Analytical equipment, analytical method, and analytical program

Abstract

In analytical instruments equipped with immersion objective lenses, analytical accuracy is ensured. [Solution] The analytical apparatus 100 for analyzing a sample C in a sample holder 2 comprises an immersion objective lens 3 provided opposite the sample holder 2, an immersion liquid supply unit 4 that supplies immersion liquid to the immersion objective lens 3, a light irradiation unit 6 that irradiates primary light onto an object including at least one of the sample C, the sample holder 2, and a sample holder that holds the sample C in the sample holder 2, a secondary light detection unit 7 that detects secondary light from the object, a sample analysis unit 81 that analyzes the sample C based on the output of the secondary light detection unit 7, and an immersion liquid amount determination unit 83 that determines the amount of immersion liquid in the immersion objective lens 3 based on the output of the secondary light detection unit 7.

Description

Technical Field

[0001] The present invention relates to an analysis device, an analysis method, and an analysis program.

Background Art

[0002] Conventionally, there is an analysis device that analyzes a sample in a sample holder via an immersion objective lens. The immersion objective lens is a lens to which an immersion liquid for filling the space between the lens and the sample holder is supplied.

[0003] As shown in, for example, Patent Document 1, this type of analysis device includes an analysis mechanism that analyzes the sample in the sample holder by supplying immersion liquid to the immersion objective lens and scanning the immersion objective lens relative to the sample holder. As a result, the amount of immersion liquid for the immersion objective lens is sufficiently ensured, so that the resolution of the analysis mechanism can be improved.

[0004] Note that in the above analysis device, when the amount of immersion liquid becomes insufficient during scanning of the immersion objective lens, the immersion objective lens is moved to an oil replenishment position to replenish the immersion liquid, and then returned to the position at which the scanning was in progress. This can prevent a shortage of the immersion liquid during sample analysis, thus enabling automatic analysis in an analysis device equipped with an immersion objective lens.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] Incidentally, the above-mentioned analytical apparatus is further equipped with a weighing mechanism separate from the analytical mechanism for determining the weight of the immersion objective lens. The weighing mechanism determines the amount of immersion liquid supplied to the immersion objective lens based on the change in the weight of the immersion objective lens when the immersion liquid is supplied to it, and determines whether additional immersion liquid needs to be supplied.

[0007] However, in the above-mentioned analytical apparatus, the weighing mechanism determines the amount of immersion liquid based solely on the weight of the immersion objective lens. Therefore, even if the weighing mechanism determines that the weight of immersion liquid is appropriate, there is a possibility that the immersion liquid is not properly supplied to the lens. For example, if the immersion liquid is supplied to only a part of the lens, and / or to a location other than the lens, if the weight of the immersion liquid is appropriate, the weighing mechanism may determine that the weight of immersion liquid is appropriate, and the analytical mechanism may analyze the sample. In this case, the analytical mechanism may analyze the sample with the immersion liquid not properly supplied to the lens, and thus the analytical accuracy of the analytical mechanism cannot be guaranteed.

[0008] Therefore, the present invention has been made in view of the above problems, and its main objective is to ensure analytical accuracy in an analytical apparatus equipped with an immersion objective lens. [Means for solving the problem]

[0009] In other words, the analytical apparatus of the present invention is characterized by comprising: an immersion objective lens provided opposite the sample holder; an immersion liquid supply unit that supplies immersion liquid to the immersion objective lens; a light irradiation unit that irradiates primary light onto an object including at least one of the sample, the sample holder, and a sample holder that holds the sample within the sample holder; a secondary light detection unit that detects secondary light from the object; a sample analysis unit that analyzes the sample based on the output of the secondary light detection unit; and an immersion liquid amount determination unit that determines the amount of immersion liquid in the immersion objective lens based on the output of the secondary light detection unit.

[0010] With such an analytical device, the sample analysis unit analyzes the sample based on the output of the secondary photodetector, and the immersion liquid volume determination unit determines the amount of immersion liquid based on the output of the secondary photodetector. Therefore, the output of the secondary photodetector for sample analysis can be used to determine the amount of immersion liquid. Consequently, by determining the amount of immersion liquid in the immersion liquid volume determination unit, the sample analysis unit can perform the analysis of the sample while the immersion liquid is appropriately supplied onto the lens, thereby ensuring the analytical accuracy of the sample analysis unit.

[0011] The secondary light detection unit includes a spectral detection unit that detects the spectrum of secondary light from the object, and the immersion volume determination unit determines the amount of liquid immersion in the immersion objective lens based on the spectral data of the object. With this configuration, the spectral data used for sample analysis is also used to determine the amount of immersion liquid, thus ensuring the accuracy of the sample analysis unit's analysis of spectral data. Specifically, by selecting spectral data from objects that are less susceptible to changes over time during analysis, such as sample holders or sample retainers, rather than spectral data from objects that are more susceptible to changes over time during analysis, such as samples, the selected spectra can be used to determine the amount of immersion liquid. Therefore, the determination of the amount of immersion liquid can be based on spectral changes associated with changes in the amount of immersion liquid, rather than spectral changes due to changes in the object over time, thus ensuring the accuracy of the sample analysis unit's analysis of spectral data.

[0012] The spectrum detection unit mentioned above includes one that detects the spectrum of Raman scattered light generated from the object. With this configuration, the chemical bonding and / or molecular structure of the target object can be identified by detecting the Raman spectrum. Specifically, the chemical bonding and / or molecular structure of objects that do not change over time during analysis, such as glass or culture media, can be identified by the Raman scattered light spectrum, allowing for more accurate selection of spectra that do not change over time. Therefore, the amount of immersion liquid can be determined based on the spectral change accompanying the change in the amount of immersion liquid, rather than the spectral change due to the change in the object over time, thus further ensuring the accuracy of the spectral data analysis performed by the sample analysis unit.

[0013] The immersion volume determination unit determines the amount of immersion liquid in the immersion objective lens based on the measured data obtained by the spectrum detection unit and reference data that serves as a basis for determining the amount of immersion liquid. With this configuration, the immersion volume determination unit determines the amount of immersion liquid in the immersion objective lens based on the measured data of the object detected by the spectrum detection unit and reference data, so it can accurately determine whether the amount of immersion liquid in the immersion objective lens is insufficient.

[0014] The aforementioned reference data may be generated from spectral data of known components constituting the object. With this configuration, reference data is already known for known components, and the immersion volume determination unit uses the measured data and reference data for known components. Therefore, when the immersion volume determination unit determines the amount of immersion, it does not need to calculate new reference data. In addition, while the spectrum of a sample may change over time during analysis, the spectrum of known components shows less change over time compared to the spectrum of the sample, allowing for accurate determination of the amount of immersion liquid.

[0015] The aforementioned reference data includes actual measurement data obtained by the spectrum detection unit under measurement conditions that serve as the basis for determining the amount of immersion liquid. With this configuration, the measurement conditions for the reference data and the actual data are the same, so by comparing the reference data and the actual data, it is possible to determine more accurately whether the amount of immersion liquid is insufficient.

[0016] The secondary light detection unit includes an image detection unit that detects an image of the object through the immersion objective lens, and the immersion liquid volume determination unit determines the amount of liquid immersed in the immersion objective lens based on the image data of the object output by the image detection unit. In this configuration, for example, the image from the image detection unit is used to analyze the shape of the sample, and the immersion volume determination unit judges the image data of the object output by the image detection unit. Specifically, by selecting an image of an object that does not change easily over time during analysis, such as a sample holder or sample holder, from the images of the image detection unit, rather than an image of an object that changes easily over time during analysis, such as a sample, the selected image can be used to determine the amount of immersion liquid. Therefore, the determination of the amount of immersion liquid can be made based on the image change accompanying the change in the amount of immersion liquid, rather than the image change due to the change in the object over time, thus ensuring the accuracy when the sample analysis unit analyzes the sample based on the image.

[0017] The immersion volume determination unit determines the amount of liquid immersion in the immersion objective lens based on the image data of the object output by the image detection unit and reference image data that serves as a reference for determining the amount of liquid immersion. With this configuration, the immersion volume determination unit determines the amount of immersion liquid based on a comparison between the image data of the object output by the image detection unit and the reference image data of the object. This allows for accurate determination of whether the amount of immersion liquid in the immersion objective lens is insufficient.

[0018] The aforementioned sample holder may be one in which multiple different storage spaces for accommodating the sample are arranged in a planar manner. With this configuration, the sample on the sample holder can be continuously analyzed by causing one of the objective lens and the sample holder to scan relative to the other.

[0019] The apparatus further comprises a scanning mechanism that causes one of the immersion objective lens and the plurality of the accommodation spaces to scan relative to the other, and while the scanning mechanism is sequentially scanning the immersion objective lens across the plurality of the accommodation spaces, the immersion liquid amount determination unit determines the amount of immersion liquid of the immersion objective lens based on the output of the secondary light detection unit. With this configuration, it is possible to prevent the supply of immersion liquid from being interrupted during analysis, so there is no need to interrupt the analysis midway, and the analysis can be reliably performed continuously, whereby the analysis time can be shortened.

[0020] The immersion liquid supply unit is detachably attached to the objective lens, and examples of the immersion liquid supply unit include those that supply immersion liquid to the immersion objective lens based on the determination of the amount of immersion liquid by the immersion liquid amount determination unit. With this configuration, the immersion liquid supply unit is detachably attached to the immersion objective lens, and the immersion liquid supply unit supplies immersion liquid to the immersion objective lens based on the determination of the amount of immersion liquid by the immersion liquid amount determination unit, so there is no need to move the immersion liquid supply unit to the position for supplying immersion liquid, and wasteful consumption of liquid can be prevented. In addition, since the immersion liquid supply unit supplies immersion liquid to the immersion objective lens based on the determination of the amount of immersion liquid by the immersion liquid amount determination unit, the sample can be automatically analyzed continuously.

[0021] Examples of the sample holder include those that store the sample and a liquid and have an open upper portion, and further comprise a lid that closes the opening of the sample holder. With this configuration, the lid closes the opening of the sample holder, so that the liquid stored in the sample holder can be prevented from evaporating through the opening. Accordingly, when analyzing the sample, it is possible to suppress the sample from changing over time. Thus, when the immersion liquid amount determination unit determines that the amount of the immersion liquid is insufficient, the insufficiency of the immersion liquid amount can be identified as resulting from a decrease in the immersion liquid amount rather than a decrease in the liquid stored in the sample holder, and the insufficiency of the immersion liquid amount can be reliably determined.

[0022] An analysis method in an analyzer that includes an immersion objective lens disposed opposite to a sample holder and analyzes a sample in the sample holder, the method comprising: supplying an immersion liquid to the immersion objective lens; irradiating a target object including at least one of the sample, the sample holder, and a sample holding member that holds the sample in the sample holder with primary light; detecting secondary light from the target object; analyzing the sample based on the detection of the secondary light; and determining the amount of the immersion liquid of the immersion objective lens based on the detection of the secondary light.

[0023] An analysis program for an analyzer that analyzes a sample in a sample holder, wherein the analyzer includes: an immersion objective lens disposed opposite to the sample holder; an immersion liquid supply unit configured to supply an immersion liquid to the immersion objective lens; a light irradiation unit configured to irradiate a target object including at least one of the sample, the sample holder, and a sample holding member that holds the sample in the sample holder with primary light; and a secondary light detection unit configured to detect secondary light from the target object, the program causing a computer to function as: a sample analysis unit configured to analyze the sample based on an output of the secondary light detection unit; and an immersion liquid amount determination unit configured to determine the amount of the immersion liquid of the immersion objective lens based on the output of the secondary light detection unit. Effects of the Invention

[0024] According to the present invention, analysis accuracy can be ensured in an analyzer provided with an immersion objective lens. Brief Description of the Drawings

[0025] [Figure 1]A schematic diagram showing an analytical device in one embodiment of the present invention. [Figure 2] A figure showing a comparison between measured data and reference data in the same embodiment. [Figure 3] A flowchart illustrating the analysis method in the said embodiment. [Figure 4] A diagram showing an analytical apparatus in another embodiment. [Figure 5] (a) an image in another embodiment where the contour of the object is clear, and (b) an image where the contour of the object is blurred. [Figure 6] A diagram showing a sample holder in another embodiment. [Modes for carrying out the invention]

[0026] The following describes an analytical apparatus and analytical method according to one embodiment of the present invention, with reference to the drawings. Note that, for the sake of clarity, the following figures may be simplified or exaggerated for illustrative purposes. The same reference numerals are used for identical components, and their descriptions are omitted as appropriate.

[0027] <Device configuration> In this embodiment, the analytical apparatus 100 analyzes sample C based on the spectrum obtained by irradiating the object with primary light and detecting the resulting secondary light. Specifically, the analytical apparatus 100 uses so-called Raman spectroscopy, in which laser light is irradiated onto sample C as primary light, and the resulting Raman scattered light is detected as secondary light. More specifically, the analytical apparatus 100 is an inverted microscope Raman spectrometer in which an immersion objective lens 3 is provided below a sample holder 2 that houses sample C, and the analysis of sample C is performed through the immersion liquid of the immersion objective lens 3.

[0028] In this embodiment, the object includes at least one of the sample C, the sample holder 2, and a sample holder that holds the sample C within the sample holder 2. The sample C is a biological sample such as cells, but is not limited to biological samples; it may also be a sample other than a biological sample, such as a battery, pharmaceuticals, emulsion, industrial product, or chemical substance. The sample holder is a liquid or solid that holds the sample C within the sample holder 2. Here it is a culture medium for the sample C, but is not limited to a culture medium; examples include water, physiological saline, buffer solution, adhesive, solid culture medium, grease, tape, etc. The state in which the sample holder holds the sample C may be a state in which the sample C is in contact with the sample holder 2, a state in which the sample C is separated from the sample holder 2, or any other state in which the sample C is held so that it can be analyzed.

[0029] Specifically, as shown in Figure 1, the analytical apparatus 100 comprises a sample holder 2 for containing the sample C, an immersion objective lens 3 provided opposite the sample holder 2, an immersion liquid supply unit 4 for supplying immersion liquid to the immersion objective lens 3, a scanning mechanism 5 for scanning the immersion objective lens 3 relative to the sample holder 2, a light irradiation unit 6 for irradiating the object with primary light, a secondary light detection unit 7 for detecting secondary light from the object, and an information processing device 8 for processing the output from the secondary light detection unit 7. The configuration of each part will be described below.

[0030] The sample holder 2 is generally plate-shaped and accommodates a sample C on one side 2a. In this embodiment, the sample holder 2 has a storage space 2S for accommodating multiple samples C together with a sample holder such as a culture medium, and the storage space 2S is arranged planarly on one side 2a of the sample holder 2. More specifically, the sample holder 2 is a well plate having different storage spaces 2S for each of the multiple samples C. Note that the sample holder 2 is not limited to accommodating a sample C, but may also be a device on which a sample C is placed, such as a slide or a petri dish.

[0031] In this embodiment, each of the multiple containment spaces 2S is composed of a surface 2a that holds the sample C and a surrounding wall that extends from the surface 2a and surrounds the sample C. In this embodiment, the top of each containment space 2S is open, and the sample C and the culture medium, which is the sample holder, are stored inside the containment space 2S. The multiple containment spaces 2S are arranged, for example, in a matrix, but they may also be arranged in a row or randomly.

[0032] The immersion objective lens 3 is a lens for supplying the immersion liquid. The immersion objective lens 3 is positioned opposite the other surface 2b of the sample holder 2 and focuses light directed toward the object and light from the object. Specifically, the immersion objective lens 3 has an opposing surface 3a that faces the other surface 2b of the sample holder 2. Light is emitted from the opposing surface 3a toward the sample C, and light from the object is incident on the opposing surface 3a. In this embodiment, the immersion objective lens 3 is a common lens for the spectrum detection unit 71 and the image detection unit 72, which will be described later, but they may be separate lenses.

[0033] The immersion liquid supply unit 4 supplies immersion liquid to the immersion objective lens 3. The immersion liquid referred to here is a liquid used to improve the resolution when analyzing the sample C by adjusting the refractive index of light directed toward the sample C and the refractive index of light from the sample C. Examples include water, oil, glycerin, and silicone oil. Specifically, the immersion liquid supply unit 4 supplies immersion liquid to the immersion objective lens 3, thereby immersing the space between the other surface 2b of the sample holder 2 and the opposing surface 3a of the immersion objective lens 3 with liquid. More specifically, the immersion liquid supply unit 4 comprises an adapter 41 for ejecting the immersion liquid and a storage unit 42 connected to the adapter 41 via a tube for storing the immersion liquid.

[0034] The adapter 41 is detachably attached to the immersion objective lens 3 and ejects the immersion liquid onto the opposing surface 3a of the immersion objective lens 3. Specifically, the adapter 41 is attached from the outside of the opposing surface 3a of the immersion objective lens 3 so as to surround the opposing surface 3a of the immersion objective lens 3. Note that the adapter 41 may also be a non-detachable type, such as a tubular shape.

[0035] The storage unit 42 is a housing having a storage space for storing immersion liquid. Specifically, the storage unit 42 has a pump (not shown) built into the storage space, and the pump is driven based on the control of the supply amount control unit 84, which will be described later, to supply immersion liquid to the adapter 41 via a tube. Note that when the immersion liquid in the immersion objective lens 3 is sufficiently filled, the pump is stopped.

[0036] The scanning mechanism 5 scans one of the immersion objective lens 3 and the sample holder 2 relative to the other in order to analyze multiple samples C in succession. Specifically, the scanning mechanism 5 scans the sample holder 2 so that the immersion objective lens 3 moves along an arrangement of multiple containment spaces 2S. In this embodiment, the scanning mechanism 5 scans the immersion objective lens 3 one row at a time across multiple containment spaces 2S arranged in a matrix.

[0037] As shown in Figure 1, the scanning mechanism 5 of this embodiment includes a stage 51 on which a sample holder 2 is placed on one side and which faces an immersion objective lens 3 on the other side, and an actuator 52 that moves the stage 51 relative to the immersion objective lens 3. An opening is formed in the stage 51, and when the sample holder 2 is placed on the stage 51, the other side 2b of the sample holder 2 covers this opening. As a result, the other side 2b of the sample holder 2 is exposed to the immersion objective lens 3, so that the immersion liquid of the immersion objective lens 3 directly immerses the other side 2b of the sample holder 2. The actuator 52 moves the stage 51 in the XY direction. The scanning mechanism 5 may also scan the immersion objective lens 3 relative to the sample holder 2.

[0038] In this embodiment, when the scanning mechanism 5 sequentially scans the immersion objective lens 3 through a plurality of housing spaces 2S, the immersion liquid amount determination unit 83, described later, determines the amount of immersion liquid in the immersion objective lens 3 based on the output of the secondary light detection unit 7. The state in which the scanning mechanism 5 scans the immersion objective lens 3 includes the state in which the scanning mechanism 5 is moving the immersion objective lens 3 along the arrangement of the plurality of housing spaces 2S, and the state in which the immersion objective lens 3 is stopped relative to the sample holder 2.

[0039] The light irradiation unit 6 irradiates the object with primary light. Specifically, the light irradiation unit 6 has a spectral light irradiation unit 61 that irradiates primary light for detecting the Raman spectrum of Raman scattered light from the object, and an imaging light irradiation unit 62 that irradiates primary light for imaging the object.

[0040] The spectral light irradiation unit 61 irradiates the object with primary light for spectral purposes. Primary light for spectral purposes, as used here, can include, for example, excitation light that excites components contained in the object. More specifically, the spectral light irradiation unit 61 is a laser light source that irradiates laser light. Furthermore, a bandpass filter (not shown) that transmits predetermined wavelength components, and / or a half-mirror that reflects laser light to the object and transmits scattered light from the object, may be provided in the optical path from the laser light source to the immersion objective lens 3.

[0041] The imaging light irradiation unit 62 irradiates the object with primary light for imaging. The primary light for imaging, as used here, is illumination light that illuminates the object. Specifically, the imaging light irradiation unit 62 has a first illumination light irradiation unit 62a located on the same side as the secondary light detection unit 7 relative to the object, and a second illumination light irradiation unit 62b located on the opposite side of the object from the secondary light detection unit 7. In this embodiment, the first illumination light irradiation unit 62a and the second illumination light irradiation unit 62b can be, for example, white LED light sources. The imaging light irradiation unit 62 may also have at least one of the first illumination light irradiation unit 62a and the second illumination light irradiation unit 62b.

[0042] In this embodiment, in order to reduce the influence of white LED light on Raman scattered light, the spectral light irradiation unit 61 and the imaging light irradiation unit 62 irradiate the object with primary light at different timings depending on the object detected by the secondary light detection unit 7. Specifically, the spectral light irradiation unit 61 irradiates the object with primary light when detecting the spectrum of Raman scattered light, and the imaging light irradiation unit 62 irradiates the object with primary light when detecting an image of the object.

[0043] The secondary light detection unit 7 detects secondary light from the object. In this embodiment, secondary light from the object includes Raman scattered light generated from the object, transmitted light that has passed through the object, and reflected light that has reflected from the object. Specifically, the secondary light detection unit 7 detects the spectrum of secondary light from the object and also detects an image of the object based on the secondary light from the object.

[0044] The secondary light detection unit 7 of this embodiment includes a spectrum detection unit 71 that detects the spectrum of secondary light from an object, and an image detection unit 72 that detects an image of the object via the immersion objective lens 3. The image referred to here may be a reflected image obtained by illumination from the first illumination light irradiation unit 62a, and / or a transmitted image obtained by illumination from the second illumination light irradiation unit 62b.

[0045] The spectrum detection unit 71 detects the Raman spectrum of Raman scattered light from an object. Specifically, the spectrum detection unit 71 spectrally analyzes the Raman scattered light from the object, outputs an intensity signal indicating the Raman spectrum corresponding to the spectrally analyzed Raman scattered light, and generates spectral data of the object. More specifically, the spectrum detection unit 71 includes a spectrometer 71a that spectrally analyzes the Raman scattered light from the object, an intensity signal output unit 71b that outputs an intensity signal based on the spectrally analyzed Raman scattered light, and a spectral data generation unit 71c that generates spectral data of the object based on the intensity signal. The intensity signal output unit 71b is, for example, a multi-channel detector such as a CCD, but any detector that can detect Raman spectra is acceptable.

[0046] The spectral data generation unit 71c generates spectral data of the object based on the intensity signal from the intensity signal output unit 71b. Spectral data, in this context, refers to the light intensity in a predetermined wavelength range from the light intensity signal detected when irradiated with a predetermined laser intensity.

[0047] In this embodiment, when analyzing the sample C in the sample holder 2, the spectral data generation unit 71c generates actual measurement data of the object. The actual measurement data in this embodiment includes the spectra of the components contained in the object. The actual measurement data is output to a display unit and / or information processing device 8, such as the display D.

[0048] Here, the components constituting the object include known components whose spectra were already known before the analysis of sample C, and the measured data includes the spectra of sample C and the spectra of the known components. Specifically, known components are components that are less likely to change over time during analysis than, for example, sample C. More specifically, known components are components derived from the material constituting sample holder 2, such as glass, components constituting the sample holder, such as water, and / or labeling components that are calibration labels attached to sample holder 2, such as standard substances, fluorescent markers, or reference microparticles. Note that if sample C is less likely to change over time during analysis, the known components may be used as sample C.

[0049] The image detection unit 72 captures an image of the object and detects the image of the object. Specifically, the image detection unit 72 is a camera that captures an image of the object while the imaging light irradiation unit 62 is irradiating the object with primary light. The image of the object detected by the image detection unit 72 is output to a display unit such as the display D and / or an information processing device 8.

[0050] The information processing device 8 is a general-purpose or dedicated computer equipped with a CPU, memory, AD converter, DA converter, etc. According to a program stored in a predetermined area of ​​memory, the CPU and / or its peripheral devices cooperate to perform at least the functions of a sample analysis unit 81, a data storage unit 82, an immersion liquid volume determination unit 83, and a supply volume control unit 84, as shown in Figure 1. The spectral data generation unit 71c and the sample analysis unit 81 may be provided in the same computer as the information processing device 8, or they may be provided in a different computer. Furthermore, the information processing device 8 may be provided outside the analysis device 100.

[0051] The sample analysis unit 81 analyzes sample C based on the output of the secondary photodetector 7. The output of the secondary photodetector 7 when analyzing sample C includes at least the spectrum of sample C and / or an image of sample C. However, if sample C is not present in the object, the sample analysis unit 81 performs an analysis indicating that sample C is not present. Specifically, the sample analysis unit 81 obtains spectral data of the object from the spectral data generation unit 71c and analyzes the physical properties of sample C. Alternatively, the sample analysis unit 81 may obtain an image containing sample C from the image detection unit 72 and analyze the shape of sample C.

[0052] The data storage unit 82 stores spectral data. Specifically, the data storage unit 82 stores at least reference data that serves as a basis for determining the amount of immersion liquid. The reference data in this embodiment is spectral data obtained under measurement conditions that serve as a basis for determining immersion liquid. The measurement conditions that serve as a basis for determining immersion liquid are measurement conditions that are the same as the measurement conditions used to generate the actual measurement data. Examples include the intensity of the primary light used to generate the actual measurement data, the measurement time used to generate the actual measurement data, and / or the amount of immersion liquid used to generate the actual measurement data. The amount of immersion liquid used to generate the actual measurement data is an amount sufficient to perform the analysis of sample C. Examples include the amount of immersion liquid that the operator deems appropriate, or the amount of immersion liquid that does not affect the analysis results when generating the actual measurement data. The reference data in this embodiment includes the spectrum of sample C and the spectra of known components, but it is sufficient if it includes at least the spectra of known components. If the analysis of sample C is performed multiple times, the actual measurement data obtained from the first analysis may be set as the reference data for the second and subsequent analyses, and this reference data may be stored in the data storage unit 82.

[0053] The immersion volume determination unit 83 determines the amount of liquid immersion into the immersion objective lens 3 based on the output of the secondary photodetector 7. The output of the secondary photodetector 7 when determining the amount of liquid immersion includes at least the spectrum of known components and / or images of known components. Specifically, the immersion volume determination unit 83 determines the amount of liquid immersion into the immersion objective lens 3 based on the measured data of the object and the reference data of the object. Specifically, the immersion volume determination unit 83 compares the measured data with the reference data obtained under measurement conditions equivalent to the measurement conditions when the measured data was generated.

[0054] More specifically, the immersion volume determination unit 83 acquires measured data from the spectral data generation unit 71c and acquires reference data obtained under the same measurement conditions as those used to generate the measured data from the data storage unit 82. Then, as shown in Figure 2, the immersion volume determination unit 83 determines whether the amount of immersion is appropriate by comparing the spectra of known components, such as the peak intensity and / or peak position of known components, between the measured data and the reference data. If the sample C is one that does not change easily over time during analysis, the immersion volume determination unit 83 may determine whether the amount of immersion is appropriate based on the spectrum of the sample C.

[0055] As shown in Figure 2, if the difference in the spectra of known components between the measured data and the reference data is greater than a predetermined value, the immersion volume determination unit 83 determines that the amount of immersion liquid in the immersion objective lens 3 is insufficient. On the other hand, if the difference in the spectra of known components between the measured data and the reference data is less than a predetermined value, the immersion volume determination unit 83 determines that the amount of immersion liquid in the immersion objective lens 3 is sufficient.

[0056] The supply amount control unit 84 controls the amount of immersion liquid supplied by the immersion liquid supply unit 4 based on the determination result of the immersion liquid amount determination unit 83. Specifically, the supply amount control unit 84 intermittently or continuously controls the operation of the pump in the storage unit 42 according to whether the amount of immersion liquid is excessive or insufficient as determined by the immersion liquid amount determination unit 83. More specifically, the supply amount control unit 84 can control the pump's operating time, control the pump to supply a set amount of immersion liquid, and / or control the pump based on the deficiency in the amount of immersion liquid obtained from the determination result of the immersion liquid amount determination unit 83.

[0057] More specifically, if the immersion volume determination unit 83 determines that the amount of immersion liquid in the immersion objective lens 3 is insufficient, the supply volume control unit 84 drives the pump in the storage unit 42 to increase the amount of immersion liquid in the immersion objective lens 3. On the other hand, if the immersion volume determination unit 83 determines that the amount of immersion liquid in the immersion objective lens 3 is sufficient, the supply volume control unit 84 does not start or stops the pump in the storage unit 42 to maintain the amount of immersion liquid in the immersion objective lens 3. This prevents the amount of immersion liquid in the immersion objective lens 3 from becoming excessive, thus preventing the supply of excess immersion liquid. In addition, since the amount of immersion liquid in the immersion objective lens 3 can be maintained, automatic and continuous analysis of sample C using the immersion objective lens 3 becomes possible.

[0058] <Analysis method> The analysis method for sample C in this embodiment will be described below with reference to Figure 3.

[0059] First, the supply control unit 84 drives the pump of the storage unit 42, causing the immersion liquid supply unit 4 to supply immersion liquid to the immersion objective lens 3 (S1).

[0060] Next, the spectral light irradiation unit 61 of the light irradiation unit 6 irradiates the object with primary light through the immersion objective lens 3 (S2). This generates secondary light, or Raman scattered light, from the object.

[0061] Next, the secondary light detection unit 7 detects secondary light from the object (S3). Specifically, the spectrometer 71a spectrally analyzes the secondary light from the object, and the intensity signal output unit 71b outputs an intensity signal based on the spectrally analyzed secondary light.

[0062] Next, the spectral data generation unit 71c generates spectral data of the object based on the intensity signal from the intensity signal output unit 71b (S4). The spectral data generated by the spectral data generation unit 71c during the analysis of sample C is measured data. The spectral data includes at least the spectrum of sample C and the spectra of known components.

[0063] Next, the immersion volume determination unit 83 acquires spectral data from the spectral data generation unit 71c and reference data from the data storage unit 82. The reference data includes at least spectral data of known components obtained under the same measurement conditions as when the actual measurement data was generated.

[0064] The immersion volume determination unit 83 then determines whether the difference between the measured data of the object and the reference data of the object is greater than or equal to a predetermined value (S5). Specifically, the immersion volume determination unit 83 determines the amount of liquid immersion in the immersion objective lens 3 by comparing the spectral data of known components between the measured data and the reference data. The spectral data of known components in the reference data is stored in the data storage unit 82 at least before the determination by the immersion volume determination unit 83.

[0065] If the difference between the measured data and the reference data in the spectrum of known components is greater than a predetermined value, the immersion volume determination unit 83 determines that the amount of immersion liquid is insufficient. In this case, the supply volume control unit 84 drives the pump of the storage unit 42 to increase the amount of immersion liquid (S6).

[0066] After the supply amount control unit 84 increases the amount of immersion liquid, the supply amount control unit 84 stops the pump from running. Then, returning to S2, the immersion liquid amount determination unit 83 determines the amount of immersion liquid again.

[0067] On the other hand, if the difference between the measured data and the reference data in the spectral data of known components is less than a predetermined value, the immersion volume determination unit 83 determines that the amount of immersion is sufficient. In this case, the analyzer 100 analyzes the sample C based on the spectral data of the object generated by the spectral data generation unit 71c and / or the image of the object captured by the image detection unit 72 (S7).

[0068] Next, the user or computer decides whether to terminate the analysis of sample C. If the analysis of sample C is to be continued, the actuator 52 of the scanning mechanism 5 moves the sample holder 2 in the XY direction so that the immersion objective lens 3 moves along the array of multiple containment spaces 2S.

[0069] As the scanning mechanism 5 sequentially scans the immersion objective lens 3 into multiple containment spaces 2S, the immersion liquid amount determination unit 83 determines the amount of immersion liquid in the immersion objective lens 3 based on the output of the secondary photodetector 7, similar to the method shown in S2 to S6. The sample analysis unit 81 analyzes the sample C based on the output of the secondary photodetector 7, similar to the method shown in S7. As a result, when the scanning mechanism 5 sequentially scans the immersion objective lens 3 into multiple containment spaces 2S, a state in which sufficient immersion liquid is supplied to the immersion objective lens 3 is maintained, making it possible to analyze multiple samples C continuously and automatically.

[0070] Furthermore, when the scanning mechanism 5 scans the immersion objective lens 3 over multiple samples C, or when the analysis of sample C is performed multiple times consecutively, it is not necessary to determine the amount of immersion liquid each time sample C is analyzed. In other words, if the amount of immersion liquid reduction is within a predetermined range, the analysis of sample C may be performed after generating the spectral data of the object without determining the amount of immersion liquid.

[0071] <Effects of this embodiment> In the analytical apparatus 100 of this embodiment, the sample analysis unit 81 analyzes the sample C based on the output of the secondary photodetector 7, and the immersion liquid volume determination unit 83 determines the amount of immersion liquid in the immersion objective lens 3 based on the output of the secondary photodetector 7. Therefore, the output of the secondary photodetector 7 for the analysis of the sample C can be used to determine the amount of immersion liquid. Accordingly, the discrepancy between the immersion determination and the analysis of the sample can be eliminated, and the analytical accuracy of the sample analysis unit 81 can be ensured. Furthermore, since there is no need to install a separate device to measure the amount of immersion liquid in the immersion objective lens 3, the device cost can be reduced, contributing to the miniaturization of the device.

[0072] <Other Embodiments> However, the present invention is not limited to the embodiments described above.

[0073] In the above embodiment, when the scanning mechanism 5 sequentially scans the immersion objective lens 3 into multiple containment spaces 2S to analyze multiple samples C in succession, the immersion liquid volume determination unit 83 determined the amount of immersion liquid. However, when analyzing one sample C continuously over a predetermined period, the immersion liquid volume determination unit 83 may also determine the amount of immersion liquid.

[0074] In the above embodiment, the immersion volume determination unit 83 determined the amount of immersion based on the output of the spectrum detection unit 71. However, as shown in Figure 4, the immersion volume determination unit 83 may determine the amount of immersion based on image data of the object output by the image detection unit 72.

[0075] Specifically, the immersion volume determination unit 83 determines the amount of immersion based on the image data of the object output by the image detection unit 72. The image data of the object referred to here is an image with a certain shape, such as the outline of sample C, the outline of sample holder 2, or, if markings are formed on sample holder 2, the outline of the markings.

[0076] More specifically, the data storage unit 82 stores reference image data, which is image data of an object used as a reference when determining the amount of immersion liquid. The reference image data is captured in advance under the same measurement conditions as those used to determine the amount of immersion liquid. The immersion liquid amount determination unit 83 then acquires the image captured by the image detection unit 72, obtains reference image data showing the outline of the object from the data storage unit 82, and compares the image data of the object output by the image detection unit 72 with the reference image data.

[0077] As shown in Figure 5(a), if the difference between the contour of the object captured by the image detection unit 72 and the contour of the object in the reference image data is less than a predetermined value, the immersion volume determination unit 83 determines that the contour of the object is clear and that the amount of immersion volume is sufficient. On the other hand, as shown in Figure 5(b), if the difference between the contour of the object captured by the image detection unit 72 and the contour of the object in the reference image data is greater than or equal to a predetermined value, the immersion volume determination unit 83 determines that the contour of the object is blurred and that the amount of immersion volume is insufficient. In addition to the contour of the object, the immersion volume determination unit 83 may also determine the amount of immersion volume based on the contrast difference of the object between the image data and the reference image data.

[0078] As shown in Figure 6, the sample holder 2 may further include a lid 22 that covers the upper opening of the storage space 2S of the sample holder 2. Specifically, the lid 22 is flat and is provided facing the entire one side 2a of the sample holder 2. As shown in Figure 6, a sealing member 23 may be interposed between the lid 22 and the sample holder 2. Furthermore, the sample holder 2 may further include a heater 24 that heats the culture medium, which is the sample held, in order to maintain a constant environment in the storage space 2S.

[0079] With this configuration, the lid 22 closes the opening of the sample holder 2, preventing the liquid stored in the sample holder 2 from evaporating from the opening. Therefore, when analyzing sample C, it is possible to suppress changes in sample C over time, and the judgment result of the immersion liquid volume determination unit 83 can be identified as a decrease in the immersion liquid, rather than a decrease in the culture medium, which is the sample holder stored in the sample holder 2, thus reliably determining a shortage of immersion liquid.

[0080] In the above embodiment, when it was determined that the amount of immersion liquid was insufficient, the pump of the immersion liquid supply unit 4 was driven to supply the immersion liquid. However, the pump of the immersion liquid supply unit 4 may be configured to be constantly driven during the analysis of sample C. In this case, if the immersion liquid amount determination unit 83 determines that the amount of immersion liquid is insufficient, the supply amount control unit 84 controls the output of the pump of the immersion liquid supply unit 4 to increase the amount of immersion liquid. If the immersion liquid amount determination unit 83 determines that the amount of immersion liquid is excessive, the supply amount control unit 84 controls the output of the pump of the immersion liquid supply unit 4 to decrease the amount of immersion liquid.

[0081] Alternatively, the immersion objective lens 3 may be housed in a storage container in which the immersion liquid supplied from the immersion liquid supply unit 4 is stored, and the immersion liquid supply unit 4 may supply the immersion liquid to the immersion objective lens 3 while the sample holder 2 and the immersion objective lens 3 are immersed in the immersion liquid in the storage container.

[0082] In the above embodiment, the analytical device 100 was a microscope using so-called Raman spectroscopy, but it may also be a fluorescence microscope that detects a fluorescence spectrum as a secondary light spectrum from an object, and / or a microscope that detects spectra other than Raman spectra and fluorescence spectra.

[0083] In the above embodiment, the analyzer 100 was a so-called inverted microscope in which the immersion objective lens 3 was provided below the sample holder 2, but it may also be an upright microscope in which the immersion objective lens 3 was provided above the sample holder 2.

[0084] Furthermore, the present invention can be modified in various ways, as long as it does not contradict its spirit. [Explanation of Symbols]

[0085] 100...Analyzer 2 ···Sample holder 3. Immersion objective lens 4...Immersion liquid supply section 5. Scanning mechanism 6 ···Light irradiation section 7. Secondary light detection unit 71 ···Spectrum detection unit 71a...Spectrometer 71b...Intensity signal output section 71c ···Spectrum data generation unit 72 ···Image detection unit 7 ···Information Processing Device 81 ···Sample Analysis Department 82 ···Data storage unit 83 ...Immersion liquid amount judgment section 84 ···Supply Control Unit

Claims

1. An analytical device for analyzing a sample in a sample holder, An immersion objective lens is provided opposite the sample holder, An immersion liquid supply unit that supplies immersion liquid to the aforementioned immersion objective lens, A light irradiation unit that irradiates primary light onto an object including the sample, the sample holder, and at least one of the sample holders that hold the sample within the sample holder, A secondary light detection unit for detecting secondary light from the aforementioned object, A sample analysis unit analyzes the sample based on the output of the secondary photodetector, An analytical apparatus comprising: an immersion liquid volume determination unit that determines the amount of immersion liquid in the immersion objective lens based on the output of the secondary light detection unit.

2. The secondary light detection unit has a spectral detection unit that detects the spectrum of secondary light from the object, The analytical apparatus according to claim 1, wherein the immersion volume determination unit determines the amount of immersion liquid in the immersion objective lens based on the spectral data of the object.

3. The analytical apparatus according to claim 2, wherein the spectral detection unit detects the spectrum of Raman scattered light generated from the object.

4. The analytical apparatus according to claim 2 or 3, wherein the immersion volume determination unit determines the amount of immersion liquid in the immersion objective lens based on the measured data obtained by the spectrum detection unit and reference data that serves as a basis for determining the amount of immersion liquid.

5. The analytical apparatus according to claim 4, wherein the reference data is generated from spectral data of known components constituting the object.

6. The analytical apparatus according to claim 4 or 5, wherein the measurement conditions for the reference data are the measurement conditions for the actual data obtained by the spectrum detection unit when analyzing the sample.

7. The secondary light detection unit includes an image detection unit that detects an image of the object via the immersion objective lens. The analysis apparatus according to any one of claims 1 to 6, wherein the immersion volume determination unit determines the amount of immersion liquid in the immersion objective lens based on the image data of the object output by the image detection unit.

8. The analysis apparatus according to claim 7, wherein the immersion volume determination unit determines the amount of immersion liquid in the immersion objective lens based on image data of the object output by the image detection unit and reference image data showing an image that serves as a basis for determining the amount of immersion liquid.

9. The analytical apparatus according to any one of claims 1 to 8, wherein the sample holder has a plurality of different storage spaces for accommodating the sample arranged in a planar manner.

10. The system further comprises a scanning mechanism that scans one of the immersion objective lens and the plurality of housing spaces relative to the other, The analytical apparatus according to claim 9, wherein when the scanning mechanism scans the immersion objective lens sequentially through a plurality of housing spaces, the immersion liquid amount determination unit determines the amount of immersion liquid in the immersion objective lens based on the output of the secondary light detection unit.

11. The immersion liquid supply unit is detachably attached to the immersion objective lens. The analytical apparatus according to any one of claims 1 to 10, wherein the immersion liquid supply unit supplies immersion liquid to the immersion objective lens based on the determination of the amount of immersion liquid by the immersion liquid amount determination unit.

12. The sample holder stores the sample and the sample holder and has an open top. The analytical apparatus according to any one of claims 1 to 11, further comprising a lid for closing the opening of the sample holder.

13. An analytical method in an analytical apparatus that includes an immersion objective lens positioned opposite a sample holder, for analyzing a sample in the sample holder, wherein The immersion liquid is supplied to the aforementioned immersion objective lens, A primary light is irradiated onto an object that includes at least one of the sample, the sample holder, and the sample holder that holds the sample within the sample holder. Secondary light from the aforementioned object is detected, Based on the detection of the secondary light, the sample is analyzed. An analytical method for determining the amount of immersion liquid in the immersion objective lens based on the detection of the secondary light.

14. An analysis program for an analytical instrument that performs analysis of a sample in a sample holder, The aforementioned analytical device is An immersion objective lens is provided opposite the sample holder, An immersion liquid supply unit that supplies immersion liquid to the aforementioned immersion objective lens, A light irradiation unit that irradiates primary light onto an object including the sample, the sample holder, and at least one of the sample holders that hold the sample within the sample holder, The system includes a secondary light detection unit that detects secondary light from the aforementioned object, Based on the output of the secondary photodetector, it functions as a sample analysis unit that analyzes the sample, An analysis program that causes a computer to function as an immersion volume determination unit, which determines the amount of immersion liquid in the immersion objective lens based on the output of the secondary light detection unit.

Images