of the scattering angle filter and the scattered light collector in the other embodiment shown in FIG. 10; It is an explanatory view explaining a scattering angle filter. FIG. 10 is an explanatory diagram when a camera with a lens is used as a scattered light detection unit; It is another example in the case of measuring a scattered light image using a camera with a lens as a scattered light detection unit. FIG. 15 is an explanatory diagram illustrating an example of the operation of the analyzer. 16 is a flowchart for performing the operation of FIG. 15; FIG. 4 is an explanatory diagram for explaining a storage state of measurement results in a storage device and a storage state of analysis results using the stored data;

1. Description of Analysis Apparatus 100 for Analysis of Sample 154 Based on Scattered Light 1.1 Introduction In the present specification, configurations denoted by the same reference numerals perform the same operations and actions and have the same effects. In order to avoid complication, there are cases where repeated explanations for configurations with the same reference numerals are omitted. Also, in the description of this specification, the terms "detection", "detection", "measurement", and "measurement" are not strictly differentiated. Described as having the same meaning. Furthermore, the terms "irradiate" and "incident" are not used strictly. Described as having the same meaning.

1.2 Overview of Analysis Apparatus 100 FIG. 1 shows the configuration of an analysis apparatus 100, which is an embodiment to which the present invention is applied. As will be described in more detail below, light source 110 produces light 112 suitable for illuminating sample 154 to be analyzed. The light 112 generated by the light source 110 is guided to the incident light adjusting section 120 , and the incident light adjusting section 120 adjusts the light 112 to generate the incident light 126 . Incident light 126 is introduced into the light irradiation section 140 . The light irradiation unit 140 generates irradiation light 148 for irradiating the sample 154 based on the introduced incident light 126 . Illumination light 148 illuminates analysis microregion 156 of sample 154 and forms a focus on analysis microregion 156 . Due to irradiation with the irradiation light 148 , scattered light is generated on the front side and the rear side of the sample 154 from the analysis minute region 156 of the sample 154 . At least one of the forward and backward scattered light 160 is converted into parallel small-angle scattered light 168 by the parallel scattered light converter 170 and detected by the scattered light detector 200 . Based on the detection result of the scattered light detection unit 200 , the sample 154 is analyzed by the analysis unit 250 and the analysis result is output from the input/output device 266 of the analysis unit 250 .

The small-angle scattered light 160 generated on the rear side and the small-angle scattered light 160 generated on the rear side are converted into parallel small-angle scattered light 168, and the parallel small-angle scattered light 168 based on the small-angle scattered light 68 on the rear side is detected. Scattered light detection unit 200 has a configuration in which small-angle scattered light 160 generated on the front side is converted into parallel small-angle scattered light 168 by parallel scattered light conversion unit 170 and detected by scattered light detection unit 200. Since they have the same configuration and substantially the same effects, description using drawings is omitted.

The irradiation light 148 generated by the light irradiation unit 140 is very important in improving the analysis accuracy of the sample 154 . In the light irradiation section 140 , the irradiation light 148 is formed based on the incident light 126 generated by the incident light adjustment section 120 . 126 is important. In addition, it is desirable to prevent stray light other than the incident light 126 from entering the light irradiation section 140 . The optical diaphragm 138 is provided for this purpose. Furthermore, the optical diaphragm 138 has the effect of preventing reflected light from the sample section 150, the parallel scattered light conversion section 170, and the like from entering the sample 154 of the sample section 150 again.

The embodiment shown in FIG. 1 describes a configuration that utilizes small-angle scattered light 160 generated in the forward direction of the small-angle scattered light 160 generated by sample 154 based on illumination light 148 . Furthermore, in the description of FIG. 1, only the small-angle scattered light 160 generated on one side of the optical axis 146 is illustrated. However, these are for avoiding complexity of explanation and complexity of illustration. Scattered light is generated from the sample 154 not only in the forward direction but also in the backward direction. Regarding the small-angle scattered light 160 among the scattered lights, the small-angle scattered light 160 generated toward the rear as well as forward can be used similarly. Also, the small-angle scattered light 160 generated forward and backward is generated not only at a specific angle with respect to the optical axis 146 but also over the entire circumference. It is possible to utilize the scattered light generated over the entire circumference. In FIG. 1, the small-angle scattered light 160 limited to only a specific angle of the scattered light is shown entering the scattered light detector 200 . However, this is described as an example, and scattered light is generated over the entire circumference of the optical axis 146 including portions other than the small-angle scattered light 160 illustrated in FIG. It can be input to the scattered light detection unit 200 as necessary and can be detected.

In the analysis device 100, it is preferable to have a degree of freedom in the positional relationship between the light irradiation unit 140 and the incident light adjustment unit 120, and when it is necessary to change the optical axis of the incident light 126 with respect to the light irradiation unit 140. occurs. In this case, it is possible to change the optical axis by providing the optical path changing unit 130 . In this embodiment, the optical path changing section 130 is provided between the incident light adjusting section 120 and the optical diaphragm 138, but this is only an example, and it can be provided in other parts as required. You can set it.

As described above, the light irradiation section 140 converts the incident light 126 from the optical path changing section 130 into a condensed beam having a focal point, and irradiates the sample 154 with the irradiation light 148 . The condenser lens 142 of the light irradiation unit 140 is set so that the focal point of the irradiation light 148 coincides with the position of the target area to be analyzed on the sample 154, that is, the focal point is positioned at the analysis minute area 156 to be analyzed. It is In other words, the positional relationship between the condenser lens 142 and the sample 154 is set based on the focal length of the condenser lens 142 .

A sample position adjustment mechanism 158 provided in the sample section 150 accommodates the sample 154 so that the focal position of the irradiation light 148 is the analysis microregion 156, which is the analysis target point to be analyzed inside the sample 154. It is possible to fine-tune the position of the sample cell 152 in three dimensions. With such a configuration, as will be described in detail below, by adjusting the sample position adjustment mechanism 158 so that the irradiation light 148 is focused on the analysis minute region 156, which is the portion of the sample 154 to be analyzed, Small-angle scattered light 160 can be generated precisely from the region one wishes to analyze. In this embodiment, the small-angle scattered light 160 generated at the target portion within the sample 154 can be used, so that the analysis result of the target portion can be obtained.

Since the relationship between the focal position of the irradiation light 148 and the sample 154 in the direction of the optical axis 146 is determined by the focal length of the condenser lens 142, the distance on the optical axis 146 between the condenser lens 142 and the sample cell 152 is By accurately maintaining the distance between lens 142 and sample cell 152 , illumination light 148 can be focused inside sample 154 . However, where on the plane perpendicular to the optical axis 146 on the sample 154 the irradiation light 148 should be focused depends on the intention of the operator. Therefore, it is important that the sample positioning mechanism 158 can move the sample cell 152 in a plane perpendicular to the optical axis 146 . As will be described again below, similar to this embodiment and other embodiments described below, before starting formal measurement, based on the detection result of the scattered light detection unit 200, the parallel small-angle scattered light 168 The image for adjustment is displayed on the input/output device 266 as an image for adjustment by the analysis unit 250 . While viewing this adjustment image, the sample position adjustment mechanism 158 moves and adjusts at least the sample cell 152 in a plane perpendicular to the optical axis 146 . This allows the illuminating light 148 to be focused on the area desired to be analyzed. The focal region of the illuminating light 148 is the desired analysis microregion 156 .

After the analysis minute area 156 determined by the focal position of the irradiation light 148 is set, the operator issues a formal measurement start instruction through the input/output device 266 of the analysis unit 250 . As a result, the analysis unit 250 acquires the measurement results necessary for analyzing the dynamic scattered light and the static scattered light from the scattered light detection unit 200 . The captured measurement result of the scattered light detection unit 200 is stored in the storage device 290 of the analysis unit 250 in association with the position of the measurement element on the measurement plane of the scattered light detection unit 200 . The analysis minute area 156 to be measured can be accurately set, and the measurement data repeatedly taken from there is stored in association with the positional relationship on the measurement plane in the photodetector 202 that is related to the angle information with respect to the optical axis 146. Therefore, the stored data enables highly accurate analysis of both dynamic light scattering and static light scattering as described below. The stored measurement results are the values measured for the common analysis minute area 156 at the same temporal timing.

A portion of the irradiation light 148 applied to the sample 154 becomes scattered light, but most of it becomes transmitted light 164 that penetrates the sample 154 . The transmitted light 164 is converted into light parallel to the optical axis 146 by the parallel scattered light conversion section 170 and blocked by the transmitted light blocking section 176 arranged behind or in front of the parallel scattered light conversion section 170 . On the other hand, of the scattered light generated by the analysis minute region 156 of the sample 154, the small angle scattered light 160, which has an angle close to the transmitted light 164, in other words, an angle smaller than the maximum angle θ0S described in FIG. It is converted into parallel small-angle scattered light 168 by the section 170 and detected by the scattered light detection section 200 .

In the embodiment illustrated in FIG. 1, the illuminating light 148 can be focused on the analysis microregion 156 of the sample 154 . Therefore, the scattered light generated in the analysis minute region 156 of the sample 154 to be analyzed can be detected in real time by the scattered light detection unit 200 for both dynamic light scattering and static light scattering. The state of particles and aggregates inside can be analyzed in real time by the analysis unit 250 .

The analysis unit 250 detects both dynamic light scattering and static light scattering, analyzes various states of particles and aggregates in the sample 154 based on these detection results, and outputs the results to the analysis unit 250. It can be output from the input/output device 266 . Information representing various states includes, for example, the diffusion coefficient of particles and aggregates, the size of particles and aggregates, the structural parameters of particles and aggregates, and the like. output.

In the embodiment shown in FIG. 1, a configuration for detecting the forward small-angle scattered light 160 among the forward and backward small-angle scattered lights 160 generated by the sample 154 is shown as a representative example. The analysis microregion 156 of the sample 154 produces small angle scattered light 160 not only forwards but also backwards, as described above. Therefore, even if the small-angle scattered light 160 that is generated toward the rear as well as the forward is used, analysis results can be obtained in the same way as when the small-angle scattered light 160 that is forward is used. Therefore, the backward small-angle scattered light 160 can be used instead of the forward small-angle scattered light 160 . In the embodiments described below, as a representative example of the embodiments based on the forward small-angle scattered light 160 or the backward small-angle scattered light 160, an embodiment using forward small-angle scattered light will be shown and described. The technical action and effect are essentially the same for both embodiments. It should be noted that the transmitted light 164 exists in the forward direction in the traveling direction of light, and does not exist in the rearward direction. If the backward small-angle scattered light 160 is used, there is no need to consider the overlapping of the transmitted light 164 and the small-angle scattered light 160 . However, it is necessary to consider the overlap with the illumination light 148 . It is also necessary to consider the relationship between the light irradiation section 140 and the parallel scattered light conversion section 170 .

1.3 Description of Each Configuration Constituting Analysis Apparatus 100 (1) Description of Light Source 110 The light source 110 described in FIG. is required to occur. Incident light 126 is formed by the incident light adjustment unit 120 based on the light 112 generated by the light source 110 , and irradiation light 148 for irradiating the sample 154 is formed by the light irradiation unit 140 based on the incident light 126 . When the irradiation light 148 is applied to the sample 154 , it is desirable that the sample 154 efficiently generates scattered light, particularly small-angle scattered light 160 . Light source 110 is required to produce light 112 with properties suitable for efficiently producing small angle scattered light 160 at sample 154 .

The light source 110 can use various lasers, such as various gas lasers, semiconductor lasers (LD), diode pumped solid state (DPSS) lasers, and light emitting diodes (LEDs). Among them, the light source 110 is preferably a high-intensity laser light source that has a high coherence length and oscillates at a single wavelength in a single longitudinal mode. As such a laser light source, for example, there is a DPSS laser. A DPSS laser is a small laser light source containing an optical resonator necessary for laser light oscillation. This DPSS laser has the advantage of being able to obtain stable, high-output laser light with low power and high efficiency. Therefore, it is suitable as the light source 110 of the analysis device 100 that is the embodiment of the present invention.

In addition, the light source 110 preferably has a compact driving power source and a light source main body, and has a size suitable for incorporation into the analysis apparatus 100 for analyzing particles and aggregates. For this reason, for example, low and medium molecules such as peptides and nucleic acids, biopolymers such as proteins, and their aggregates, as well as fine particles such as nano to submicron metals, plastics, and inorganic substances, and their aggregates, are subject to analysis. , it is preferable to use a laser light source of several mW to several tens of mW.

The power of the light 112 output by the light source 110 is determined by the intensity of the scattered light of the sample 154 to be analyzed, the sensitivity of the photodetector 202 of the scattered light detection unit 200, the sensitivity of the light detection camera 204, and the scattered light intensity measurement. Adjusting according to one or more of the amplification of the amplifier used, the exposure time of the scattered light intensity measurement by the photodetector 202, or the exposure time of the scattered light intensity measurement by the photodetector 202, etc. is preferred. The intensity of the incident light 126 to the sample 154 can be adjusted to an appropriate intensity range by adjusting the intensity of the light 112 from the light source 110 with the light amount adjusting element 122 of the incident light adjusting section 120.

It is preferable that the beam size of the light 112 emitted from the light source 110 is as small and thin as possible. That is, it is preferable to use a spot-shaped light beam. Also, the light 112 from the light source 110 is preferably parallel light, that is, collimated light. Furthermore, it is preferable that the cross section of the light 112 is circular from the viewpoint of being able to widely analyze various types of samples and to obtain high detection accuracy. Regarding the irradiation light 148 from the light irradiation unit 140, it is important that the light can be accurately focused on the analysis minute region 156, which is the analysis portion of the sample 154. From this point of view, in this embodiment, the cross-sectional size of the light 112 is That is, the beam size is preferably 1.6 mm or less in diameter. More preferably, the cross-sectional size of the light 112 is 1 mm or less. The cross-sectional size of the incident light 126 incident on the light irradiation unit 140, that is, the beam size, is adjusted by the incident light adjustment unit 120. If the beam size of the light 112 is 1.6 mm or less in diameter, particularly 1 mm or less, The beam size of the incident light 126 is determined with little adjustment of the beam size in the incident light adjustment unit 120, and the beam size of the incident light 126 can be easily maintained at a diameter of 1.6 mm or less, particularly 1 mm or less. becomes.

The spread angle of the light 112 emitted from the light source 110 greatly affects the narrowing accuracy of the irradiation light 148 emitted from the light irradiation unit 140 at the focal position. Reducing the size of the irradiation light 148 at the focal position as much as possible is very important for improving analysis accuracy. From this relationship, it is preferable that the spread angle of the light 112 is 1.5 mrad or less, particularly 1 mrad or less in terms of the total radiation angle. When the spread angle of the light 112 emitted from the light source 110 is large, a small spot-like light beam necessary for detection is incident on the sample 154 by a group of condensing lenses, such as an objective lens, of the light irradiation unit 140. becomes difficult. Furthermore, the relationship between the small-angle scattered light 160 and the scattering angle is disturbed, and it is difficult to accurately convert the scattered light from the sample into parallel light within a predetermined radiation scattering angle range by the lens group of the parallel scattered light conversion unit 170. becomes. These cause a decrease in analysis accuracy.

The polarization of the light 112 emitted from the light source 110 is not particularly limited. Linearly polarized light, circularly polarized light, elliptically polarized light, or other polarized light can be used. It may also be non-polarized. Among these, linearly polarized light such as TE polarized light and TM polarized light is easy to use.

Using the depolarized light scattering method, when analyzing each motion element such as the particles to be measured of the sample 154 and their aggregates, such as translational motion and rotational motion, or the orientation of crystal grains in thin films such as polymers When analyzing the properties, light of a known polarization state, such as linearly polarized light, is used as the incident light. A scattered light component polarized parallel to the incident light and a scattered light component polarized perpendicular to the incident light are separately detected using a polarizer or the like. For example, when scattered light in the H polarization state, which is the horizontal polarization state, is detected with respect to the V polarization state, which is the polarization state of the incident light which is vertically polarized, is denoted as “VH”. be. "VV" and "VH", as well as "HH" and "HV" are measured and evaluated. The orientation of crystal grains in thin films such as molecules can be analyzed. In addition, the stability of incident light is required for highly accurate scattered light measurement. Therefore, it is preferable that the output stability of the light source 110 is several percent or less per hour. Furthermore, in measuring scattered light, it is possible to avoid the influence of variations in incident light by monitoring changes in the intensity of incident light.

In the measurement method of the scattered light from the sample 154, especially the DLS measurement method, the time autocorrelation of the scattered light itself from the scatterer of the sample 154 is taken to obtain the time correlation function, and the homodyne measurement method to obtain the time correlation function. There is a heterodyne measurement method in which light is mixed with a reference light modulated at a certain frequency and a time correlation function is obtained for it. The homodyne measurement method used in DLS measurement requires a relatively simple optical system, enables measurement with a high S/N ratio, and easily obtains the time correlation function of scattered light. On the other hand, the heterodyne measurement method is often used when the scatterers of the sample 154 are in constant motion, such as in electrophoresis measurement.

In the DLS measurement using the homodyne measurement method, light having a constant intensity over time is used as the light 112 generated by the light source 110, and a time correlation function can be obtained from the temporal fluctuation of the scattered light itself. Further, as the light 112 generated by the light source 110, modulated light modulated at a certain frequency may be output. Alternatively, for example, an optical modulator such as a light chopper that rotates or oscillates at a certain frequency or an optical modulation element such as an optical modulation device is used to modulate the light 112 emitted from the light source 110 at a certain predetermined frequency. It may be used as incident light 126 .

On the other hand, in the DLS measurement using the heterodyne measurement method, light modulated at a certain predetermined frequency can be used as the reference light to be mixed with the scattered light from the scatterers of the sample 154 . For example, modulated light from the light source 110 can be used as the modulated reference light as described above. In this case, the light 112 emitted from the light source 110 is split into, for example, two optical paths, and the light on one optical path is introduced into the light irradiation unit 140 as the incident light 126, and the irradiation light 148 is emitted from the light irradiation unit 140. The sample 154 is irradiated with . Scattered light from the sample 154 based on the irradiation of the irradiation light 148 is guided to the photodetector 202 of the scattered light detection section 200 via the parallel scattered light conversion section 170 . Further, the light on another optical path is modulated to a predetermined frequency by the above optical modulator, optical modulation element, etc., and the modulated light is made incident on the photodetector 202 as it is. The photodetector 202 measures the dynamically scattered light from the scatterers of the sample 154 and the modulated reference light mixed together. In this way the time correlation function of the scattered light with respect to the modulated reference light can be obtained.

(2) Description of Incident Light Adjusting Section 120 The incident light adjusting section 120 has a function of adjusting the state of the irradiation light 148 with which the sample 154 provided in the sample section 150 is irradiated. Specifically, for example, the incident light adjustment unit 120 is composed of a group of optical elements, and adjusts the amount of light, the polarization state, and the light beam in order to convert the light 112 from the light source 110 into the irradiation light 148 suitable for the sample 154 . Adjust the shape, size, etc. of The incident light adjusted by the incident light adjusting section 120 is guided to the light irradiation section 140 after the optical path is changed by the optical path changing section 130 as necessary. In addition, in this embodiment, the incident light adjusting section 120 may be provided after the optical path changing section 130 .

The incident light adjusting section 120 has, for example, a light quantity adjusting element 122 and a polarizing element 124 . The light amount adjusting element 122 receives the light 112 from the light source 110 and adjusts the intensity of the irradiation light 148 that irradiates the sample 154 to be analyzed. The light quantity adjusting element 122 is, for example, a neutral density (ND) filter or the like.

Specifically, various optical filters such as a metal thin film filter and a dielectric filter can be used as the light quantity adjusting element 122 . By using an ND filter or the like having a transmittance of 0.1% to 50%, for example, as the light amount adjusting element 122, the light intensity, which is the amount of incident light, can be easily adjusted.

The transmittance of the light amount adjusting element 122 may be adjusted according to the intensity of the scattered light of the sample to be analyzed, the detection sensitivity and exposure time of the photodetector 202 of the scattered light detection unit 200, and the amplification degree of the detection signal. Here, the amplification factor is an amplification factor, a gain, or a gain. Scattered light from the sample 154 to be analyzed by the photodetector 202 has a good SN ratio, that is, a ratio of the signal signal to the noise signal, with respect to the light amount of the irradiation light 148 emitted from the light irradiation unit 140. The amount of incident light 126 is preferably set so that

The polarization element 124 of the incident light adjustment unit 120 has a function of adjusting the polarization state of the light 112 from the light source 110 and setting the polarization state of the incident light 126, and is an optical element that adjusts the polarization state. It is configured. If the light 112 from the light source 110 is unpolarized light, various polarizing plates and polarizing filters can be used as the polarizing element 124 to set the polarization of the incident light 126 .

If the light 112 from the light source 110 is linearly polarized light, a half-wave plate or the like may be used as the polarizer. By using this, the direction of polarization of the incident light 126 can be set to any azimuth angle, and TM polarized light (p) polarized light whose electric field vector is horizontal to the optical system or TE (s) polarized light perpendicular to the optical system is used. can get. Furthermore, if the light 112 from the light source 110 is linearly polarized light, the polarization of the light 112 from the light source 110 is converted into circularly polarized light or elliptically polarized light using a quarter-wave plate or the like, and the light is irradiated. It can also be used as incident light 126 in section 140 if desired.

Furthermore, if the intensity of the forward or backward small-angle scattered light 160 from the sample 154 being analyzed is strongly dependent on the polarization of the incident light 126, the polarizing element 124 may be It is desirable to adjust the polarization state of the incident light 126 by .

(3) Description of Optical Path Changing Unit 130 In order to actually manufacture the analysis device 100, it may be preferable to change the optical axis of the optical path from the light source 110 to the light irradiation unit 140. FIG. In such a case, the optical path changing section 130 can be used. The configuration shown in FIG. 1, in which the light 112 from the light source 110 is adjusted as described above by the incident light adjustment unit 120 and then the optical path for the incident light 126 is changed, is an example. The optical path can be changed by providing the optical path changing unit 130 at a place where the optical path needs to be changed.

The optical path changing unit 130 is configured by optical elements such as a group of mirrors, lenses, prisms, and the like, for example. For example, the optical path changing unit 130 is arranged such that the plane mirror 132 is arranged at an incident angle of 45 degrees with respect to the optical axis of the light 112 from the light source 110 , or the incident angle of 45 degrees with respect to the reflected light from the plane mirror 132 . It has a plane mirror 134 which is arranged in a state of being in a degree. With this configuration, the incident light 126 in which the traveling direction of the light 112 is reversed can be incident on the light irradiation section 140 . Reference for placing the optical axis of the irradiation light 148 from the light irradiation unit 140, the optical axis of the parallel scattered light conversion unit 170, and the photodetector 202 of the scattered light detection unit 200 on the optical axis 146 of the incident light 126. The axis of , is set.

This facilitates adjustment of the arrangement relationship among the sample unit 150 , the parallel scattered light conversion unit 170 , and the scattered light detection unit 200 . Furthermore, it helps to reduce the size of the analyzer 100 . In addition, the operation of adjusting the sample position adjustment mechanism 158 of the sample section 150 is facilitated so that the focal position of the illumination light 148 generated on the optical axis 146 of the incident light 126 matches the position of the sample 154 to be analyzed. This also leads to an improvement in adjustment accuracy. Using the optical path changing unit 130 to set the optical path to an optimum state is useful not only for reducing the size of the analysis device 100 but also for improving analysis accuracy.

It may be effective to irradiate the sample 154 with the irradiation light 148 of the light irradiation unit 140 having different wavelengths and analyze the difference in the small-angle scattered light 160 due to the difference in the wavelength of the irradiation light 148 . In this case, a configuration in which the optical axis of the incident light 126 to the sample 154 is set constant and a plurality of laser light sources with different wavelengths are arranged is an effective configuration. By providing a half mirror, a total reflection mirror, a prism, or the like for each laser light source or the like in the optical path changing unit 130, a configuration in which different wavelengths are incident on the sample 154 described above is possible.

(4) Description of Optical Diaphragm 138 As shown in FIG. The optical diaphragm 138 has a function of preventing stray light other than the irradiation light 148 from the light irradiation section 140 from entering the sample 154 . Furthermore, it also has a function of preventing reflected light from the sample unit 150 , the parallel scattered light conversion unit 170 , and the like from entering the sample 154 again. The optical diaphragm 138 comprises two diaphragms 139 arranged in parallel in this embodiment. Each aperture of the diaphragm 139 has a slit having a shape larger than the cross-sectional shape of the incident light 126 for passing the incident light 126 . In this embodiment, the slit has a circular shape because the cross-sectional shape of the incident light 126 is circular. The diameter of the aperture, which is a slit formed in each diaphragm 139, is larger than the diameter of the incident light 126, and has an aperture diameter suitable for preventing the above-described stray light and reflected light. If the diameter of the light beam of the incident light 126 is, for example, 1 mmφ, the diameter of the diaphragm 139 is preferably 1.2 mm or more and 2 mm or less.

Even if the optical diaphragm 138 is composed of a single diaphragm 139, it has the effect of preventing the above-described stray light and reflected light. However, a greater effect can be obtained by arranging a plurality of optical apertures which are arranged perpendicular to the optical axis 146 of the illumination light 148 on the sample 154 and spaced apart in parallel. In particular, as the optical diaphragm 138, a configuration in which two diaphragms having apertures of the same size are spaced apart is preferable. As the diaphragm 139, for example, for the incident light 126 having a diameter of 1 mmφ, two diaphragms having a diameter of about 1.2 mm or more and about 2 mm or less are arranged at an interval of about 5 cm or more and 30 cm or less in the optical axis direction of the incident light. The configuration in which they are arranged in parallel with each other produces a very large effect.

(5) Description of Light Irradiation Unit 140 Operations of the light irradiation unit 140 and the sample unit 150 will be described with reference to FIGS. 2 and 3 in addition to FIG. The light irradiation section 140 converts the incident light 126 into a light beam focused within the sample 154 and irradiates the sample 154 of the specimen section 150 as irradiation light 148 . Specifically, the light irradiation unit 140 includes an optical element for condensing the incident light 126 adjusted by the incident light adjustment unit 120, a spot size of the irradiation light 148 applied to the sample 154, and an irradiation amount in the sample 154. and a lens adjustment mechanism 144 that adjusts the focal position of the light 148 and the like.

Depending on the size of the light beam of the incident light 126 incident on the light irradiating section 140, the type and size of the objective lens, which is the condensing lens of the light irradiating section 140, and the shape and size of the sample cell 152 are determined. Furthermore, depending on the light beam size of the incident light 126, the types and sizes of lenses constituting a parallel scattered light conversion unit 170, which will be described later, or the types and sizes of lenses constituting a scattered light collecting unit 190, scattered light detection When the photodetector camera 204 is used as the photodetector 202 or the scattered light detector 200 of the unit 200, the shape and size of the light receiving portion of the photodetector camera 204 are determined. The scattered light detection unit 200 has a function of detecting an image and converting it into an electric signal, and may include a light detection camera 204 . Photodetection camera 204 can be used as an alternative to photodetector 202 .

As already mentioned above, the sample section 150 includes a sample cell 152 containing a sample 154 and a sample positioning mechanism 158 for moving the sample cell 152 in the X, Y and Z directions. The sample cell 152 is moved by the sample position adjustment mechanism 158 so that the minimum beam spot 157, which is the irradiation position of the irradiation light 148, coincides with the minute analysis area 156, which is the portion to be analyzed in the sample 154 stored in the sample cell 152. , along the X, Y, and Z axes, which are three-dimensional axes. As a result, the irradiation light 148 is formed and irradiated so that the minimum beam spot 157 overlaps the analysis microregion 156 at the target position in the sample 154, and the analysis microregion 156 generates the small-angle scattered light 160 suitable for detection. can be done. As described above, the position of sample cell 152 can be finely moved relative to minimum beam spot 157 by sample positioning mechanism 158 along a three-dimensional axis. However, it is not necessary to limit the adjustment along the three-dimensional axis, and it is also effective to configure the sample cell 152 so that it can be moved only along the optical axis 146, for example. Further, fine adjustment of the position may be made possible on a plane perpendicular to the optical axis 146 .

The relationship between the minimum beam spot 157 in the sample 154 of the irradiation light 148 from the light irradiation unit 140 and the generation of the small-angle scattered light 160 will be described with reference to FIG. The maximum angle of incidence of the illumination light 148 incident on the sample 154 is defined as the maximum angle of incidence θi, the maximum transmission angle of the transmitted light 164 passing through the sample 154 is defined as the maximum transmission angle θ0, and the maximum small-angle scattering emitted from the sample 154 and measured is Let the radiation angle of the light 167 be radiation angle θ0S. Each of these angles is represented by an angle from the optical axis 146 which is the central axis of the irradiation light 148 to the sample 154 .

As shown in FIG. 3, the irradiation light 148 incident on the sample 154 of the sample unit 150 is scattered by scattering objects such as particles, aggregates, and crystal grains to be measured while passing through the sample 154. The light is transmitted and transmitted light 164 is emitted from the sample 154 . Since the illuminating light 148 on the sample 154 is illuminating light 148 that is incident at various incident angles within a given range of incident angles θi, the transmitted light 164 from the sample 154 is various within a range of radiation angles θ0. Radiation at an angle.

Here, the area (θ<θ0) of the transmitted light 164 of the sample 154 includes not only the transmitted light 164 but also the small-angle scattered light 160 from the sample 154 . Scattered light from a scatterer to be measured, such as a sample containing particles or aggregates or a solid thin film, is generally much weaker than transmitted light. It is practically difficult to measure it separately from the transmitted light 164 . Therefore, the small-angle scattered light 160 that can be measured is the scattered light that appears outside the region of the transmitted light 164, that is, when θ>θ0. Therefore, as the angle of the small-angle scattered light of the sample 154, in addition to the angle θ0S from the optical axis, the measured scattering angle θS measured from the maximum transmission angle θ0 is given by the following equation.
[Number 1]
θS=θ0S−θ0 (Formula 1)

As the condenser lens 142 of the light irradiation unit 140 that forms the irradiation light 148 to be irradiated to the sample 154, various lenses such as a spherical plano-convex lens, a spherical bi-convex lens (hereinafter simply referred to as a "spherical convex lens"), an aspherical lens, and a cylindrical surface can be used. It is possible to use single lenses such as plano-convex lenses and cylindrical biconvex lenses (hereinafter simply referred to as "cylindrical lenses"), and various achromatic lenses made by bonding two or more single lenses with different optical properties. is. The light irradiation unit 140 is configured to form a minute spot of irradiation light on the sample 154 of the sample unit 150 and efficiently irradiate the irradiation light 148 with the above-described single lens, achromatic lens, or any of these lenses. It is composed of an optical system etc. combining lenses.

When the light from the light source 110 is parallel (collimated) beam light such as laser light, for example, the light beam size is formed by an optical diaphragm or the like, and is incident on the light irradiation unit 140, so that the irradiation is changed to a minute size. Light 148 is incident on sample 154 . Here, the minimum beam spot 157 of the irradiation light 148 obtained by condensing, for example, a circular parallel beam light by the condensing lens 142 configured by the above-described single lens, achromatic lens, or a combination of these various lenses The size W is represented by the following formula. Note that the focal length of the condenser lens 142 is f. Also, the size W of the minimum beam spot 157 is the diameter.
[Number 2]
W=2λ/(π・NA) (Formula 2)

Here, the size W is the diameter of the minimum beam spot 157 of a circular beam within 1/e2 (=0.135) of the central maximum value of a laser beam having a Gaussian intensity distribution, and λ is the wavelength of light. , NA (numerical aperture) is the numerical aperture of the condenser lens system. Furthermore, the numerical aperture NA of the condenser lens 142 of the light irradiation section 140 is NA=D0/(2f). Here, D0 is the diameter of the light beam incident on the condenser lens system (range within 1/e2 of the central maximum value). Therefore, the size W of the minimum beam spot 157 obtained at the focal length f of the condenser lens 142 is expressed by the following equation.
[Number 3]
W=4fλ/(π・D0) (Formula 3)
For example, if the condenser lens 142 of the light irradiation unit 140 is composed of a condenser lens with a focal length f=10 mm, the incident light 126 entering the condenser lens 142 has a diameter D0 of 0.7 mm and a wavelength λ of 473 nm. When the laser light beam is condensed, a minimum beam spot of 8.6 μm is obtained as the diameter W of the minimum beam spot 157 .

Here, for example, when the light from the light source 110 is laser light, the incident light 126 incident on the condenser lens 142 becomes larger than the beam diameter at the exit port of the light source 110 due to the spread angle of the laser light itself. That is, the diameter D0 of the incident light 126 entering the condenser lens 142 is expressed by the following equation.

[Number 4]
D0=Dout+ωL (Formula 4)
Here, Dout is the beam diameter at the exit port of the light source 110, ω is the spread angle (full angle) of the laser light beam, and L is the distance from the exit port of the light source 110 to the incident side of the light irradiation section 140. The light beam diameters of D0 and Dout indicate beam diameters within a range of 1/e2 (=0.135) of the central maximum value of laser light having a Gaussian intensity distribution.

For example, the value of the diameter D0 of the incident light 126 incident on the light irradiation unit 140 is Dout=0.7 mm from the laser light source of ω=1.2 mrad, and for example, the distance L from the laser light source to the condenser lens 142 is 665 mm. In some cases, D0=1.5 mm calculated from the above equation.

Therefore, for example, a laser beam having a diameter Dout of 0.7 mm output from the light source 110 of λ=473 nm and ω=1.2 mrad using a condenser lens 142 having a focal length of f=10 mm is condensed. When the distance L to the optical lens 142 is 665 mm, the diameter W of the minimum beam spot 157 obtained by condensing the light with the condensing lens 142 is W=4.0 μm, which is smaller than the above value (8.6 μm). , and the spread of the laser light beam results in illumination light 148 forming a smaller size minimum beam spot 157 .

Here, as described above, the size W of the minimum beam spot 157 of the irradiation light 148 obtained by condensing the laser beam or the like with the condensing lens 142 of the light irradiation unit 140 depends on the medium around the condensed spot being air ( It is a calculated value when the refractive index is n=1.00). The size of the minimum beam spot 157 in the sample 154 of the light irradiation unit 140 is because the medium has a larger refractive index than air (for example, n=1.33 for water), so the actual value of the size W is , is slightly larger than these calculated values. Therefore, as shown in FIG. 3, the size of the minimum beam spot 157 of the irradiation light 148 irradiated onto the sample 154 from the condenser lens 142 of the light irradiation section 140 is slightly larger than the above calculated value.

The light irradiation unit 140 includes a lens adjustment mechanism 144 that finely adjusts the distance between a condenser lens 142 and the sample 154 for irradiating the sample 154 of the sample unit 150 with the irradiation light 148 . That is, the light irradiation section 140 has a mechanism for finely adjusting the focal position (distance) of the condenser lens 142 to the sample 154 . For fine adjustment of the focal position (distance) of the condensing lens 142, for example, an optical stage with a micrometer that finely moves the condensing lens 142 with a micrometer can be used. More specifically, for example, a precision optical stage having a moving distance of ±3 mm on which the condenser lens 142 is mounted and a minimum scale of a micrometer of 10 μm can be used, which is suitable.

As shown in FIGS. 1 to 3 as an example, the maximum incident angle θi when the sample 154 is irradiated with the irradiation light 148 formed by the condenser lens 142 of the light irradiation unit 140 is Using the diameter D0 of the circular light beam of the incident light 126 and the focal length f of the condenser lens 142, it can be approximately determined as follows.
[Number 5]
θi=tan−1(D0/2f) (Formula 5)

For example, when a laser beam of D0=1.5 mm is focused using a condenser lens 142 with a focal length of f=10 mm and irradiated onto the sample 154 of the sample unit 150, the maximum incident angle θi shown in FIG. is 4.3 degrees, and the sample 154 is irradiated with the irradiation light 148 having an incident angle θ<4.3 degrees.

Further, for example, when a laser light beam of D0=1.5 mm is condensed using a condenser lens 142 of focal length f=4 mm and irradiated onto the sample 154 of the sample section 150, the maximum incident angle θi is 10 .8 degrees and the incident angle .theta.<10.8 degrees. As the focal length f of the condenser lens 142 decreases, the maximum incident angle θi increases, and the maximum angle θ0 of the transmitted light 164 also increases. This results in a decrease in the measured scattering angle .theta.S. On the other hand, as described above, the size W of the minimum beam spot 157 can be narrowed, and the minute analysis area 156 is narrowed.

The maximum incident angle θi of the irradiation light 148 irradiated onto the sample 154 by the light irradiation unit 140 is preferably in the range of 0.5 degrees to 20 degrees, and more preferably in the range of 0.5 degrees to 10 degrees. It is desirable to set the maximum incident angle θi of the irradiation light 148 under the above-described conditions and allow the irradiation light 148 at an incident angle equal to or less than the maximum incident angle θi to enter the sample 154 to generate scattered light from the sample 154 .

More precisely, the maximum incident angle θi of the irradiation light 148 that irradiates the sample 154 is determined by the focal length f of the condenser lens 142 of the light irradiation unit 140 and the size of the light beam of the incident light 126 that is incident on the condenser lens 142. (diameter), the distance between the condenser lens 142 and the sample 154, the refractive index of the sample 154 itself and the length (optical path length) of the transmitted light passing through the sample 154 in the optical axis direction, and the sample housing the sample 154 It is determined by the shape of the cell 152 and the material (refractive index) of its constituent members.

The maximum incident angle θi shown in FIG. 3 may be smaller than 0.5°. θi=0°, that is, the light incident on the sample 154 of the measurement sample section 4 may be parallel light (collimated light). In this case, the size of the light beam irradiated to the sample 154 is adjusted using an optical element group for beam light formation, which is a combination of optical elements such as a lens, a pinhole, and an optical diaphragm, which constitutes the light irradiation unit 140. to make parallel light (collimated light) incident on the sample 154 .

As a specific condenser lens 142, an objective lens having a short focal length and a high NA is particularly suitable. More specifically, an objective lens that has a relatively long working distance WD and is corrected for infinity is particularly preferable. As the condenser lens 142, it is possible to use a finite system type microscope objective lens that can form an enlarged image even without an imaging lens.

For example, the NA values of these objectives, which are suitable as the condenser lens 142, are respectively 0.13 for a 5× objective lens, 0.3 for a 10× objective lens, and 0 for a 20× objective lens. 0.4, 0.55 for a 50x objective and 0.8 for a 100x objective. Further, for example, the working distance WD of these objective lenses as the condenser lens 142, which is preferable, is about 2 mm (for a 100-fold objective lens) to 11.6 mm (for a 5-fold objective lens), which is relatively large. long.

As the condenser lens 142 of the light irradiation unit 140, for example, an objective lens for a long working distance with infinity correction, a 20-fold objective lens (focal length f is 10 mm, working distance WD is 11.1 mm, NA is 0.4, resolution of 0.7 μm) or a 50× objective lens (f=4 mm, WD=8.2 mm, NA=0.55, resolution=0.5 μm).

(6) Description of sample section 150 The sample section 150 will be described. The sample unit 150 includes a sample cell 152 that stores a sample 154 and a sample position adjustment mechanism 158 that supports the sample cell 152 and finely moves and adjusts the position of the sample cell 152 with respect to the optical axis 146 of the irradiation light 148 . have. A sample position adjustment mechanism 158 irradiates a sample 154 with irradiation light 148 formed by a condenser lens 142 of a light irradiation unit 140 , and a sample position adjustment mechanism 158 irradiates the analysis microregion 156 on a two-dimensional plane perpendicular to the optical axis 146 or in a three-dimensional direction. In addition, it has the function of being able to move minutely. Furthermore, although not shown, it has a function of controlling the temperature of the sample 154 .

The sample cell 152 is a container that can contain the liquid and transmit scattered light from the sample 154 when the sample 154 is liquid. If the sample 154 is a solid such as a thin film, the sample 154 may contain and fix the solid and have a structure that allows the scattered light from the sample 154 to pass through. Furthermore, for example, when a thin film or the like formed on a transparent substrate such as a glass substrate is to be measured, the substrate itself on which the thin film or the like is formed functions as the sample cell 152, and the thin film formed on the substrate functions as the sample cell 152. etc. act as samples 154 .

The sample cell 152 is adjustable in installation position by a sample position adjustment mechanism 158 . After the adjustment is completed, the measurement operation is started. It is desirable to find a desired measurement spot for analysis on the sample 154 , measure data that can be used for both DLS measurement and SLS measurement at the desired measurement spot by the scattered light detector 200 , and store the data in the storage device 290 . For this reason, as will be described below, the irradiation light 148 is emitted from the light irradiation unit 140 for adjustment, and the small-angle scattered light 160 generated by the irradiation of the irradiation light 148 is measured by the scattered light detection unit 200, and the measurement result is The based image is displayed as an adjustment image in the input/output device 266 of the analysis unit 250 . While viewing this adjustment image, the sample cell 152 can be moved, for example, on a plane perpendicular to the optical axis 146 to find an irradiation position where the image displayed in the adjustment image is a desired image. . This adjustment image allows the adjustment to be set very efficiently as an optimal region, the analysis micro-region 156 .

When the sample 154 is a liquid, the sample cell 152 is preferably a rectangular cell in which the incident surface 151 and the transmission surface 153 of the sample cell 152 shown in FIG. 3 are flat. In order to irradiate the liquid sample 154 with the irradiation light 148 from the light irradiation unit 140 in a spot shape and to efficiently generate and measure the small-angle scattered light 160, the direction of the optical axis of the irradiation light 148 passing through the sample 154 should be The optical path length LS of the sample 154, which is the length, is preferably as short as possible. The shorter the optical path length LS of the sample 154, the less the spot beam of the irradiation light 148 formed by the condenser lens 142 of the light irradiation unit 140 spreads in the sample 154, and the more scattered light is scattered from the local portion of the sample 154. It can be ejected, allowing localized analysis within the sample 154 .

Therefore, the sample cell 152 containing the sample 154 preferably has a short optical path length LS, which is the length in the optical axis direction, that is, the sample cell 152 as thin as possible. For example, the optical path length LS of the sample 154 is preferably 1 mm or less, more preferably 0.5 mm or less. Therefore, the length between the entrance surface 151 and the transmission surface 153 of the sample cell 152 is preferably about the same size, that is, 1 mm or less, more preferably 0.5 mm or less. Moreover, the volume of the sample 154 to be used is preferably several hundred μl or less to several tens of μl or less. The above l stands for liters.

Therefore, as the sample cell 152 for the liquid sample 154, for example, two transparent flat substrates sandwich a flat spacer having a specific thickness, and the sample 154 is injected between the substrates formed by the spacer. It is preferred to use a sandwich-type sample cell 152 that holds and holds the sample. In the case of forward small-angle scattered light measurement, the irradiation light 148 from the light irradiation unit 140 is incident from one substrate side (incidence side) of the two transparent flat plate substrates, passes through the sample 154, The scattered light emitted from one substrate side is measured. Further, in the case of the small angle backward scattered light measurement, the irradiation light 148 from the light irradiation unit 140 is incident from one of the two transparent flat plate substrates, and the scattered light emitted from the same substrate side to measure. That is, on one substrate side of the sample 154, light is incident on the sample 154 and scattered light is emitted.

Specifically, it is preferable to use an optical material having excellent transparency in the wavelength region of the light to be measured on the incident light side and the scattered light emission side as the transparent flat substrate. Therefore, various transparent glasses, transparent plastics, and the like are suitable.

When the sample 154 is a solution containing biomolecules such as proteins, peptides, nucleic acids, or aggregates thereof, the scattered light is remarkably large in a small scattering angle range of 10 degrees or less, particularly in a scattering angle of 8 degrees or less. Scattered light is noticeable. Scattered light from the sample 154 passes through the transparent sample cell 152 and is emitted to the outside, so that it is affected by refraction at the incident surface 151 and the transmission surface 153 .

Further, as described above, the condensing lens 142 of the light irradiation unit 140 allows the incident light with a preferable maximum incident angle θi in the range of 0.5 degrees to 20 degrees, particularly a maximum incident angle θi in the range of 0.5 degrees to 10 degrees. Preferably, the illumination light 148 is incident on the sample 154 at an angle. The incident angle on the incident light side of the sample 154 is also preferably small, as is the scattering angle of the small angle scattered light 160 . In this embodiment, since the entrance surface 151 and the transmission surface 153 of the sample cell 152 are flat, if the thickness is thin to some extent, the incident angle is 20 degrees or less, especially 10 degrees or less. In addition, when the scattering angle is as small as 10 degrees or less, there is no particular need to correct the refraction angle associated with refraction as described above. That is, if the sample cell 152 is thin to a certain degree, it is particularly necessary to correct the scattering angle in consideration of the refraction angle in the sample cell 152 in measuring the small-angle scattered light for the irradiation light 148 with a small incident angle. no longer.

Therefore, in the measurement of the small-angle scattered light 160, it is preferable that the flat plates of the incident surface 151 and the transmission surface 153 that constitute the sample cell 152 are as thin as possible. Specifically, in the case of measurement of small-angle scattered light 160 in irradiation light 148 of small-angle incidence, the thickness tin of the incident surface 151 flat plate of the sample cell 152 and the thickness tout of the flat plate of the transmission surface 153 of the light irradiation unit 140 are The range of 1/10 to 1/5 or less of the diameter D0 of the incident light 126 entering the condenser lens 142 is preferable. That is, the thickness tin<D0/10 to D0/5 and the thickness tout<D0/10 to D0/5 are preferable. In particular, thickness tin<D0/10 and thickness tout<D0/10 are more preferable.

In addition, in solutions containing biomolecules such as proteins, peptides, and nucleic acids and their aggregates, and in solid thin films such as polymers such as plastics that form crystal grains, where the generation of small forward scattered light with a small scattering angle is remarkable. , the scattered light may be emitted to the same side as the incident side of the irradiation light 148 depending on the structure and optical arrangement of the sample 154 . In this case, scattered light from sample 154 is detected on the incident side of illumination light 148 . That is, depending on the sample 154, the backward small-angle scattered light may appear more prominently than the forward small-angle scattered light. Backward small-angle scattered light is scattered at a scattering angle of 170 degrees or more from the optical axis 146 of the transmitted light 164 of the irradiation light 148 and up to 180 degrees (reversal direction of the incident light), particularly from 172 degrees to 180 degrees. Scattered light at corners.

Specifically, the small angle backward scattered light is, for example, the irradiation light 148 to the sample 154 is reflected by the opposite wall of the sample cell 152 containing the sample 154, and the reflected light is the small angle scattered light from the sample 154. , and scattered light is emitted on the same side as the incident side of the illumination light 148 . In this case, backward small-angle scattered light is to be measured. Similar to the measurement of , thin is preferred. However, the other flat plate on the opposite side may be thick as long as it can efficiently reflect the transmitted light 164 passing through the measurement sample. In the sample cell 152 used for measuring small-angle backward scattered light, the flat plate provided in front of the irradiation light 148 may be made of any material that reflects light, such as glass and various plastics. The front flat plate used should have a smooth surface so as not to emit scattered light from itself, and preferably reflect the transmitted light well.

In addition, the flat plates of the entrance surface 151 and the transmission surface 153 forming the sample cell 152 may be transparent members of the same material and the same thickness, or may be members of different materials and different thicknesses. If the flat plate-shaped substrates on the incident side and the scattered light emitting side are made of transparent members of the same material and thickness, there is no distinction between the entrance surface 151 and the transmission surface 153 in the sample cell 152 . That is, the irradiation light 148 may be incident from either side. In this case, as the flat plate of the sample cell 152, it is possible to use, for example, a cover glass of the same standard with a thickness of 0.2 mm or less.

As the flat plate spacer of the sample cell 152, glass, polystyrene, polypropylene, fluororubber, silicon rubber, and the like are suitable. Silicon rubber is particularly preferred, and a sheet or film is used. Silicon rubber has good adhesion to a glass substrate or the like, and when the sample cell 152 is analyzed by a batch method, the concentration of various solutes in the sample 154 hardly changes due to solvent evaporation from the gap within several hours. It doesn't matter. However, when the measurement sample solution is held in the sample cell 152 for a long period of several tens of hours or longer, silicon grease or fluorine is used between the spacer and the flat substrate to further prevent liquid leakage. Sealing with grease or the like is preferred.

When the solvent of the sample 154 is an organic solvent such as ethanol or acetonitrile, the silicon rubber used as the spacer of the sample cell 152 has weak adhesion to the glass substrate or the like. In that case, the space between the spacer and the flat substrate is sealed with silicone grease, fluorine grease, or the like, or the flat plate of the sample cell 152 is held with a certain amount of pressure using a clip or the like. Improves adhesion between rubber and glass substrates, etc., and prevents solvent evaporation and liquid leakage from gaps. Alternatively, the sample position adjustment mechanism 158 that holds the sample cell 152 applies a certain amount of pressure to the flat substrate to hold it.

As described above, the spacers that make up the sample 154 and the two flat plates function as the sample cell 152 that holds the sample 154 . For this reason, for example, a slot of any shape such as circular, elliptical, or rectangular is formed in a flat plate spacer, and the sample 154 is placed in the slot space formed by sandwiching the spacer between two flat plates. can be stored. That is, the sample cell 152 has a sample 154 injected and held in this slotted space.

Analysis of a sample 154, such as a solution or solid thin film, is often performed homogeneously and uniformly. Therefore, from the analysis of the sample 154 by measuring the small-angle scattered light 160, some or all of the sample 154 irradiated with light has information about the movement and structure of particles, aggregates, etc. of the sample 154. information is obtained. However, the following analysis of sample 154 requires not only average information as described above, but also local analysis. That is, for example, samples in which concentration differences of constituent components occur locally in a liquid, such as liquid-liquid phase separation, and crystallization solutions of proteins, peptides, nucleic acids, etc., aggregates and crystal nuclei are formed in the solution. Samples in which crystal grains are formed unevenly in a thin film such as a polymer film, etc., are analyzed. When such a sample 154 is to be analyzed, the scattered light is measured to measure the distribution within the measurement sample on a microscope scale.

The spacer thickness tsp used in the sample portion 150 corresponds to the optical path length in the sample 154 . The irradiation light 148 from the condenser lens 142 is allowed to pass through the sample 154 while maintaining an appropriate minute spot without expanding it to the sample 154 as much as possible, and the scattered light from the minute irradiation portion of the sample 154 is efficiently emitted. Therefore, it is preferable that the optical path length of the sample 154 to be measured is short and the volume of the sample 154 is very small. Specifically, the volume of the sample 154 to be measured is preferably as small as, for example, 25 μl or less. In this case, the spacer thickness tsp is preferably about the diameter of the incident light 126 incident on the condenser lens 142 of the light irradiation unit 140, for example, 1 mm or less, particularly 0.5 mm or less.

In addition to scattered light measurement in a state where the sample 154 is sealed in the sample unit 150, that is, sample analysis in a batch method, a sample liquid, a flocculating agent, or a crystallization agent is transferred from the outside to the sample cell 152 during analysis. It is also possible to configure so that a solution such as the like can be injected and discharged. In this case, the sample cell 152 may be provided with an insertion port or a discharge port for injecting or discharging a liquid. In that case, it is possible to perform analysis by adjusting or changing the concentration of each substance during the analysis of the sample 154 by scattered light measurement.

As the sample cell 152, it is conceivable to use a commercially available rectangular or cylindrical solution cell for light absorption measurement. Many commercially available solution cells have a long optical path length LS of, for example, 5 mm to 1 cm and require a sample liquid of several ml or more. However, if the optical path length of a commercially available solution cell is particularly short, for example, about 1 mm or less, it can also be used. However, in this case, in the measurement of the small-angle forward scattered light or the measurement of the small-angle backward scattered light, the refraction in the sample cell 152 is It is necessary to correct the incident angle and the scattering angle of the condensed light in consideration of the angle.

A sample position adjusting mechanism 158 that supports the sample cell 152 includes a rotating mechanism and a tilting mechanism for adjusting the angle of incidence on the sample 154, and a mechanism for performing positional movement with respect to the optical axis, which is the central axis of the incident light beam.

The incident angle of the center of the incident light to the sample cell 152 is perpendicular to the central axis of the irradiated light 148 by the condenser lens 142 of the light irradiation unit 140, that is, the optical axis 146 of the incident light 126 (incidence angle 0°). is particularly preferred. In addition, a slight angle near the incident angle of normal incidence, for example, an incident angle of 1 to 2° or less is sufficient, and the reflected light of the sample cell 152 becomes stray light, which is received by the photodetector 202 of the scattered light detection unit 200. The incident angle is adjusted by the sample position adjustment mechanism 158 so that the sample position adjustment mechanism 158 does not impinge on the part. A rotating stage, a tilting stage, or the like can be used to adjust the incident angle of the condenser lens 142 in this manner. In addition, when the sample cell 152 is fixed to the sample position adjusting mechanism 158, the incident angle can be set at, for example, 1 to 2° or less without using a rotating stage, tilting stage, or the like. It is good if there is

A sample position adjustment mechanism 158 that supports the sample cell 152 has a structure in which the sample cell 152 is finely moved two-dimensionally in a direction perpendicular to the optical axis 146 of the irradiation light 148, and the incident light of a fine spot in the in-plane direction of the sample 154 is adjusted. The beam is relatively scanned to measure scattered light from a minute area of sample 154 . This allows microscopic scale analysis of the sample 154 to be performed.

Specifically, the sample cell 152 is finely moved, for example, by placing the support holder on a two-axis stage of the axis and the XY axes and using the two-axis stage of the X axis and the Y axis. Furthermore, the support holder is installed on the Z-axis stage in the optical axis direction, that is, the support holder is installed on the three-axis stage of the X-, Y-, and Z-axes, and the sample cell 152 is moved minutely. good too. In addition, in the case of investigating a certain one-dimensional distribution in the local analysis of the sample 154, for example, the support holder is installed on the X-axis stage or the Y-axis stage, and the sample cell 152 is used using this single-axis stage. may be moved slightly.

Also, an XY two-axis stage or an XYZ three-axis stage for finely moving the sample cell 152 may be used as the support holder. In this case, the sample cell 152 is placed and held on an XY two-axis stage or an XYZ three-axis stage.

As the sample position adjusting mechanism 158, which is a mechanism for finely moving the sample cell 152, as described above, for example, an X-axis and Y-axis biaxial stage having a through hole through which light can pass, or an X-axis and Y-axis stage. A 3-axis stage of axis and Z axis can be used. As a multi-axis stage with 2 axes of X axis and Y axis, or 3 axes of X axis, Y axis, and Z axis, it is a manual type stage that moves linearly with a micrometer. A multi-axis stage with a volume of ±20 mm and a micrometer minimum scale of 10 μm can be used. When the minimum beam spot 157 of the irradiation light 148 irradiated onto the sample 154 by the condenser lens 142 of the light irradiation unit 140 is, for example, 10 μm or less in diameter, by moving the stage by a micrometer, for example, a minimum of 10 μm, The illumination positions within the sample 154 can be scanned. Scattered light measurements thus allow microscopic scale local analysis of the sample 154 .

Further, when the sample 154 is subjected to microscopic motion scanning for microscopic analysis with a resolution of about 10 μm or less, for example, an ultra-precision stage driven by a piezoelectric element can be used as the sample position adjusting mechanism 158 . As an ultra-precision stage driven by a piezoelectric element, for example, a table surface size of 120 mm×120 mm, a movement amount of 100 μm, and a minimum positioning resolution of 0.01 μm can be used. Here, in scanning the sample 154, a stage with a micrometer and a stage driven by a piezoelectric element may be combined to constitute a multi-axis stage and be used. In this case, for example, when scanning the sample 154, a stage with a micrometer is used for fine movement of 10 μm or more, and a piezo element driving stage is used for very fine movement of about 10 μm or less.

Low and medium molecules such as peptides and nucleic acids, biopolymers such as proteins, particles such as inorganic substances such as metals and plastics, and their aggregates, which are the targets of analysis, are subject to thermal motion by solvent molecules in liquids, for example water molecules in aqueous solutions. Under collision, they undergo Brownian motion, which is random diffusion. Such Brownian motion is sensitive to the temperature T of the liquid in which the particles and aggregates are present. For example, the diffusion coefficient D [m 2 /s] of the Brownian motion of spherical particles is represented by the Stokes-Einstein equation as shown below and is proportional to the liquid temperature T [K].
[Number 6]
D=kBT/(6πηa) (6)
Here, η [kg/m s] is the viscosity of the liquid medium (viscosity coefficient, viscosity), a [m] is the radius of the particles or aggregates, kB is the Boltzmann constant (kB = 1.380 × 10-23 J /K).
By estimating the diffusion coefficient D of the scatterers as spherical particles from dynamic light scattering (DLS) measurements, the size (radius) a of the spherical particles can be calculated by the Stokes-Einstein equation above as a=kBT/(6πηD ) can be estimated from Here, the viscosity η of the liquid medium and the temperature T of the liquid are measured by another appropriate method and their measured values are used. The temperature of the liquid sample 154 is preferably adjusted to a predetermined constant temperature and the scattered light is measured.

On the other hand, there is a need to change the temperature of the sample 154 while measuring the scattered light and analyze the state of particles and aggregates in the liquid in real time. Specifically, for example, in a solution of proteins, peptides, etc., the process of forming aggregates depending on temperature is analyzed. For example, while measuring the scattered light from the sample, the temperature of the sample is increased or decreased to analyze and evaluate the state of aggregate formation in real time. In the case of such analysis, the scattered light is measured while changing the temperature of the liquid sample 154 at a predetermined heating rate or a predetermined cooling rate. Therefore, the temperature of the liquid sample 154 can be changed at a predetermined heating rate or a predetermined cooling rate, and the scattered light generated by the sample 154 can be measured while the temperature of the sample 154 is adjusted and controlled. It is possible to do

Also, in the case of thin films forming crystal grains of polymers such as plastics, which are another object of analysis, the formation of crystal grains and the like changes sensitively to temperature. Therefore, similarly to the liquid sample 154, the scattered light generated by the sample 154 is measured while the temperature of the sample 154 such as a thin film is controlled to be maintained at a predetermined constant temperature. In the case of the sample 154 such as a thin film, the temperature of the sample 154 is changed at a predetermined temperature increase rate or a predetermined temperature decrease rate, and the scattered light generated by the sample 154 under such an environment is measured. This makes it possible to analyze a sample 154 such as a thin film.

The light irradiation section 140 has a mechanism for adjusting and controlling the temperature of the sample 154 . Various temperature controllers, cooling/heating stages, and the like can be used as devices for adjusting and controlling the temperature of the sample 154 . In these temperature controllers, for example, the temperature is changed by heating with a heater, cooling with a refrigerant, heating or cooling with a Peltier element, etc., and the temperature of an object is measured with a temperature sensor such as a platinum resistor or a semiconductor. , with feedback to the heating and cooling mechanisms, the temperature is regulated and controlled, for example, by a PID control method. Since the temperature of the sample 154 is adjusted and controlled via the sample cell 152 that houses the sample section 150, the thermal properties (heat capacity, thermal conductivity, etc.) of the sample section 150 including the sample 154 are taken into consideration. use a suitable temperature controller.

When the temperature controller cools the temperature of the sample 154 below room temperature, it is necessary to prevent condensation. Water droplets adhere to the sample cell 152 due to condensation due to cooling, and the scattered light due to the water droplets becomes strong back noise, making it difficult to measure the scattered light of the sample 154 . Therefore, it is preferable to introduce an inert gas such as nitrogen or argon into the outer peripheral portion of the sample cell 152, and an inlet for the inert gas is provided at a portion where the sample cell 152 of the temperature controller is attached. In addition, the introduction of inert gas has the effect of preventing oxidation that may occur during heating of samples such as thin films that form crystal grains such as polymers such as plastics, so it is not suitable for use at high temperatures. Introduction of active gas is preferred.

A cooling/heating stage using a Peltier element or the like as a device for adjusting and controlling the temperature of the sample 154 is compact because heating and cooling can be performed with a small-sized Peltier element or the like. Especially preferred. More specifically, for example, a Peltier cooling and heating stage for microscopes is suitable. Microscopic Peltier Cooling Heating Stages heat and cool samples, for example, by combining Peltier elements and water circulation units.

When the liquid sample 154 is to be measured, the temperature of the sample can be adjusted and controlled with an accuracy of ±0.05°C within a temperature range of -20°C to 120°C, for example. Also, the temperature rising/falling rate can be set at, for example, 0.01 to 20° C./min.

In addition, when a solid object such as a thin film is to be measured, for example, in the temperature range of -190°C or room temperature to 600°C, the temperature accuracy is ±0.2°C (-190°C to room temperature), ±0.05°C ( The temperature of the sample can be adjusted and controlled from room temperature to 600°C. Also, the temperature rising/falling rate can be set at, for example, 0.01° C./min to 20° C./min (−190° C. to room temperature) and 0.01° C./min to 150° C./min (room temperature to 600° C.).

The microscope Peltier cooling/heating stage is provided with an optical window that enables microscopic observation of the sample 154, and is compatible with an objective lens for microscopic observation. A Peltier type cooling and heating stage for a microscope corresponds to objective lenses for long working distances of, for example, 20x, 50x, and 100x, and enables microscopic observation of a sample under temperature control of the sample. is suitable as a temperature controller for the sample 154 in the particle/aggregate analyzer according to

A sample cell 152 containing a liquid sample 154 has a structure for adjusting and controlling the temperature of the sample 154 . The temperature of the sample 154 may be controlled by directly attaching and holding it to a cooling/heating portion such as a cooling/heating stage. That is, in this case, the cooling/heating stage itself functions as a support holder for the sample section 150 and holds the sample cell 152 . When the sample 154 is a solid object such as a thin film, the temperature of the sample 154 may be controlled by directly attaching and holding the sample 154 to a cooling/heating portion such as the cooling/heating stage.

A device for adjusting and controlling the temperature of the sample 154, such as a cooling and heating stage, finely scans the sample cell 152 for microscopic analysis of the sample 154 by scattered light measurements. It may be mounted on a multi-axis stage with three axes, X, Y, and Z axes. Further, the cooling/heating stage or the like may be provided with a mechanism for finely moving the sample cell 152 or the sample 154 itself in, for example, the X-axis and Y-axis directions. Specifically, for example, the sample cell 152 or the measurement sample is finely scanned by a micrometer stage built into a cooling/heating stage or the like, and scattered light from a local region of the sample 154 is measured to perform microscopic analysis. can.

(7) Description of Parallel Scattered Light Conversion Unit 170 The parallel scattered light conversion unit 170 converts the small-angle scattered light 160 out of the scattered light from the sample 154 emitted from the beam of the irradiation light 148 irradiated to the sample unit 150 into A range of measured scattering angles .theta.S are captured and converted into parallel small-angle scattered light 168 parallel to the optical axis 146. FIG. Although this embodiment describes the case of using the forward small-angle scattered light 160, the concept is the same for the backward small-angle scattered light, and similar actions and effects can be obtained. Specifically, the sample 154 is irradiated with irradiation light 148 condensed to a predetermined size by the condenser lens 142 of the light irradiation unit 140 . In addition to transmitted light 164 from sample 154, small angle scattered light is emitted forward. At this time, small-angle scattered light is also emitted backward at the same time. A representative example of an analyzer for analysis will be described. Assuming that the minimum beam spot 157 of the sample 154 is irradiated with the irradiation light 148, the small-angle scattered light 160 emitted within the range of the measurement scattering angle θS from the minimum beam spot 157 is incident on the lens 172, and the lens 172 It is converted into parallel small-angle scattered light 168 that is parallel light (collimated light). The diameter of the minimum beam spot 157 is, for example, in the range of several tens of μm to several hundreds of μm, preferably in the range of 10 μm or more and 100 μm or less.

The parallel scattered light conversion unit 170 converts the parallel small angle scattered light 168 parallel to the optical axis 146, and the parallel small angle scattered light 168 enters the scattered light detection unit 200, where the light intensity of the parallel small angle scattered light 168 is detected. be done. In the case of using the small-angle scattered light generated in the rear, the scattered light is converted into light parallel to the optical axis 146 by the parallel scattered light conversion unit 170 (not shown) arranged behind, and the intensity of the light is detected by the scattered light detection unit 200. Detected by equipment similar to Specifically, the scattered light detection unit 200 measures the parallel small-angle scattered light 168 as an intensity distribution in a predetermined scattering angle range at once, or continuously measures the intensity distribution in a predetermined time range. . The scattered light detection unit 200 may capture moving images of parallel forward small-angle scattered light or parallel backward small-angle scattered light as a parallel forward small-angle scattered light image or a parallel backward small-angle scattered light image, respectively.

The measurement result by the scattered light detection unit 200 is sent to the analysis unit 250, and the analysis unit 250 determines the state of particles and aggregates of the sample 154 from the intensity distribution of the parallel small-angle scattered light 168 measured by the scattered light detection unit 200. To analyze. For this reason, the analysis unit 250 temporarily stores the measurement data of the parallel small-angle scattered light 168 measured by the scattered light detection unit 200 in the storage device 290 of the arithmetic processing unit 270, and stores the measurement data in the storage device 290 as necessary. read from and analyze.

The parallel scattered light conversion section 170 has a lens 172 and a support device 174 that supports the lens 172 . The support device 174 has, for example, a mechanism for supporting the lens 172 and adjusting the position and tilt angle of the lens 172 .

As the lens 172 of the parallel scattering light conversion unit 170, various lenses, for example, a single lens such as a spherical plano-convex lens, a spherical bi-convex lens, a cylindrical plano-convex lens, and a cylindrical bi-convex lens (hereinafter simply referred to as a "cylindrical lens"). , and various achromatic lenses in which two or more single lenses having different optical properties are bonded together can be used. The lens 172 may be the single lens, the achromatic lens, or an optical system combining these various lenses so as to efficiently take in scattered light spread over a predetermined scattering angle range and convert it into parallel light. consists of

The lens 172 of the parallel scattered light conversion unit 170 is arranged between the condenser lens 142 of the light irradiation unit 140 and the photodetector 202 of the scattered light detection unit 200, and further converts the small-angle scattered light 160 from the sample 154 into the optical axis. It is placed in a position suitable for converting into parallel small angle scattered light 168 parallel to 146 . The scattered light detector 200 is arranged so that the parallel small-angle scattered light 168 converted by the lens 172 is perpendicularly incident on the detection surface of the photodetector 202 of the scattered light detector 200 . That is, the photodetector 202 of the scattered light detection unit 200 is arranged perpendicular to the optical axis 146 .

An achromatic lens with little spherical aberration is suitable for the lens constituting the parallel scattered light conversion unit 170 . For example, when a circular achromatic lens is used, the scattered light emitted from the irradiation area of the minute circular spot-like light of the sample 154, the minimum beam spot 157 shown in FIG. The parallel small-angle scattered light 168 can be obtained relatively easily. Also, as the lens 172 of the parallel scattered light conversion unit 170, a shape other than circular, for example, a cylindrical achromatic cylinder lens can be used. When using a single-lens cylindrical lens or an achromatic cylinder lens, the collimated small-angle scattered light 168 is obtained only in the curved surface direction of the cylinder.

In the configuration shown in FIG. 2, when the distance from the sample cell 152 of the sample section 150 to the lens 172 of the parallel scattered light conversion section 170 is the distance LB, the distance LB is the focal length f of the lens 172 and the incident angle of the incident light. It depends on the range, the beam size of the incident light, the size of the illuminating light 148 at the sample 154, the structure of the sample cell 152, and the like. Among these factors, it largely depends on the focal length f of the lens 172 used in the parallel scattered light conversion section 170 . Assuming that the small-angle scattered light 160 is emitted from the minute analysis area 156 of the sample 154, the position where the lens 172 of the parallel scattered light conversion unit 170 is arranged is such that the distance LB is theoretically equal to the focal length f.

However, the most appropriate placement of the lens 172 of the parallel scattered light conversion section 170 is the position where the lens 172 causes the small angle scattered light 160 from the sample 154 to be parallel (collimated) to the optical axis 146 . Specifically, the scattered light passing through the lens 172 of the parallel scattered light conversion unit 170 becomes a parallel image, and is arranged at a position where it becomes a parallel small-angle scattered light image. That is, the position of the lens at which the parallel small-angle scattered light 168 is obtained is the position where the lens 172 of the parallel scattered light conversion section 170 is arranged.

More specifically, the lens 172 constituting the parallel scattered light conversion section 170 can be, for example, a circular achromatic lens with a diameter of 50 mmφ and a focal length f in the range of 50 mm to 150 mm. For circular lenses of the same diameter, a shorter focal length can capture a wider range of scattering angles from the sample 154 .

For example, a 20× infinity correction objective lens, for example, is used as the condenser lens 142 of the light irradiation unit 140 for the sample 154 whose measurement target is latex particles with a diameter of 0.1 μm dispersed in pure water. The objective lens has a focal length f of 10 mm and a numerical aperture NA of 0.4. In FIG. 2, the distance LA from the condenser lens 142 to the sample 154 is arranged to be 10 mm, for example. Irradiation light 148 having a maximum incident angle θi of 4.3 degrees shown in FIG. Here, the sample cell 152 for storing the sample 154 is composed of two pieces of 0.15 mm thick microscope cover glass sandwiching a 0.5 mm thick silicone rubber sheet with a 7 mm diameter hole. are doing. In this case, as the lens 172 of the parallel scattered light conversion unit 170, for example, a circular achromatic lens with a diameter of 50 mm and a focal length f of 75 mm is arranged at a position where the distance LB is 68 mm, which is slightly shorter than the focal length of 75 mm. The lens 172 can capture forward small-angle scattered light from the sample 154 in an angular range where the emission angle θ0S is less than 20 degrees and convert it into parallel small-angle forward scattered light.

In this embodiment, an objective lens having a short focal length and a large NA can be used as the lens 172 of the parallel scattered light conversion section 170 . In particular, an objective lens that has a relatively long working distance WD and is corrected for infinity is suitable. When an objective lens is used as the lens 172 of the parallel scattered light conversion unit 170, the small angle scattered light 160 from the sample 154 of the sample unit 150 is accurately converted into the small parallel scattered light 168 by the objective lens. Compared to lenses with a long focal length f, such as an achromatic lens with a focal length f of 75 mm, for example, the short focal length of the objective lens makes it difficult to adjust the position of the objective lens. However, a precision optical stage or the like, for example, a multi-axis precision optical stage using a micrometer or the like is used in the support device 174 having a mechanism for adjusting the position of the lens 172 of the parallel scattered light conversion unit 170, so that the objective lens can be finely adjusted. Small-angle scattered light 160 from sample 154 in sample section 150 can be captured by a structure that allows fine adjustment of the position, particularly the position of the objective lens that defines the distance between sample cell 152 and the objective lens used in lens 172. , can be accurately converted by the objective lens. That is, accurate parallel small-angle scattered light 168 can be obtained.

Alternatively, the small-angle scattered light 160 may be directly guided to the light receiving section of the photodetector 202 of the scattered light detection section 200 without using the parallel scattered light conversion section 170 . That is, without using the parallel scattered light conversion section 170, the scattered light detection section 200 can measure the small-angle scattered light 160 from the light irradiation section 140 all at once.

(8) Description of Transmitted Light Blocking Portion 176 The transmitted light 164 that has passed through the sample 154 has an extremely high intensity compared to the scattered light from the sample 154 . Therefore, in order to measure weak scattered light with the photodetector 202 of the scattered light detection unit 200 , it is necessary to avoid scale over of the photodetection signal due to strong transmitted light entering the scattered light detection unit 200 . In addition, the transmitted light causes stray light to the scattered light detection unit 200 and interferes with weak scattered light measurement. Therefore, it is preferable to prevent the transmitted light 164 from the sample 154 from entering the scattered light detection section 200 by the transmitted light shielding section 176 as shown in FIGS. That is, the transmitted light blocking section 176 blocks the transmitted light 164 of the sample 154 from entering the scattered light detection section 200 or attenuates the intensity of the transmitted light 164 .

The transmitted light 164 from the sample 154, like the scattered light from the sample 154, particularly the forward small-angle scattered light 160, passes through the lens 172 of the parallel scattered light conversion unit 170, and thus proceeds under the lens action of the lens 172. . That is, the illumination light 148 formed by the condenser lens 142 of the light illumination unit 140 is incident on the sample 154, and the transmitted light 164 spreads at various radiation angles within the range of the predetermined maximum transmission angle θ It radiates from 154 and is captured by lens 172 in the same manner as scattered light from sample 154 . Then, the lens 172 converts the transmitted light 164 into parallel transmitted light 166 as well as the scattered light from the sample 154, and parallels the scattered light collimated by the lens 172, especially the parallel small-angle scattered light 168, to generate the scattered light. Proceed to photodetector 202 of detector 200 . Therefore, the transmitted light blocker 176 blocks the parallel transmitted light 166 collimated by the lens 172 so as to prevent the transmitted light 164 from the sample 154 from entering the photodetector 202 of the scattered light detector 200 . Mounting on shaft 146 is preferred. The transmitted light shielding part 176 is arranged, for example, between the lens 172 and the detection surface of the scattered light detection part 200, or between the sample cell 152 and the lens 172, especially when the small-angle scattered light 160 from the sample 154 is measured. do.

Various absorbers, mirrors, and the like can be used for the transmitted light shielding portion 176 . Furthermore, the transmitted light shielding part 176 must not generate scattered light. The material of the transmitted light shielding portion 176 is not particularly limited as long as it has a function of blocking transmitted light or attenuates the intensity of transmitted light and does not generate scattered light. For example, paper, wood, metal, plastic, glass, etc. can be used as the material of the transmitted light shielding portion 176 . In order to enhance the effect of blocking transmitted light or the effect of attenuating transmitted light, these surfaces are preferably matte, particularly black. Various optical elements can also be used as the transmitted light shielding portion 176 . For example, filters, mirrors, half mirrors, beam splitters, optical path changing optical elements, and the like can be used. More specifically, various filters such as metals, metal films, and dielectric multilayer films, mirrors and half mirrors, polarizing elements such as polarizing plates and wavelength plates, absorption ND filters containing light absorbing substances, metal films, etc. A reflective ND filter, an optical path changing optical element such as various prisms and lenses, and the like may also be used.

The shape and size of the transmitted light from the sample 154 depends on the shape and range of incident angles of the irradiation light 148 incident on the sample cell 152 or the sample 154 of the sample unit 150, the structure of the sample cell 152, and the irradiation light 148 on the sample 154. It depends on the spot size, the shape and focal length f of the condenser lens 142, and the like. Therefore, the shape and size of the member used for the parallel scattered light conversion section 170 are selected according to the shape and size of the transmitted light 164 from the sample 154 . For example, in the case of circular transmitted light, a disk-shaped member having a size larger than that is used. When the transmitted light shielding part 176 is arranged between the lens 172 and the scattered light detection part 200, the parallel transmitted light 166 is collimated or diffused by the lens 172. Therefore, the arrangement of the transmitted light shielding part 176 is A member corresponding to the shape and size of the transmitted light at the position where the light is transmitted is used.

The transmitted light blocker 176 blocks or attenuates the parallel transmitted light 166 from the sample 154 . However, not only that, but also the scattered light from the sample 154 is blocked or attenuated. That is, the transmitted light shielding part 176 becomes a shadow in measurement of the scattered light by the photodetector 202 of the scattered light detection part 200, and the scattering angle corresponding to the part of the shadow appearing on the light receiving surface of the scattered light detection part 200 is At range, the measurement of scattered light is difficult. Therefore, it is desirable to accurately measure the parallel small-angle scattered light 168 from the sample 154 to the lower angle side. is preferred. For this reason, it is preferable to use a member slightly larger than the size of the transmitted light beam to be blocked for the transmitted light blocking portion 176 .

For example, a 20x infinity corrected objective lens (f = 10 mm, NA 0.4) is used as the condenser lens 142 of the light irradiation unit 140 for the pure water dispersion sample 154 of latex particles with a diameter of 0.1 µm. Then, as shown in FIG. 2, it is placed at a position where the distance LA from the condenser lens 142 to the sample 154 is 10 mm. Irradiation light 148 is incident on the sample cell 152 at a maximum incident angle θi of 4.3 degrees shown in FIG. It is arranged at a position where the distance LB is 68 mm. Here, the sample cell 152 for storing the sample 154 has a structure in which a silicon rubber sheet with a thickness of 0.5 mm and a hole with a diameter of 7 mm is sandwiched between two microscope cover glasses with a thickness of 0.15 mm. are doing. In the above case, the transmitted light 164 from the sample 154 is collimated by 172 and the diameter D1 of the circular collimated beam of transmitted light is 10 mm. Therefore, in this case, it is preferable to use, for example, a metallic disc or rod with a diameter of 12 mm and which is matted black as the transmitted light shielding portion 176 .

Further, the transmitted light shielding part 176 is arranged either between the parallel scattered light conversion part 170 and the scattered light detection part 200 or between the sample 154 and the parallel scattered light conversion part 170 to obtain the effect. . When the transmitted light shielding part 176 is installed between the sample 154 and the parallel scattered light conversion part 170, the transmitted light from the sample cell 152 of the sample part 150 or the sample 154 is a minute irradiation light beam spot on the sample 154. Since the transmitted light is from the minimum beam spot 157, the size of the member of the transmitted light blocking portion 176 can be made smaller than in the case of blocking the parallel transmitted light 166 that has passed through the lens 172. The light shielding portion 176 can be made smaller. The transmitted light shielding portion 176 having a smaller size makes it possible to measure scattered light at a smaller scattering angle.

In addition, the transmitted light shielding part 176 prevents the transmitted light from entering the photodetector 202 of the scattered light detection part 200 on the optical axis of the transmitted light between the sample part 150 and the scattered light detection part 200. Deploy. Moreover, the transmitted light shielding part 176 may be arranged on the optical axis (advancing direction) of the transmitted light, and the position (distance) between the sample 154 and the scattered light detection part 200 is not limited. It should be noted that the transmitted light shielding part 176 may not be arranged in the configuration for measuring the rear small-angle scattered light.

(9) Description of Configuration and Effects of Scattered Light Detector 200 The scattered light detector 200 includes a photodetector 202 having a light receiving surface 203. Parallel small angle scattered light 168 is detected. A light receiving surface 203 of the photodetector 202 is composed of a large number of photodetection elements. Each photodetector 11 outputs an electrical signal corresponding to the light intensity of the incident parallel small-angle scattered light 168 , and the electrical signal is amplified by the amplification/cooling section 212 and sent to the analysis section 250 .

It is important that the parallel small-angle scattered light 168 is perpendicularly incident on the light receiving surface 203 of the scattered light detection unit 200 . For this reason, the scattered light detection unit 200 includes a light receiving surface 203 of the photodetector 202 for adjusting the light receiving surface 203 of the photodetector 202 to be perpendicular to the optical axis 146 , that is, the parallel small-angle scattered light 168 . A position adjustment mechanism 220 is provided with an angle adjustment mechanism for adjusting the angle of surface 203 with respect to optical axis 146 . The scattered light detection unit 200 includes an amplification/cooling unit 212 that amplifies the signal from each photodetection element 11 of the photodetector 202 and cools the scattered light detector 202 . A control interface (not shown) for electrically transmitting and receiving output signals and control signals to the photodetector 202 and the amplifier/cooling unit 212, a driving power supply (not shown) for the photodetector 202, etc. are doing.

An example of using the photodetector 202 for detecting the image of the forward small-angle scattered light image of the small-angle scattered light 160 by the scattered light detection unit 200 will be described below. However, the image of the forward small-angle scattered light image based on the small-angle scattered light 160 can be detected in the same way by using the light detection camera 204, and the following description can be applied as it is. That is, the image of the forward small-angle scattered light image based on the small-angle scattered light 160 can be similarly detected using the light detection camera 204 having the camera lens 206 shown in FIGS. The examples shown in FIGS. 13 and 14 specifically describe the image of the forward small-angle scattered light image produced by the action of the ring-shaped aperture 182 of the scattering angle filter 180 . However, as described above, it can also be used in place of the scattered light detection unit 200 in the detection of the small-angle forward scattered light image described with reference to FIGS.

Photodetector 202 is a photodetector suitable for detecting scattered light from sample 154 . Specifically, the photodetector 202 is a multichannel photodetector (consisting of a multi-pixel sensor) including a large number of photo sensor element arrays such as a multichannel photodiode array and a linear image sensor, a CCD image sensor, a CMOS image sensor, and the like. a photodetector). In this embodiment, as the photodetector 202, it is preferable to use a multi-channel photodetector, which is a large-area photodetector in which a large number of microphotodetection elements are arranged or integrated at high density. In a configuration using a multi-channel photodetector, the intensity distribution of the parallel small-angle scattered light 168 can be measured, for example, in a spatial pattern at once in a short time while the photodetector 202 is fixed and stationary. That is, in this case, the scattered light image drawn by the scattered light by the multichannel photodetector is measured as a still image. Furthermore, by continuously photographing the parallel small-angle scattered light 168 with a multi-channel photodetector, in other words, by photographing moving images, temporal changes in the spatial pattern described by the intensity distribution can be measured.

In this case, the small-angle parallel scattered light 168 is continuously photographed as images by the multichannel photodetector, and the moving images of the scattered light images obtained include the SLS and DLS regarding the sample 154 . That is, the position dependence of the scattered light intensity photographed as an image, that is, the scattered light intensity distribution corresponds to the SLS. On the other hand, focusing on a specific point or a specific position in the captured scattered light image, the time change of the scattered light intensity at that point corresponds to the DLS. By continuously photographing scattered light images at predetermined time intervals, it is possible to acquire temporal changes in scattered light intensity at a point of interest on the scattered light image. Based on this acquired data, if necessary, at each point on the scattered light image, in other words, at a plurality of parts, the analysis unit 250 analyzes the frequency power spectrum or changes in the frequency power spectrum with respect to the time change of the scattered light intensity. can be output from

It is preferable that the photodetector 202 measures the scattered light in, for example, a predetermined scattering angle range at a time of, for example, 100 ms or less, more preferably 50 ms or less. It is preferable that the photodetector 202 can continuously shoot the parallel small-angle scattered light 168 from the parallel scattered light conversion unit 170 as scattered light images, that is, to shoot moving images. As the photodetector 202, for example, a multi-channel photodetector including a CMOS image sensor (hereinafter simply "CMOS camera") is particularly suitable. With a CMOS camera, for example, continuous shooting is possible with an exposure time of 10 ms and a frame rate of 100 f/s or 50 f/s with a length of continuous shooting (the number of shot images) of 150 to 300 shots.

The photodetector 202 of the scattered light detection unit 200 preferably has high light receiving sensitivity near the light wavelength of the light source 110 used for scattered light measurement. Scattered light from molecules strongly interacting with solvent molecules, such as low and medium molecules such as peptides and nucleic acids in solution, and biopolymers such as proteins, and their aggregates, is dispersed in the liquid and is relatively strong against the solvent molecules. The interactions are generally weak compared to particles and aggregates thereof, such as nano- to sub-micron metals, plastics, and inorganic substances. Even forward small-angle scattered light or backward small-angle scattered light, which has a relatively large scattering intensity, has an extremely small scattered light intensity compared to the scattered light from the particles and aggregates dispersed in the solvent, so the scattered light A photodetector with high detection sensitivity is preferably used for the photodetector 202 of the detection unit 200 .

The photodetector 202 itself needs to have a high quantum efficiency near the light wavelength of the light source 110, and the photodetector 202 is used in a highly sensitive state in order to measure weak scattered light. For this purpose, the light receiving part of the photodetector 202 is cooled in order to suppress the dark current noise of the photodetector 202 as much as possible. The light-receiving part of the photodetector can be cooled by blowing air, water flow circulation, electronic elements, etc. In particular, when using an electronic element such as a Peltier element, the element is small, so it is possible to cool the photodetector. can be built in. For example, it is preferable to use a photodetector with a quantum efficiency of 50% or more, and use a Peltier element to cool the light receiving part of the photodetector to −10° C. or less to reduce dark current noise.

As the photodetector 202 suitable for the scattered light detection unit 200, a CCD camera with a CCD image sensor or a CMOS camera with a CMOS image sensor, which has a particularly large light receiving surface, is suitable. A CCD camera or CMOS camera for weak light measurement is particularly suitable, and a linear image sensor with a high quantum efficiency of 50% to 70% or more, a very small dark current, a large saturation charge amount, and a wide dynamic range is used. A face-to-face camera is preferred. In order to suppress dark current, these cameras may incorporate an electronic cooler such as a Peltier device in the amplifier/cooling unit 61, etc., and may be configured to cool the light receiving surface to -10°C or less, for example. .

As the photodetector 202 of the scattered light detection unit 200, for example, the size of the light receiving unit is 14.0 mm wide×16.6 mm high, and the minimum pixel size is 6.5 μm (quantum efficiency of 50% or more) effective pixels 2160×2560. A CMOS camera with a 5.5 megapixel CMOS area image sensor of channels (pixels) can be used. A built-in Peltier element allows the CMOS sensor to be cooled down to -10°C, and the dark current noise is extremely low at 0.02e-/(pixels/sec). Also, the dynamic range is as wide as 3300:1.

With this CMOS camera, in continuous shooting (video shooting) of scattered light images, for example, when the effective light receiving surface for shooting is 2160 × 2560 pixels in the entire area, the shooting frame rate is 49 f / s with the global shutter mechanism, rolling shutter The mechanism can measure images continuously at 100 f/s. When shooting moving images at a higher speed, for example, the effective light receiving surface area is set to, for example, 1080×1920 pixels, and moving images such as scattered light images are shot at 97 f/s with a global shutter and 200 f/s with a rolling shutter. becomes possible. However, the camera suitable for the photodetector 202 of the scattered light detection unit 200 is not limited to the specifications of the CMOS camera described above.

(10) Description of Configuration and Effect of Analysis Unit 250 When the operator inputs measurement conditions for the scattered light detection unit 200 from the input/output device 266, the scattered light detection unit 200 is controlled based on the input measurement conditions. , the position and angle of the photodetector 202 are set and fixed based on the input conditions. After the photodetector 202 is fixed, the intensity distribution of the parallel small-angle scattered light 168 is measured based on the input conditions. If the input condition is measurement of a still image, the still image is measured. For example, if the instruction content is to shoot a still image a plurality of times at specified intervals, shooting is performed according to the instruction content. Also, if the input condition is measurement of moving images, the intensity distribution of the parallel small-angle scattered light 168 is continuously photographed as moving images.

The shooting timing and shooting time of still image shooting and moving image shooting are often controlled according to the passage of time. For this reason, among the control conditions input from the input/output device 266, the conditions related to the passage of time are once converted into conditions based on absolute time by the time management device 280 and stored. Based on the correspondence with the passage of absolute time made by the time management device 280, the related configuration of the analysis device 100 is controlled. Each component is controlled by a control signal 264 which is output from the controller 260 to the relevant component based on the operation of the CPU 276 . Measurement results from each component and signals representing the state of each component are captured by the analysis unit 250 via the controller 260 . Data measured by the scattered light detection unit 200 is the same. Specific analysis operations and the like by the analysis unit 250 will be described later.

1.4 Measurement of dynamic light scattering (DLS) and static light scattering (SLS) (1) Overview
As described above, one light scattering measurement method is dynamic light scattering (DLS), which detects light scattering from the sample 154 at a predetermined scattering angle and measures the temporal variation, or fluctuation, of the light scattering. : Dynamic Light Scattering) measurement method. This DLS measurement provides, for example, information about the movement of the scatterers of the sample 154 to be analyzed.

At a predetermined scattering angle θ0, that is, at a certain scattering vector _q0 , _the frequency power spectrum I(ω) and the correlation A function G(τ) and a scattering factor S(q ₀ , ω) are obtained. where ω is the angular frequency and τ is the relaxation time.

Furthermore, in DLS measurement analysis, the diffusion coefficient D of the scatterer is often calculated from the correlation function G(τ) to evaluate the size of the scatterer, ie, the value of the hydrodynamic particle size Rh. Here, the evaluation value of the particle diameter size Rh of the scatterer evaluated from the diffusion coefficient D is effective only when a spherical scatterer model can be applied to the analysis target such as fine particles and aggregates. It should be noted. In addition, to calculate Rh, it is necessary to use the viscosity coefficient (viscosity) η of the sample solution containing fine particles and aggregates, and the measured value of the temperature T. In particular, the value of the viscosity coefficient η will be measured.

Another light scattering measurement method is a static light scattering (SLS) measurement method that measures the dependence of the scattering angle on the average intensity of light scattering from an analyte. The SLS measurement method mainly provides information about the structure of the scatterers of the sample 154 to be analyzed.

At various scattering angles θ (scattering vector q), the average intensity I(q, ω ₀ ) of light scattering averaged at a certain time t ₀ is measured, and the structure factor S(q, ω ₀ ) in the scatterer is obtained. be done. where ω ₀ is the angular frequency with respect to t ₀ . In the SLS measurement method, the scattered light intensity is often measured in dilute solutions such as polymers and colloids in which the concentration of the scatterers is sufficiently thin so that the interference effect between the scatterers can be ignored, and the intensity of the scattered light is measured in samples of different concentrations. In the SLS analysis, the molar mass (molecular weight), the radius of gyration, and the second virial coefficient are analyzed and evaluated, for example, from Zim's plot, Berry's plot, and the like.

In addition, in the SLS measurement method, the scattering angle dependence of the average light scattering intensity is measured particularly at a low angle forward or backward, and the fractal dimension, which is an index of the structural parameter (structural factor) of the scatterer, is analyzed and evaluated. That _is , the _structure factor S(q , ω ₀ ) is the spatial Fourier transform of the average local density function, so the factors directly related to the fine structure of the scatterer can be evaluated.

That is, the average intensity I(q,ω ₀ ) of forward scattered light or backscattered light measured for particles or aggregates exhibits a power function type. That is, I(q, ω ₀ )=I ₀ q ^−α . Here, _I0 is a constant, α is a power number, and indicates a value of 0<α<3 for a measurement object in a three-dimensional space. Then, the fractal dimension Df of the aggregate or agglomerate of the measured particles is evaluated by evaluating the power number α from curve fitting with the power function of the measured average intensity I(q,ω ₀ ).

Conventionally, in the analysis of particles by light scattering measurement, the DLS measurement method was used for analytical evaluation of particle size and its distribution, while the SLS measurement method was used for analytical evaluation such as molecular weight. In other words, depending on the purpose of analysis and evaluation, analytical instruments employing either one of the light scattering measurement methods have been used separately. There has been no analyzer capable of measuring both the DLS measurement method and the SLS measurement method with the same analyzer and performing both analyses.

In addition, the DLS measurement method and the SLS measurement method greatly differ in the method of measuring light scattering itself as described above, and also greatly differ in the method of analyzing the measurement data. If two aspects of characteristic light scattering, that is, temporal measurement (DLS measurement) and spatial measurement (SLS measurement) of light scattering can be measured simultaneously for the same analysis object, analysis objects such as particles and aggregates It is possible to simultaneously analyze and evaluate the knowledge about the motion and structure of , with one light scattering measurement device, and the effect is very large.

In addition, the formation of micron-order droplets by liquid-liquid phase separation, which has recently attracted particular attention for polymer gelation, crystallization of proteins and peptides, and intracellular metabolic reactions, and heterogeneity in solution. There is a great need for microstructural analysis and evaluation of solutions on a submicron scale for the aggregation of proteins, peptides, etc. that occur in microscopic scales. The analyzer 100 of the embodiment can sufficiently meet such needs.

(2) Description of Scattered Light Detection in Analysis Apparatus 100 of Embodiment As described above, in the analysis apparatus 100, the DLS measurement method and the SLS measurement method are performed by the scattered light detection unit 200 on the sample 154 to be analyzed. measurement data that can be used for both Before describing the configuration for measurement, the results measured by the configuration of the analyzer 100 will be described with reference to FIGS. 4 and 5. FIG.

FIG. 4 is a photographed image 230 obtained by measuring and analyzing parallel small-angle scattered light 168 based on small-angle scattered light 160 generated from the minimum beam spot 157 by the photodetector 202 of the scattered light detection unit 200 . A small-angle scattered light image by the small-angle scattered light 160 from the minimum beam spot 157 is magnified on a microscope scale and displayed as a microscope image on the photodetector 202 of the scattered light detection unit 200. This microscope image is stored in the storage device 290 as the measurement result. It is a photographed image created based on data of a microscope image that is stored and is a measurement result. On the side closer to the optical axis 146 is a dark portion 232 that is generated because the small-angle scattered light from the sample 154 is shielded by the transmitted light shielding portion 176 . An image portion 234 based on the measurement of the parallel small-angle scattered light 168 spreads on the outer peripheral side of the dark portion 232 .

(3) Explanation of Analysis of DLS Analysis Region R1 in Image Unit 234 In FIG. Measurements are repeated every time. In this case, in this embodiment, it is possible to specify measurement data relating to the DLS analysis region R1 obtained by repeated measurements, and to implement the DLS measurement method.

For example, when the operator specifies the DLS analysis region R1 as an analysis target by the DLS measurement method, as shown in FIG. It is obtained from the operation in The analysis unit 250 can specify the data regarding the DLS analysis region R1 for each measurement of the X-coordinate value xp and the Y-coordinate value yp in the obtained coordinates (xp, yp). Therefore, by reading the data at each measurement of the DLS analysis region R1 from the measurement data obtained by repeatedly measuring continuously at predetermined time intervals, and displaying the change as a graph with time as a parameter, for example, the DLS Analysis results based on the measurement method can be output from the input/output device 266 .

(4) Description of Method of Specifying Measurement Data Concerning DLS Analysis Region R1 FIG. 6 is a partially enlarged view of an image detection surface in an example of the photodetector 202 in the scattered light detection unit 200. FIG. Each photodetector is arranged to form a two-dimensional plane. For example, photodetector 11 to photodetector 12, photodetector 13, . Photodetector elements 11 to 21, photodetector elements 31, . . . are arranged. The detection result of each photodetector is transmitted from the scattered light detection unit 200 to the analysis unit 250 in a predetermined order along the X-axis direction and the Y-axis direction, and stored in the storage device 290 of the analysis unit 250, for example, in the order of addresses. retained. The order of addresses held in the storage device 290 has a predetermined correspondence with each photodetector. Therefore, by specifying the address of the storage device 290, the measurement data of the photodetector to be used can be read. Therefore, the image based on the small-angle scattered light 160 detected by the photodetector 202 is finely divided in two dimensions of the X axis and the Y axis, and the detection data for each divided area is stored in the storage device 290 for each of the divided areas. The area and the storage position in the storage device 290 are stored while maintaining the correlation.

Furthermore, under conditions set by the operator, the photodetector 202 maintains the operation of detecting an image based on the small-angle scattered light 160, and the photodetector elements 11 to 34 for each area, in other words, are exemplified. The detection results for each area are repeatedly stored in the storage device 290 under the above conditions. That is, the temporal change of the image based on the small-angle scattered light 160 is stored in the storage device 290 . Based on the detection results stored in the storage device 290, it becomes possible to analyze changes in the measurement results over time for the set area, and the DLS analysis by the analysis unit 250 becomes possible.

The forward small-angle scattered light image detected by the photodetector 202 includes an area corresponding to the optical axis 146 and a plane perpendicular to the optical axis 146 from the optical axis 146, as shown in FIGS. contains the area associated with the radial image at . The forward small-angle scattered light image of the area extending in the radial direction from the optical axis 146 including the area specified by the operator is finely divided, the finely divided measurement unit area is measured, and the measurement result is divided into the finely divided areas. Each area of the specified measurement unit is stored in the storage device 290 in a state in which the position can be designated. Therefore, the detection results necessary for SLS analysis in the specified region can be read out and used, thereby enabling SLS analysis related to the specified region.

When the positional relationship between the optical axis 146 and the photodetector 202 of the scattered light detection unit 200 is specified, the coordinates (x0, y0) of the optical axis 146 and the coordinates (xp, yp) of the DLS analysis region R1 shown in FIG. ) and the photodetector elements shown in FIG. 6 are associated with each other. Therefore, the address of the storage device 290 holding the detection data is determined from the associated photodetection element, and the measurement results of the coordinates (x0, y0) desired to be used and the coordinates (xp, yp) of the DLS analysis region R1 are wired. 205 can be read out. Here, each photodetector element corresponds to the area of each measurement unit described above.

When performing an analysis based on the DLS measurement method, it is necessary to store the data measured at predetermined time intervals in the DLS analysis region R1 each time, and to read out the stored data at different measurement points when performing the analysis. Become. FIG. 7 shows a state in which data measured at predetermined time intervals are stored. When measuring N times at measurement time points T1, T2, . . . Secure locks B2, . . . TN. The detection result of each photodetector element of the photodetector 202 at the measurement time T1 is stored in the area of the block B1 of the storage device 290 at the address associated with each photodetector element. Similarly, the detection result of each photodetector element of the photodetector 202 at the measurement time T2 is stored in the address associated with each photodetector element in the area of block B2. By repeating this N times, the detection result of each photodetector element of the photodetector 202 can be stored by associating the detection result up to the measurement time TN with the address in the associated block.

When performing an analysis based on the DLS measurement method in the DLS analysis region R1, the measurement data of the DLS analysis region R1 at different detection times T1 . . . TN can be read from the blocks B1 . , it is possible to perform analysis based on the DLS measurement method from these measurement data.

It is very important to set the DLS analysis area R1 to the position to be analyzed. By creating and displaying the photographed image 230 shown in FIG. 4 by the analysis unit 250 and setting the DLS analysis region R1 based on the photographed image 230, a very useful DLS analysis result can be obtained. A DLS analysis region R1 is set on the captured image 230, for example, by the method described above. The photodetector element in the photodetector 202 corresponding to the DLS analysis region R1 is specified by the calculation of the analysis unit 250, the data corresponding to the DLS analysis region R1 is read for each block in FIG. DLS analysis can be performed on the region desired to be analyzed.

(5) Description of method for specifying measurement data relating to SLS analysis region R2 Next, the SLS measurement method in the analysis device 100 will be described with reference to FIGS. 8 and 9. FIG. The photographed image shown in FIG. 8 is the photographed image described above with reference to FIG. is a photographed image obtained by measuring and analyzing the parallel small-angle scattered light 168 based on . The SLS measurement method is a measurement method for measuring the scattering angle dependence of the low-angle light scattering intensity, and the measurement of the SLS analysis region R2 shown in FIG. 8 will now be described as an example. The parallel small-angle scattered light 168 is measured by the photodetector 202 shown in FIG. In FIG. 8, of the small-angle scattered light 160, a dark portion 232 near the optical axis 146 is a portion where the parallel small-angle scattered light 168 and the parallel transmitted light 166 are blocked by the transmitted light blocking portion 176. FIG. The image portion 234 represents the intensity of the small-angle scattered light that depends on the scattering angle with respect to the optical axis 146 of the parallel small-angle scattered light 168 .

Once the SLS analysis region R2 in the image portion 234 is identified, the photodetector elements of the photodetector 202 corresponding to the SLS analysis region R2 are identified as described above. The photodetector elements of the photodetector 202 corresponding to the X-axis direction of the SLS analysis area R2 are, for example, the photodetector elements 21 to 2N shown in FIG. The photodetector elements corresponding to the width of the SLS analysis region R2 in the Y-axis direction are the photodetector element 11, the photodetector element 21, and the detector element 31, respectively. Actually, the width of the SLS analysis region R2 in the Y-axis direction is wider than the photodetector elements 21 and 2N, and there is a possibility that a plurality of or many photodetector elements are included in the Y-axis direction. In that case, the average value of the measurement results of the photodetectors included in the width in the Y-axis direction may be used.

In the above description, the SLS analysis region R2 is assumed to be in the X-axis direction for the sake of simplicity. However, when determining the scattering angle, angles other than the X-axis direction can be freely determined. For example, the image portion 234 shown in FIGS. 4 and 8 can be displayed by the analysis portion 250, and the operator can set the SLS analysis region R2 based on the image portion 234 shown in FIGS. When the SLS analysis region R2 is set, the photodetector element in the photodetector 202 corresponding to the set SLS analysis region R2 is determined, and the measured values of the corresponding photodetector element of the photodetector 202 are shown in FIG. It becomes possible to read from the address of the storage device 290 described. By analyzing the measurement data read from the address of the storage device 290 according to the distance from the photodetector corresponding to the optical axis 146, the analysis result of the SLS analysis region R2 can be obtained.

2. Description of Other Embodiments (1) Overview of Other Embodiments An analyzer 102 in which functions are added or changed to the embodiments shown in FIGS. 1 to 9 is shown in FIGS. 14. In the configuration of the analysis apparatus 102 shown in FIG. 10, the light source 110, the incident light adjustment section 120, the optical path changing section 130, the optical diaphragm 138, the light irradiation section 140, the sample section 150, the parallel scattered light conversion section 170, the scattered light detection section 200 , the analysis unit 250 , etc. have already been described as components of the analysis device 100 . Components with the same reference numerals as those shown in FIGS. 1 to 9 have the same configuration and the same effects unless otherwise specified. A major difference between the analyzer 102 and the analyzer 100 is that the scattered angle filter 180 and the scattered light collector 190 are provided between the parallel scattered light converter 170 and the scattered light detector 200 . Differences in analysis content and improvements due to this will be explained below.

(2) Description of Scattering Angle Filter 180 and Scattered Light Condensing Section 190 The scattering angle filter 180 and the scattered light condensing section 190 will be described with reference to FIGS. 11 and 12. FIG. A scattered angle filter 180 having an opening 182 is arranged between the parallel scattered light conversion unit 170 and the scattered light collecting unit 190 . The specific-angle parallel small-angle scattered light with the selected angle is incident on the scattered light collector 190 as the arcuate specific-angle scattered light 186 described below. The incident circular arc-shaped specific-angle scattered light 186 is converted into converged small-angle scattered convergent light 196 by the scattered light collector 190, guided to the photodetector 202 of the scattered light detector 200, and its intensity and the like are measured.

The scattered light collector 190 includes a lens 192, a support holder 194, and the like. The support holder 194 has a mechanism for holding the lens 192 and adjusting the position and tilt angle of the lens 192 . Moreover, the scattering angle filter 180 transmits the arc-shaped light centered on the optical axis 146 whose radius is the length corresponding to the set angle, out of the parallel small-angle scattered light 168 . This arc-shaped light selected by the scattering angle filter 180 is shown in FIGS. 10 and 11 and FIGS. The scattering angle filter 180 has a function of selecting and transmitting arcuate specific angle scattered light 186 from parallel small angle scattered light 168 . In this embodiment, the scattered angle filter 180 also has the function of the transmitted light blocker 176 in the previous embodiment, which prevents the parallel transmitted light 166 from the sample 154 from entering the scattered light detector 200. .

The scattering angle filter 180 has, as shown in FIG. 12, a light blocking plate 181 having a ring-shaped opening 182 formed therein. A parallel transmitted light 166 based on the transmitted light 164 from the sample 154 is blocked by the light shielding plate 181 and can be prevented from entering the photodetector 200 . The light shielding plate 181 is arranged perpendicular to the optical axis 146 , and the light shielding plate 181 transmits only light of a specific radius in a plane perpendicular to the optical axis 146 . Since the specific radius corresponds to the scattering angle of the specific scattered light, it is possible to selectively transmit only the specific angle parallel small angle scattered light of the set specific scattering angle. As will be described below, the aperture 182 formed in the scattering angle filter 180 has an arcuate shape that forms at least a portion of the circumference of a circle with a radius determined by the specific angle with respect to the optical axis 146. . Therefore, the light transmitted through the opening 182 of the scattering angle filter 180 has an arcuate shape. 180 to enter the scattered light collector 190 .

12, the light shielding plate 181 includes a circular inner light shielding portion 183 centered on the optical axis 146, an opening 182, and a surrounding light shielding portion 185. As shown in FIG. Arc-shaped specific angle scattered light 186 at a set scattering angle is selectively passed through aperture 182 . For this reason, the inner light shielding portion 183 provided on the optical axis 146 side of the ring-shaped opening 182 shields the parallel transmitted light 166 inside the specific scattering angle, and the surrounding light shielding portion 185 further shields the parallel small-angle scattered light 168 . The parallel small-angle scattered light 168 on the outer peripheral side of the opening 182 is blocked. Scattering angle filter 180 has a plurality of bridges 187 connecting peripheral light shield 185 and inner light shield 183 to form a ring-shaped aperture 182 and support inner light shield 183, as shown in FIG. ing.

The aperture 182 of the scattering angle filter 180, the inner light shielding portion 183, and the surrounding light shielding portion 185 are, as described above, the shape of the parallel small angle scattered light 168, the shape of the arcuate specific angle scattered light 186 at the selected scattering angle, and the selection thereof. It is determined according to the range of the scattering angle, the shape of the parallel transmitted light 166 to be shielded, the shape of the photodetector 202 of the scattered light detection unit 200, and the like. When the parallel small-angle scattered light 168 is a circular image, the shape of the aperture 182 of the scattering angle filter 180 is preferably ring-shaped. For example, when the parallel small-angle scattered light 168 is a rectangular image, the shape of the aperture 182 of the scattering angle filter 180 is preferably a linear slit.

The material of the scattering angle filter 180 is not particularly limited as long as it is a material through which the transmitted light 164 from the sample 154 of the sample section 150 does not pass. For example, various metals, plastics, wood, etc. can be used. It is also desirable to prevent the transmitted light 164 from the sample 154 from illuminating the scattering angle filter 180 to generate stray light. For example, it is preferable that the surface of the scattering angle filter 180, particularly the side irradiated with the transmitted light 164, is matted black.

Specifically, as the scattering angle filter 180, as shown in FIG. A ring-shaped opening 182 with a gap of 1.0 mm is formed between the inner light shielding portion 183 and the circular surrounding light shielding portion 185 . Using this scattering angle filter 180 with a ring-shaped aperture 182 of 1.0 mm, for example, as the lens 172 of the parallel scattered light conversion unit 170, a 20-fold infinity correction objective lens (focal length f is 10 mm, numerical aperture NA is 0.4), and the distance LB from the sample 154 to the objective lens 172 of the parallel scattered light conversion unit 170 is set at, for example, 15 mm. The angle θ0S (see FIG. 3) is selected in the range of scattering angles from 16.7 degrees to 20.2 degrees to allow passage through the ring aperture 182 of this scattering angle filter 180 . Further, when the beam diameter of the transmitted light 164 from the sample 154 is, for example, 1.5 mm in radius by the objective lens 172 of the parallel scattered light conversion unit 170, the maximum angle θ0S (see FIG. 3) is calculated to be the maximum angle Since θ0S is 5.7 degrees, the parallel small-angle scattered light with the measured scattering angle θS in the range of 11.0 degrees to 14.5 degrees is selected by the scattering angle filter 180 and scattered. It is introduced into the lens 192 of the light condensing section 190 .

The parallel small-angle scattered light in the predetermined narrow scattering angle range selected by the scattering angle filter 180 is focused by the lens 192 of the scattered light collecting unit 190 as a scattered light image, and is detected by the photodetector 202 of the scattered light detection unit 200. detected. Therefore, since the focused scattered light image detected by the scattered light detection unit 200 is the arc-shaped specific angle scattered light 186 that has passed through the aperture 182 of the scattering angle filter 180, the shape of the aperture 182 of the scattering angle filter 180 is The image is reduced or enlarged. In this embodiment, the aperture 182 has a nearly circular shape in order to produce an ideal image. However, if the opening 182 is an arc, an image corresponding to the arc can be created. For example, even if the arc is about 1/6 of the circumference, an image corresponding to the arc is produced. However, if the aperture is a point, the amount of light passing through it is so inadequate that virtually no image is produced.

The arc-shaped specific angle scattered light 186 that passes through the scattering angle filter 180 and enters the lens 192 of the scattered light collector 190 is collimated by the lens 172 of the parallel scattered light converter 170. In particular, for example, when the focal position of the lens 192 of the scattered light collecting section 190, that is, the distance L1 between the lens 192 and the light receiving surface of the scattered light detecting section 200 is arranged to be equal to the focal length of the lens 192, the arc-shaped specific The focused scattered light image of angularly scattered light 186 is of minimal size. In particular, if it is assumed that the scattered light is emitted from an infinitesimal point on the sample 154, the scattered light image is theoretically an infinitesimal point.

However, in practice, as described above, the irradiation light 148 incident on the sample 154 has a finite minute size of, for example, a circular incident beam diameter of several μm to several tens of μm. Even when the distance from the light receiving surface of 200 is arranged equal to the focal length of the lens 192, the focused scattered light image of the parallel small-angle scattered light 168 is not as a point but as a circular image of finite size. It is detected by the detection unit 200 .

Further, when the light receiving surface of the scattered light detection unit 200 is arranged before and after the focal position of the lens 192 of the scattered light collector 190, the parallel small-angle scattered light 168 that passes through the scattering angle filter 180 and is focused by the lens 192 is The scattered light image is obtained by projecting the shape of the opening 182 of the scattering angle filter 180 onto the light receiving surface of the scattered light detection unit 200, and reducing or enlarging the light image passing through the opening 182. FIG.

Irradiation light 148 formed by the condenser lens 142 of the light irradiation unit 140 is incident on the sample 154, and scattered light is emitted from the minimum beam spot 157 (see FIG. 3), which is a minute irradiation light area. Scattered light emitted from slightly different positions within the irradiated light region of the sample 154, for example, two scatterers separated by several μm or less in the sample 154, is emitted from slightly different positions. Follow different optical paths. Therefore, the scattered light has its optical path changed by the lens 172 of the parallel scattered light conversion unit 170, a part of it is selected by the scattering angle filter 180, and the optical path is changed by the lens 192 of the scattered light collecting unit 190, A scattered light image is formed.

The scattered light image detected by the scattered light detection unit 200 is formed by including the following two types of scattered light. That is, one is that scattered light is radiated by a scatterer from an irradiation light region that can be regarded as a minute point on the sample 154, and the scattered light is collimated by the lens 172 of the parallel scattered light conversion unit 170. Small-angle scattered light 168 is obtained. A scattering angle filter 180 selects a portion of the parallel small-angle scattered light 168 within a predetermined scattering angle range, and the small-angle scattered convergent light 196 is focused by a lens 192 to form a scattered light image.

The other is that scattered light is radiated from a group of scatterers at different positions within an irradiation light region that can be regarded as a finite size in the sample 154, and the optical path of the scattered light is changed by the lens 172 of the parallel scattered light conversion unit 170. , the scattered light is substantially collimated by the lens 172 . A scattering angle filter 180 selects a part of the scattered light, and a lens 192 changes the optical path of the scattered light to form a scattered light image. Therefore, the scattered light image formed by the latter scattered light reflects the positional distribution of the scatterers in the irradiation light region in the sample 154 . That is, the scattered light image formed by the latter scattered light represents a microscopic image of the sample 154, which is a microscopic image of the scattered light.

With respect to the measurement sample 154, the scattered light images formed by the two types of forward small-angle scattered light and backward small-angle scattered light follow different optical paths and are formed into images. As for the scattered light image, the shape of the aperture of the scattering angle filter 180 appears at a position different from the projected scattered light image. Specifically, for example, when using a scattering angle filter 180 with a ring-shaped aperture as described above, the parallel forward small-angle scattered light or the parallel backward small-angle scattered light focused by the lens 192 forms a scattered angle filter A bright ring-shaped scattered light image onto which the ring-shaped aperture of 180 is projected appears near the center. A scattered light image reflecting the position distribution appears. That is, the scattered light microscopic image appears in a dark field area different from the bright scattered light image projected by the ring-shaped aperture of the scattering angle filter 180 . Thus, for example, the arrangement shown in FIG. 11 allows simultaneous dynamic and static small angle forward scattered light measurements to be made on the sample 154 at the microscope scale.

In addition, in FIG. 11, the scattered light detection unit 200 has a photodetector 202 for detecting a microscope image. In the embodiment shown in FIG. 13, a light detection camera 204 having a translucent screen 208 and a camera lens 206 is used as the scattered light detection section 200 . Further, in the embodiment shown in FIG. 14, a light detection camera 204 having a camera lens 206 is used as the scattered light detection section 200 . The detection result of the light detection camera 204 is the same as the operation and effect of the scattered light detection unit 200 described above. The measurement results are taken into the analysis unit 250, stored in the storage device 290, and the stored results are used for DLS analysis and SLS analysis.

In the use example of the photodetector 202 described above, a microscopic image is created on the measurement surface of the photodetector 202 by the parallel small-angle scattered light 168, and the image is subdivided by the photodetector 202 and measured. The same is true for the light detection camera 204 shown in FIGS. 13 and 14, and the microscopic image created by the small-angle scattered convergent light 196 captured through the camera lens 206 is divided into subdivided areas and sequentially captured and stored in storage device 290 .

The embodiment shown in FIG. 13 displays an image based on small-angle scattered convergent light 196 on a screen, and the microscope image displayed on the screen is photographed by a light detection camera 204 having a camera lens 206, and photographed by a microscope. The image is stored in the storage device 290 of the analysis unit 250 . In FIG. 13, a translucent screen 208 is used as the screen. By doing so, the microscopic image can be taken from the rear side of the semi-transparent screen 208 , which is the back side of the semi-transparent screen 208 . In this case, it is easy to arrange the camera lens 206 of the photodetection camera 204 so as to be aligned with the optical axis 146, and there is an effect that the entire device can be made compact.

The embodiment shown in FIG. 14 is an example in which the camera lens 206 of the photodetection camera 204 is arranged behind the focal position of the small-angle scattered convergent light 196 . The size of the microscope image created by the small-angle scattered convergent light 196 can be changed by arranging it behind or in front of the focus position of the small-angle scattered convergent light 196 .

13 and 14, the amplification/cooling unit 212 is a cooling device for amplifying the captured signal and for reducing the level of electrical noise generated inside the photodetection camera 204. FIG. It has the same effect as the amplifying/cooling section 212 shown in FIG.

3 Description of Analysis Unit 250 3.1 Configuration of Analysis Unit 250 The analysis unit 250 includes a controller 260, an input/output device 266, a CPU 276, a storage device 290, and the like. The analysis unit 250 controls the overall operation of the analysis device 100 or the analysis device 102, and also detects measurement results of the scattered light detection unit 200 including the photodetector 202 and the light detection camera 204 that operates as the scattered light detection unit 200. , performs DLS analysis and SLS analysis, and outputs the results from the input/output device 266 . A controller 260 is provided to control each component of the analysis device 100 and the analysis device 102 , and a control signal is sent from the controller 260 to each component according to a command from the CPU 276 . Measurement results from the photodetector 202 of the scattered light detection unit 200 and the photodetection camera 204 operating as the scattered light detection unit 200 are captured via the controller 260 and recorded in the storage device 290 as described above. .

When the operator inputs the instruction content, the instruction content is input from the input/output device 266 . Measurement results and analysis results are output from the input/output device 266 . The input/output device 266 has a keyboard, a display device, a printer, and the like to perform these operations.

3.2 Description of Operation of Analysis Unit 250 FIG. 15 is an explanatory diagram illustrating an example of the operation of the analysis device 100 and the analysis device 102 . For example, in this embodiment, before entering the measurement analysis mode M2 for capturing measurement results for performing DLS analysis and SLS analysis and executing DLS analysis and SLS analysis based on the captured measurement results, adjustment mode M1 is performed. action is performed. An adjustment mode M 1 for adjusting the position of the sample cell 152 by the sample position adjustment mechanism 158 of the sample section 150 is started when an instruction to start the adjustment mode M 1 is input from the input/output device 266 . In the adjustment mode M1, the position of the sample cell 152 is adjusted by the sample position adjusting mechanism 158 so that the irradiation position of the irradiation light 148 matches the position to be analyzed. Actually, an image of the small-angle scattered light 160 from the current irradiation position of the irradiation light 148 is captured by the analysis unit 250 from the scattered light detection unit 200 and the light detection camera 204 that operates as the scattered light detection unit 200, The image is displayed on the input/output device 266 as an adjustment image. The operator can adjust the position of the sample cell 152 by the sample position adjustment mechanism 158 while viewing the adjustment image displayed on the input/output device 266 . When the position to be analyzed is found based on the image displayed on the input/output device 266 and the position of the sample cell 152 is set by the sample position adjustment mechanism 158, the adjustment mode M1 ends. The control and operation of the analysis unit 250 for this end may be performed by the operator's instruction to end, or may be performed by the operator's instruction to start the measurement analysis mode M2.

In the adjustment mode M1, the input/output device 266 displays an image based on data taken in from the scattered light detector 200 or the photodetector 202 as an adjustment image. However, calculations for DLS analysis and SLS analysis based on the detection result of the scattered light detection unit 200 and the light detection camera 204 used as the scattered light detection unit 200 are not executed. In addition, the operation of continuously and repeatedly taking in the measurement results of the scattered light detection unit 200 or the light detection camera 204 used as the scattered light detection unit 200 into the storage device 290 is also stopped.

The light detection camera 204 used as the scattered light detection unit 200 or the diffuse light detection unit 200 based on the small angle scattered light 160 from the irradiation position of the irradiation light 148 determined by the current set position of the sample cell 152 experimentally. The measurement results may be taken repeatedly on a trial basis, the results may be used to perform DLS analysis or SLS analysis on a trial basis, and the position of the sample cell 152 may be readjusted based on these results. In this embodiment, the test DLS analysis and SLS analysis are performed in the measurement analysis mode M2 described below, and the sample cell 152 is readjusted by the sample position adjustment mechanism 158 based on the results. It is possible.

After the adjustment of the positioning of the sample cell 152 in the adjustment mode M1 is completed, the operator's instruction to start measurement is input from the input/output device 266, and the measurement analysis mode M2 is started. An example of the measurement analysis mode M2 will be described with reference to FIG. Based on an initial setting to be described later, imaging is performed from imaging times 1 to imaging times N, that is, measurement results from the scattered light detection unit 200 and the photodetector 202 are captured. In order to perform DLS analysis at the number of imaging times 1 or to perform video imaging, the measurement results of the scattered light detection unit 200 and the light detection camera 204 operating as the scattered light detection unit 200 are repeatedly and continuously captured and stored. Hold in device 290 . Here, one-time acquisition of measurement results from the scattered light detection unit 200 and the photodetector 202 is described as sampling.

When the photographing time T1 is set for each of the photographing times 1 to the photographing times N, as shown in FIG. The measurement results are continuously loaded into the storage device 290 . The sampling execution cycle is determined by the sampling frequency input in the initial setting. The number of samplings at the imaging time T1, which is the number of times of repeated capture to the storage device 290, may be set by directly inputting the number of samplings, or may be set by inputting the duration of repetition, which is the imaging time T1. good. Sampling operation is performed a set N times in a set imaging time T1, or is performed during the set imaging time T1, which is the duration of continuous acquisition of the measurement results. The sampling operation based on is continuously repeated. When the imaging time T1 of the sampling operation ends, for example, the operation described in FIG. 15 as the number of imaging times 1 ends.

The sampling operation is stopped during the set shooting interval T2. The time management device 280 of the analysis unit 250 has a function of managing the timing of the start and end of the control operation. Issue an instruction to start the second operation. The operation described above as the operation for the number of times of photographing 1 is performed for the number of times of photographing 2 . After that, the sampling operation is stopped again during the imaging interval T2.

The above operation is repeated, and when the number of times of photographing N is completed, the measurement analysis mode M2 is terminated, and the acquisition of data for DLS analysis and SLS analysis is terminated. Calculations for DLS analysis and SLS analysis are performed based on this data, and analysis results are output from the input/output device 266 . Note that the method of ending the operation of the measurement analysis mode M2 by repeating the number of times of photographing N times is an example. That is, instead of setting the number N of times of photographing, a method of setting the total photographing time TA for photographing may be used. The conditions for the photographing time T1 and the subsequent photographing interval T2 in which the operation of continuously and repeatedly acquiring the measurement results of the scattered light detection unit 200 are the same. , the total photographing time TA, and the total photographing time TA can be set as an initial setting. In this case, after the total imaging time TA has elapsed, the acquisition operation of the measurement result of the scattered light detection unit 200 for the new imaging time T1 is not performed. Thereby, the end of the measurement analysis mode M2 can be controlled.

The operation shown in FIG. 15 describes the acquisition of measurement results from the scattered light detection unit 200 and the light detection camera 204 that is another specific example of the scattered light detection unit 200 . The content shown in FIG. 15 is an example, and changes depending on the content of the operator's instruction. Further, calculations for DLS analysis and SLS analysis can be partially analyzed based on the captured data without waiting for the end of the number of times N of photographing. That is, in parallel with the operation of loading the measurement results of the scattered light detection unit 200 in the measurement analysis mode M2, calculations for the DLS measurement method and the SLS measurement method can be performed based on the loaded measurement results.

FIG. 16 is a flow chart for the CPU 276 to execute the operation of the measurement analysis mode M2 shown in FIG. FIG. 17 is an explanatory diagram for explaining the storage state of measurement results in the storage device 290 and the storage state of analysis results using the stored data. FIG. 16 shows the operation of the measurement analysis mode M2 in which the adjustment mode M1 has already ended. When the operation is started in step S300, the initial setting described above is performed by the operator in step S302. In the initial settings, for example, the shooting time T1 for each of the number of shootings 1 to N, the sampling frequency executed for each of the number of shootings 1 to N, and the time interval of intermittent shooting are set. The setting contents shown in FIG. 15, such as T2, the total repetition number N of the number of times of imaging, the DLS analysis area, the SLS analysis area, the total imaging time TA, etc., are input from the input/output device 266 . After that, the execution of the CPU 276 moves to step S304, and the acquisition of the measurement result from the scattered light detection unit 200 at the number of times of photographing 1 shown in FIG. 15 is executed. is executed and stored in the memory block B1 shown in FIG.

When the continuous operation of capturing the measurement results for the number of times of imaging 1 is completed, step S310 is executed, and the measurement results corresponding to the DLS analysis area and the SLS analysis area are selectively extracted from the measurement results stored in the memory block B1. and stored in the addresses reserved as DD1 and SD1 of the storage device 290 shown in FIG. Next, in step S312, calculation for analysis of static scattered light is performed based on the data stored in the address SD1, and the calculation result is stored in the storage device 290 in the address ARS1 in step S314.

Further, in step S322, calculations for analysis of dynamic scattered light are executed, and the results are stored in addresses ARD10 and ARD11 in step S324. After the execution of step S324, it is determined in step S330 whether or not the execution is finished, and based on the end of the number of times of photographing 1, the operation related to the number of times of photographing 2 is started in step S304. Although the operation of step S304 is started after the photographing interval T2 has elapsed, the description of the passage of the photographing interval T2 is omitted in the flowchart of FIG. Actually, the time management device 280 described above manages the time, performs interrupt processing, etc., and controls the timing of starting the operation of the number of times of photographing 2, and the operation of step S304 is started.

Instead of setting the execution time of the measurement analysis mode M2, that is, the end condition of the measurement analysis mode M2, by the number of times of photography 1 to the number of times of photography N, if the end condition of the measurement analysis mode M2 is set by the total photography time TA, In step S330, it is determined whether the elapsed time in the measurement analysis mode M2 has reached the total imaging time TA based on the elapsed time in the measurement analysis mode M2, and if the elapsed time in the measurement analysis mode M2 has reached the total imaging time TA , the execution of the CPU 276 transitions to step S340 instead of starting the execution of step S304. At step S340, the task execution end operation from S300 is performed, and the measurement analysis mode M2 ends. When it is determined in step S330 whether or not the total shooting time TA has been reached, if it is determined that the total shooting time TA has not been reached, the execution of step S304 is started again.

When the acquisition of the measurement results based on sampling from the number of times of photography 1 to the number of times of photography N-1 shown in FIG. Stored in block BN of storage device 290 . When step S310 is executed, the data of the DLS analysis area are stored in the address DDN of the storage device 290. FIG. Also, data in the SLS analysis area is stored in the storage device 290 at the address SDN. Based on the data stored at the address SDN in step S312, operations for analysis of static light scattering are performed. This calculation result is stored in the address ARSN in step S314. Furthermore, in step S322, analysis of dynamic light scattering is performed, and the calculation result is stored in address ARDN0 and address ARDN1 in step S324.

Execution of CPU 276 transfers from step S324 to step S330. In step S330, when it is determined that the termination condition of the measurement analysis mode M2 shown in FIG. Execution continues to step S340. At step S340, the acquisition of measurement data in the measurement analysis mode M2 ends, and the analysis operation based on the acquired measurement results ends.

In the embodiment shown in the flowchart of FIG. 16, the CPU 276 executes steps S312 and S314 before executing steps S322 and S324. This is an example, and the order of execution of steps S312 and S314 and execution of steps S322 and S324 may be reversed. Further, it is not always necessary to continuously perform steps S312 and S314 and steps S322 and S324, and they may be performed separately. Alternatively, only one of them may be performed. By executing step S304, the measurement results captured from the scattered light detection unit 200 or from the light detection camera 204, which is an example of the scattered light detection unit 200, can be used for both static scattered light analysis and dynamic scattered light analysis. can be used for Therefore, the operator can use the captured measurement results to analyze both the static scattered light and the dynamic scattered light, or selectively perform either one. It is possible.

INDUSTRIAL APPLICABILITY The sample analysis apparatus and analysis method based on the present invention can be used industrially.

DESCRIPTION OF SYMBOLS 11... Photon detection element, 100... Analysis apparatus, 102... Analysis apparatus, 110... Light source, 120... Incident light adjustment part, 122... Light quantity adjustment element, 124... Polarization Element 126 Incident light 130 Optical path changing unit 132 Plane mirror 134 Plane mirror 138 Optical diaphragm 139 Diaphragm 140 Light irradiation Part 142 Condensing lens 144 Lens adjustment mechanism 146 Optical axis 148 Irradiation light 150 Sample part 151 Entrance surface 152 Sample cell 153 Transmitting surface 154 Sample 156 Micro area to be analyzed 157 Minimum beam spot 158 Sample position adjustment mechanism 160 Small angle scattered light 162 . Part 172 Lens 174 Support device 176 Transmitted light shielding part 180 Scattering angle filter 182 Aperture 184 Specific angle parallel small angle scattered light 185 Surrounding light shielding part 186 Circular specific angle scattered light 187 Bridge 188 Radius 189 Center radius 190 Scattered light collecting part 192 lens 194 support mechanism 196 small-angle scattered convergent light 200 scattered light detector 202 photodetector 203 light receiving surface 204 for photodetection Camera 205 Wiring 206 Camera lens 208 Translucent screen 212 Amplification/cooling unit 220 Position adjustment mechanism 230 Photographed image 232 Dark part 234 Image part 250 Analysis part 260 Controller 266 Input/output device 270 Arithmetic processing part 276 CPU 280 Time Management device, 290... Storage device.

Claims (14)

a light source for generating light used for measuring the sample;
a light irradiation unit that irradiates the sample with irradiation light that forms a focal point in the sample based on the light generated by the light source;
a parallel scattered light conversion unit that receives forward or backward small-angle scattered light generated in the analysis microregion, which is the region where the focal point of the irradiation light is formed, and generates parallel small-angle scattered light;
a scattered light detector for measuring the state of the small-angle scattered light from the sample based on the parallel small-angle scattered light;
an analysis unit equipped with a storage device that analyzes the sample based on the measurement result of the scattered light detection unit;
The analysis unit repeatedly stores the measurement result of the scattered light detection unit in the storage device,
Based on the measurement result of the scattered light detection unit stored in the storage device, the analysis unit detects the dynamic scattered light component, the static scattered light component, or the dynamic scattered light component and the static scattered light. Analyzing both of the components to analyze the minute analysis area of the sample. In the analysis device according to claim 1,
The scattered light detection unit includes a photodetector,
generating a microscope image on the photodetector, which is a magnified image of the state of the sample present in the analysis microregion;
The analysis device, wherein the analysis unit repeatedly takes in the microscope image as the measurement result of the scattered light detection unit and stores it in the storage device. In the analysis device according to claim 2,
The analysis unit repeatedly captures the microscope image from the scattered light detection unit, thereby storing a moving image of the microscope image in the storage device,
The analyzing apparatus, wherein the analysis unit analyzes the dynamic scattered light component and the static scattered light component based on the stored moving image. In the analyzer according to any one of claims 1 to 3,
A scattering angle filter and a scattered light collecting section are provided between the parallel scattered light conversion section and the scattered light detection section, and the scattering angle filter detects the parallel small angle scattered light from the parallel scattered light conversion section. Arc-shaped specific-angle scattered light having a specific angle and forming an arc shape on a plane perpendicular to the optical axis of the parallel small-angle scattered light is detected through the scattered-light collecting unit. incident on the photodetector of the part,
2. An analysis apparatus according to claim 1, wherein said photodetector detects a microscopic image in which a state of a sample in said minute analysis area is enlarged and displayed by said photodetector when said parallel small-angle scattered light is incident on said photodetector. In the analysis device according to claim 4,
The analysis unit repeatedly acquires and stores the microscopic image, which is an enlarged display of the state of the sample in the microanalysis area, detected by the photodetector, thereby reducing the quality of the sample in the microanalysis area. The analyzing apparatus, wherein the moving image of the microscopic image in which the state is displayed in an enlarged manner is stored in the storage device. In the analysis device according to claim 4 or claim 5,
The scattering angle filter is made of a material that blocks light, and has an arcuate opening with a radius corresponding to the specific angle with respect to the optical axis of the parallel small-angle scattered light. An analyzer, characterized in that: In the analyzer according to any one of claims 1 to 6,
The analyzer according to claim 1, wherein the incident light incident on the light irradiation unit is a light beam having a circular cross-sectional shape, and the diameter of the light beam of the incident light is 1.6 mm or less. In the analyzer according to any one of claims 1 to 7,
The light irradiation unit includes a condenser lens for condensing the irradiation light, a lens adjustment mechanism for moving the condenser lens along the optical axis of the incident light incident on the light irradiation unit, and
The analyzer according to claim 1, wherein the lens adjusting mechanism has a movable distance of ±10 mm or less along the optical axis, and a minimum length of movable adjustment of 50 μm or less. In the analyzer according to any one of claims 1 to 8,
An analysis apparatus characterized in that a diameter of a minimum beam spot formed inside the sample by irradiating the sample with the irradiation light from the light irradiation unit is in the range of 10 μm or more and 100 μm or less. . In the analyzer according to any one of claims 1 to 9,
having a sample section,
The sample unit includes a sample cell for holding the sample and a sample position adjustment mechanism for adjusting the position of the sample cell,
The analysis unit outputs an adjustment image for adjusting the position of the sample cell by the sample position adjustment mechanism based on the measurement result of the scattered light detection unit,
The analysis unit repeatedly stores the measurement result of the scattered light detection unit in the storage device based on a measurement instruction for performing the analysis,
The analysis device, wherein the analysis unit performs the analysis based on the measurement results of the scattered light detection unit repeatedly stored in the storage device. 11. The analyzer according to claim 1, wherein the maximum incident angle θi with respect to the optical axis of the irradiation light irradiated from the light irradiation unit to the sample is from 0.5 degrees to 20 degrees. An analyzer, characterized in that it is a range. In the analyzer according to any one of claims 1 to 11,
a first step in which the analysis unit acquires the measurement results of the repeated scattered light detection unit based on set sampling conditions;
a second step of extracting the measurement result of the dynamic scattered light analysis region and the measurement result of the static scattered light analysis region from the measurement results of the scattered light detection unit that are repeatedly captured;
performing calculations for dynamic scattered light analysis based on the measurement results of the dynamic scattered light analysis area and calculations for static scattered light analysis based on the measurement results of the static scattered light analysis area; 3 steps and
An analysis method characterized by having In the analysis method according to claim 12,
After the first step of acquiring repeated measurement results from the scattered light detection unit based on the set sampling conditions, the analysis unit suspends execution of the first step for a set imaging interval,
The analysis unit restarts execution of the first step after the end of the set imaging interval,
According to the set number of times of execution of the first step or the set total imaging time, which is the time during which the execution of the first step and the subsequent stop of execution are continued repeatedly, the first An analysis method characterized by repeating an operation of executing a step and stopping the execution of the first step during the imaging interval. In the analysis method according to claim 12 or claim 13,
The analysis unit performs an operation in an adjustment mode and an operation in a measurement analysis mode based on the instruction,
The adjustment mode operation is characterized in that the third step of performing the calculation for the dynamic scattered light analysis and the calculation for the static scattered light analysis based on the measurement data from the scattered light detection unit is stopped. and the analysis method.