Judgment device, control method for judgment device

Abstract

To provide a determination device that allows for the determination of cell status using a minimally invasive method with few restrictions on sample morphology. [Solution] The determination device comprises an acquisition unit that acquires a Raman spectral image captured by an imaging device that captures stimulated Raman scattered light from biological cells irradiated with excitation light; a generation unit that generates a spectral difference image of a pair of images corresponding to different wavenumber shift bands of the Raman spectral image; and a determination unit that determines the state of the cell based on the spectral difference image.

Description

[Technical Field]

[0001] The present invention relates to a determination device for determining the state of cells, and a method for controlling the determination device. [Background technology]

[0002] Unstained cell observation devices are known that use microspectroscopy to observe cell samples, allowing for the identification of cell states and conditions without staining. One such microspectroscopy technique is the stimulated Raman scattering microscopy (SRS) microscope.

[0003] SRS microscopy acquires signals originating solely from the imaginary part (Imχ(3)) of the third-order nonlinear susceptibility (χ(3)) of the sample. Therefore, because SRS microscopy reflects both the real and imaginary parts (Reχ(3) and Imχ(3)) of the stimulated Raman signal, it is unaffected by non-resonant signals and has a low level of background signal originating from water surrounding the cells. In this respect, SRS microscopy is better able to ensure contrast of the cell image against non-resonant signals that reflect both the real and imaginary parts of χ(3).

[0004] There is a method for non-invasively evaluating biological specimens using SRS light (stimulated Raman scattering light) generated by such nonlinear optical effects. Non-patent document 1 discloses a method for quantifying intracellular density by separating sample-induced aberrations and signal loss due to light scattering in multicellular specimens using an SRS signal ratio obtained by normalizing the protein-derived SRS signal with the water-derived SRS signal. Non-patent document 1 also discloses the detection of cellular states such as apoptosis using the quantified fluctuations in intracellular density. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] The Journal of Physical Chemistry B.2022,126,39:7595―7603 [Overview of the project] [Problems that the invention aims to solve]

[0006] Non-Patent Document 1 discloses an evaluation method for assessing the state of cells in a sample by obtaining the normalized mass density distribution of cells from the ratio IRatio, which is the ratio of the amount of protein-corresponding component (CH group) to the amount of water-corresponding component (OH group). Non-Patent Document 1 uses a stimulated Raman scattering (SRS) microscope to obtain and utilize the Raman spectrum of cells by scanning the wavelength of the irradiation laser. Non-Patent Document 1 discloses that this evaluation method shows low and high mass densities between living cells and dead cells.

[0007] On the other hand, the method for determining the state of cells described in Non-Patent Literature 1 uses the amount of components equivalent to water inside the cell, so when observing cells under a liquid containing water, such as a cell culture medium, the influence of the water component in the liquid is unavoidable. In Non-Patent Literature 1, the dry mass is evaluated for cells on a glass slide that has been immobilized to eliminate the influence of the cell culture medium.

[0008] In view of the above constraints, the present invention aims to provide a determination device that determines the state of cells using a minimally invasive method with fewer limitations on the sample form. [Means for solving the problem]

[0009] A determination device according to an embodiment of the present invention includes: an acquisition unit that acquires a Raman spectral image captured by an imaging device that captures stimulated Raman scattered light from a biological cell irradiated with coherent primary light; a generation unit that generates spectral difference information that emphasizes the other of two pairs of Raman scattered light corresponding to different wavenumber shift bands of the Raman spectral image; and a determination unit that determines the state of the cell based on the spectral difference information.

[0010] A control method for a determination device according to an embodiment of the present invention comprises: an acquisition unit for acquiring a Raman spectral image; a generation unit for generating spectral difference information by emphasizing the Raman signal of a first component with respect to the Raman signal of a second component; a cell region identification unit for identifying cell regions; and a determination unit for determining the state of cells contained in a sample. The process of acquiring the Raman spectroscopic image of the sample obtained by spectral imaging using the acquisition unit, Using the generation unit, a step is made to generate spectral difference information from the Raman spectral image in which the Raman signal corresponding to the protein is emphasized relative to the Raman signal corresponding to the lipid contained in the sample. A step of obtaining cell identification information by identifying cell regions in the imaging field of the Raman spectroscopic image using the cell region identification unit, The method includes a step of determining the state of the cell based on the spectral difference information and the cell identification information using the determination unit. [Effects of the Invention]

[0011] According to the present invention, it is possible to provide a determination device that determines the state of cells using a minimally invasive method with fewer restrictions on the sample form. [Brief explanation of the drawing]

[0012] [Figure 1] This is a diagram showing a determination device according to the first embodiment. [Figure 2] This is a process diagram showing the control method of the determination device according to the first embodiment. [Figure 3] This figure shows a determination device according to the second embodiment. [Figure 4] This is a process diagram showing the control method of the determination device according to the third embodiment. [Figure 5] This figure shows a determination device according to the fourth embodiment. [Figure 6] This is a process diagram showing the control method of the determination device according to the fourth embodiment. [Modes for carrying out the invention]

[0013] <First Embodiment> Using FIGS. 1, 2, Table 1, Table 2, and Table 3, the determination device 100 according to the first embodiment and the control method 10000 of the determination device will be described. FIG. 1 is a diagram showing the determination device 100 according to the first embodiment. Further, FIG. 1 can be rephrased as a diagram showing a determination system 1000 including a determination device 100 that acquires a Raman spectroscopic image from an SRS microscope 600 and determines the state of cells contained in a sample. FIG. 2 is a process diagram showing the control method of the determination device according to the first embodiment.

[0014] (Determination System) The determination system 1000 includes an SRS microscope 600 (stimulated Raman scattering microscope) and a determination device 100 that acquires a Raman spectroscopic image from the SRS microscope 600 and determines the state of cells contained in a sample 620. The determination device 100 of the present embodiment is configured independently of the SRS microscope 600 so that signal processing or image processing for determining the state of cells can be performed. The determination device 100 can be modified into a form incorporating an SRS microscope, a form incorporating a reconstruction unit 614 of the SRS microscope 600 described later, and the like.

[0015] (SRS Microscope) Using FIG. 1, the SRS microscope 600 applied to the determination device 100 according to the present embodiment will be described. The SRS microscope 600 can be rephrased as an imaging device 600 that captures a Raman spectroscopic image and can output it to the determination device 100. The SRS microscope 600 includes a placement unit 607 on which a sample 620 including biological cells is placed, and a pair of objective lenses 630 (606, 609) that face each other sandwiching the placement unit 607 so that at least a part of the focal point fs overlaps the sample 620. The SRS microscope 600 includes an irradiation optical system 650 that irradiates the sample 620 with coherent primary light Ip through the objective lens 606, and a detection optical system 660 that detects a part of the secondary light Is emitted from the sample 620 due to a non-linear optical effect through the objective lens 609. The pair of objective lenses 630 can be rephrased as an objective lens pair 630, and the objective lenses 606 and 609 may be rephrased as one and the other of the pair of objective lenses 630, respectively.

[0016] (Irradiation optical system) The irradiation optical system 650 includes an excitation light source 601 that emits an excitation light pulse train 6011 and a probe light source 602 that emits a probe light pulse train 6021. The irradiation optical system 650 further includes an adjustment unit 6032 that adjusts the time difference of one of the excitation light pulse train 6011 and the probe light pulse train 6021 with respect to the other, and a sweeping unit 6031 that sweeps the wavelength of the emitted coherent primary light Ip. The irradiation optical system 650 further includes a mirror 604 and a multiplexing unit 605 that are optically coupled to one objective lens 606 and multiplex the excitation light pulse train 6011 and the probe light pulse train 6021 coaxially. When multiplexing in the multiplexing unit 605, the optical path length of the probe light pulse train 6021 is adjusted using the adjustment unit 6032 so that the pulses of the excitation light pulse train 6011 and the probe light pulse train 6021 coincide temporally. The excitation light pulse train 6011 and the probe pulse train light 6021 are condensed into a sample 620 containing a culture solution and living cells by an irradiation objective lens 606. The secondary light including the excitation light pulse train 6011 and the probe light pulse train 6021 that passes through and diverges from the sample 620 containing the culture solution and cells is converted into substantially parallel light by a condenser objective lens 609. Further, the excitation light pulse train 6011 and the probe light pulse train 6021 are selectively transmitted through the excitation light pulse train 6011 by a filter 610, condensed onto a photodetector 612 by a lens 611, and the light intensity of the excitation light pulse train 6011 is detected. Note that the filter 610 may selectively transmit only the probe 6021, be condensed onto the photodetector 612 by the lens 611, and detect the light intensity of the probe light pulse train 6021.

[0017] (Detection optical system) The detection optical system 660 includes an optical filter 610 that selectively transmits the wavelength component of excitation light or probe light from secondary light Is collected through the other objective lens 609, and a focusing lens 611 that focuses the transmitted light that has passed through the optical filter 610. The detection optical system 660 further includes a detection unit 612 that detects a portion of the secondary light Is guided through the optical filter 610 and the focusing lens 611 and outputs a detection signal.

[0018] Here, the output signal from the photodetector 612 is transmitted to the control unit 613. The scanning signal and coordinate position of the sample stage 608 are also transmitted to the control unit 613, and together with the output signal from the photodetector 612, the image is reconstructed in the reconstruction unit 614 to acquire the Raman spectroscopic image Psrs. At this time, the probe light pulse train 6021 is rapidly swept across wavelengths from 1015 nm to 1030 nm, so each pixel of the image is scanned at 2800 cm⁻¹. -1 From 2950cm -1 Up to 150cm -1 A spectral image is obtained in which the Raman spectrum is densely acquired within the specified range. To detect the output signal of the photodetector with high sensitivity, lock-in detection can be performed using the pulse repetition frequency signal of the probe light pulse train 6021 as a reference input, although this is not shown in the diagram.

[0019] (Scanning system) The SRS microscope 600 has a scanning unit 628 that changes the relative position of the focal point fs with respect to the sample 620. The scanning unit 628 comprises a stage scanning unit 608 that scans the mounting unit 607 in a direction intersecting the optical axis of the primary light Ip, and an optical axis scanning unit 618 that scans the optical axis OA in a direction intersecting the optical axis OA of the primary light Ip.

[0020] (Control Unit) The SRS microscope 600 includes a control unit 613 that coordinately controls a scanning unit 628, a sweeping unit 6031, an excitation light source 601, and a probe light source 602.

[0021] (Reconstruction part) The SRS microscope 600 includes a reconstruction unit 614 that reconstructs an image based on the position of the focal point fs and the detection signal detected by the detection unit 612, and generates the Raman spectroscopic image. The reconstruction unit 614 may also be referred to as an imaging unit 614, an image generation unit 614, or an image formation unit 614.

[0022] (Excitation light source) The excitation light source 601 is positioned to emit an excitation light pulse train 6011 toward the multiplexer 605. The excitation light pulse train 6011 is preferably short-pulse light with a short pulse width to efficiently produce a nonlinear optical effect in the sample, and the pulse width is preferably on the order of femtoseconds to picoseconds. Furthermore, the repetition frequency of the pulse train is preferably 1 MHz or higher from the viewpoint of mitigating the effects of intensity fluctuations in the excitation light pulse train 6011. The excitation light source 601 is a titanium-sapphire laser oscillating in the near-infrared band, which satisfies the pulse width and repetition frequency requirements of the excitation light pulse train 6011. When a titanium-sapphire laser is used as the excitation light source 601, the wavelength of the excitation light is set between 700 nm and 1000 nm.

[0023] (Probe light source) The probe light source 602 is arranged to emit a probe light pulse train 6021 toward the wavelength sweeping section 6031 or the adjustment section 6032 that adjusts the delay time. Similar to the excitation light source 601, the probe light source 602 employs a pulse light source that emits a short pulse train of the probe light pulse train 6021. Also, the pulse width of the probe light pulse train 6021 is on the order of femtoseconds to picoseconds. The probe light source 602 may be an optical fiber laser such as a Yb fiber laser or an Er fiber laser. Also, the pulse repetition frequency of the probe light pulse train 6021 is configured to be 1 / 2 of the pulse repetition frequency of the excitation light pulse train 6011. By configuring it in this way, modulation can be applied to the excitation light pulse train 6011 and the probe light pulse train 6021 at the same pulse repetition frequency as the probe light pulse train 6021 due to the non-linear optical effect of the sample. The probe light pulse train 6021 passes through the optical delay system 6031 and is swept between wavelengths of 1015 nm and 1030 nm in the sweeping section 6031.

[0024] The wavelength of the probe light pulse train 6021 is set according to the type of cell specimen to be subjected to state determination and the wavelength of the excitation light pulse train 6011. That is, the wavelength of the probe light pulse train 6021 is set to a wavelength that is different by approximately the wavelength corresponding to the Raman shift (wavenumber shift) of the molecular information to be detected with respect to the wavelength of the excitation light pulse train 6011. For example, the excitation light source 601 and the probe light source 602 are configured to obtain Raman spectra with wavenumbers of 2800 cm -1 to 2950 cm -1 or less as the Raman shift for obtaining information regarding proteins and lipids. The wavenumber shift band corresponding to lipids is included in the wavenumber shift band of 2800 cm -1 to 2900 cm -1 or less, and the wavenumber shift band corresponding to proteins is 2920 cm -1 to 2940 cm -1It falls within the following wavenumber shift band. In a configuration where a titanium-sapphire laser is used as the excitation light source 601 and the wavelength of the excitation light pulse train 6011 is 790 nm, the wavelength of the probe light pulse train 6021 from the probe light source 602, controlled by the sweeping unit 6031, is swept between 1015 nm and 1030 nm.

[0025] (judgment device) Next, using Figure 1, we will describe the determination device 100 that determines the state of cells based on Raman spectroscopic images captured by the SRS microscope 600 according to this embodiment.

[0026] The determination device 100 includes an image acquisition unit 110 that acquires a Raman spectral image Imr captured by an imaging device 600 that captures stimulated Raman scattered light from biological cells irradiated with excitation light. The determination device 100 further includes a generation unit 120 that generates spectral difference information Ifd, which emphasizes the Raman scattered light signal Sot corresponding to one of the different wavenumber shift bands of the Raman spectral image Imr with respect to the other wavenumber shift band Son. The determination device 100 further includes a cell region identification unit 150 that acquires cell identification information ifc that identifies the cell region in the imaging field of view (FOV) of the Raman spectral image Imr, and a determination unit 130 that determines the state of the cell based on the spectral difference information Ifd and the cell identification information Ifc.

[0027] (Judgment Department) The determination unit 130 determines the state of the cells based on the comparison result between a predetermined reference value and spectral difference information.

[0028] (spectral difference information) The spectral difference information Ifd is configured to include Ifad, which is information about coordinate values ​​in the imaging field of view (FOV), and Ifdv, which is difference value information corresponding to the difference between one Raman scattered light signal Ione and the other Raman scattered light signal Iote. Different wavenumber shift bands correspond to wavenumber shift bands corresponding to proteins and wavenumber shift bands corresponding to lipids, which do not overlap with each other.

[0029] (Control method for the judgment device) By executing the control method for the determination device 100 according to this embodiment, it is possible to determine the state of cells. The control method 10000 for the determination device 100 for determining the state of cells will be explained with reference to Figure 2.

[0030] The determination device 100 controlled by the control method 10000 includes, as shown in Figure 1, an acquisition unit 110 that acquires a Raman spectral image and a generation unit 120 that generates spectral difference information by emphasizing the Raman signal of the first component with respect to the Raman signal of the second component. Furthermore, the determination device 100 targeted by the control method 10000 includes, as shown in Figure 1, a determination unit 130 that determines the state of cells contained in the sample based on the spectral difference information.

[0031] (Step S110: Acquiring Raman spectroscopy images) The control method 10000 includes a step S110, which is performed using the acquisition unit 110 to acquire a Raman spectral image Psrs of the spectrally imaged sample 620, as shown in Figure 2. The Raman spectral image Psrs acquired in this step is reconstructed by the reconstruction unit 614 and transmitted from the SRS microscope 600 to the determination device 100, where it is passed on to the next step, step S120, which generates spectral difference information. The Raman spectral image Psrs imaged by the reconstruction unit 614 is stored in the second storage unit 640 and transmitted to the acquisition unit 110 of the determination device 100 based on a predetermined read command. The predetermined read command is commanded to the second storage unit 640 by the acquisition unit 110, the control unit 613, etc.

[0032] Here, we will explain the data format of the Raman spectroscopic image Psrs acquired by the SRS microscope 600. The data format of the Raman spectroscopic image Psrs has multiple Raman spectroscopic signals Isrs with different wavenumber shift values ​​for the coordinate values ​​(x, y, z), as shown in Table 1. As shown in Table 1, the data format of the Raman spectroscopic image Psrs may include supplementary information such as the date and time of acquisition (scanning time, acquisition time of the SRS Raman signal), specimen information, and other supplementary information.

[0033] Table 1 shows an example of a dataset of Raman spectroscopic images Psrs obtained in step S110. Such a dataset is stored in the first storage unit 140 in a readable format. Table 1 is sorted in ascending order by spot number, which corresponds to the scanning position order of the excitation light pulse train 6011 and probe light pulse train 6021 of the SRS microscope 600, with the first priority being spot number, and the second priority being wavenumber shift Δk. As is clear when considering Table 1 sorted in ascending order with wavenumber shift Δk as the first priority and scan number as the second priority, the Raman spectroscopic image Psrs can be rephrased as being composed of a set of multiple Raman spectroscopic images psrs with different wavenumber shift values.

[0034] [Table 1]

[0035] (Step S120 to generate spectral difference information) The control method 10000 includes a step S120 which is performed using the generation unit 120 to generate spectral difference information I(p / f) for each coordinate value from the Raman spectral image Psrs acquired via the acquisition unit 110.

[0036] The spectral difference information I(p / f) is a signal obtained by emphasizing the Raman signal Ip corresponding to proteins in sample 620 with respect to the Raman signal if corresponding to lipids. The spectral difference information I(p / f) obtained in this process is, for example, the signal obtained by dividing the Raman signal Ip corresponding to the protein concentration by the Raman signal If corresponding to the lipid concentration.

[0037] Table 2 shows an example of a dataset including spectral difference information I(p / f) obtained in step S120. Such a dataset includes at least coordinate values ​​and spectral difference information I(p / f), and is stored in the first storage unit 140 in a readable format.

[0038] [Table 2]

[0039] (S130: Cell identification information is obtained based on the coordinate values ​​in the Raman spectroscopy image.) The control method 10000 includes a step S130, which is performed using a cell region identification unit 150 to acquire cell identification information Ics for coordinate values ​​in a Raman spectroscopic image. This step S130 includes a region determination step S130a, which acquires the outer contour of a cell of interest, which serves as a criterion for determining whether the coordinate values ​​constituting the Raman spectroscopic image Psrs are inside or outside a predetermined cell. Furthermore, this step 130 includes a region identification step S130b, which assigns a unique cell identification code CN to the cell region determined in step S130a in the imaging field (FOV). This step S130 utilizes the cell region and cell identification code determined based on the Raman spectroscopic image Psrs, the Raman spectroscopic image psrs corresponding to a specific wavenumber shift, a morphological image (not shown), etc. The cell identification information Ics can be rephrased as having information about the contour of the cell region and the cell identification code CN.

[0040] Table 3 shows an example of a dataset containing the cell identification code CN obtained in step S130. Such a dataset containing cell identification information Ics includes at least coordinate values ​​and cell identification information Ics, and is stored in readable format in the first storage unit 140.

[0041] [Table 3]

[0042] (S140 determines the state of the cell based on spectral difference information and cell identification information) The control method 10000 includes a step S140 in which the state of a cell is determined based on spectral difference information I(p / f) and cell identification information Ics, which is performed using the determination unit 130.

[0043] Step S140 uses the coordinate values ​​having a common identification code CN obtained in step S130 and the spectral difference information I(p / f) values ​​corresponding to those coordinate values ​​obtained in step S120 to determine the state of the cells for each cell region having a common identification code CN. Step S140 uses the average spectral difference information Iave(p / f) value, which is obtained by averaging the spectral difference information I(p / f) corresponding to the coordinate values ​​having a common identification code CN, to determine the state of the cells in the cell region having a common identification code CN.

[0044] Table 4 shows an example of a dataset obtained in step S140, including coordinate values, cell identification code CN, and spectral difference information I(p / f). Similarly, Table 5 shows an example of a dataset obtained in step S140, including cell identification code CN, average spectral difference information Iave(p / f), and judgment result. Each of these datasets described in Tables 4 and 5 is stored in the first storage unit 140 in a readable format.

[0045] [Table 4]

[0046] [Table 5]

[0047] By performing step S140 only on the intracellular coordinate values ​​of interest based on the cell identification information Ics, it is possible to reduce the computational load of cell state determination and the decrease in the throughput of the determination process.

[0048] If the average spectral difference information Iave(p / f) value is greater than or equal to a predetermined lower threshold Ith(p / f), the cell of interest is determined to be a living cell. The lower threshold Ith(p / f) is preferably calibrated for each type of cell of interest, but is set to be between 0.4 and 0.8. Two lower thresholds are set as the criteria for determining the cell state, which are adopted in the dataset shown in Table 5. The cell state includes at least one of the following: cell activity, vitality or death, cause of death, or pharmacological effect. A first lower threshold Ith1(p / f) is set to determine that the cell state is normal (living cell), and a second lower threshold Ith2(p / f) is set to determine that the cell state is not yet dead, but is in a boundary state where the probability of it being a living cell is low. In this embodiment, the first lower threshold Ith1(p / f) and the second lower threshold Ith2(p / f) are set to 0.67 and 0.50, respectively. The predetermined lower threshold Ith(p / f) may be set to three or more levels.

[0049] The dataset in Table 5 includes an additional information column that indicates whether a cell region is partial or partial, for cell regions present in the field of view (FOV) and for extracted cell identification codes (CN). This makes it possible for users or detection devices to distinguish between partial cell regions and whole cell regions where the entire cell region is included in the field of view (FOV).

[0050] <Second Embodiment> The determination device 200 according to the second embodiment is a determination device that determines the state of cells in a sample using Raman spectroscopic images from an SRS microscope 600 and a storage device 305. The determination device 200 differs from the determination device 100 of the first embodiment in that the SRS microscope 600, storage device 305, CPU 501, RAM 502, input / output interface, etc. are connected via an internet line 503 (network).

[0051] Figure 3 shows a schematic configuration of the determination device 200 of this embodiment. The determination device 200, together with the SRS microscope 600, constitutes a determination system 2000 for determining the state of cells. The determination device 200 is configured to have mutual access to the storage device 305 that stores multiple Raman spectroscopic images captured by multiple SRS microscopes, thus improving the statistical reliability of the basis for the determination compared to the determination device 100.

[0052] <Third Embodiment> The control method 30000 of the determination device in this embodiment differs from the control method 10000 of the determination device in the first embodiment in that, as a step prior to step S110, it includes step S105 to acquire cell identification information Ics for coordinate values ​​of a Raman spectroscopic image based on a morphological image.

[0053] Morphological images can be obtained using known microscopic observation methods that acquire secondary light linear to primary light. Known microscopic observation methods include optical arrangement, wavelengths of primary and secondary light, transmitted light images, reflected light images, etc.

[0054] <Fourth Embodiment> <Cell sorter unit> The determination system 4000 of this embodiment differs from the determination system 1000 of the first embodiment in that it includes a cell sorter unit 900 that separates cells 906 in a cell suspension 9013 into living cells and dead cells. The SRS microscope 670 of this embodiment includes a flow channel mounting section 902 as part of the cell sorter unit 900, configured such that the cell suspension 9013 passes through the focal points of the objective lens pair 606, 609 of the SRS microscope 670. At least a portion of the flow channel mounting section 902 is translucent, allowing for spectral imaging.

[0055] <Fluid Sample Supply Unit> The cell sorter unit 900 includes, upstream of the flow path mounting section 902, a sample container 901 in which a cell sample 906 is contained, and a sheath fluid container 920 in which a sheath fluid 9012 is contained.

[0056] The flow rate of the cell suspension 0011 supplied from the sample container 901 and the flow rate of the sheath fluid 9012 supplied from the sheath fluid container 920 are controlled by the control unit 613 so that the cells 906 in the cell suspension 9011 flow one cell at a time in an aligned manner through the flow channel mounting section 902. The sample container 901, the sheath fluid container 920, and the control unit 613 constitute a fluid sample supply section 940 that supplies a fluid sample 9013 in which the cell suspension 9011 is diluted by the sheath fluid 9012 and has fluidity. The sample container 901 and the sheath fluid container 920 that constitute the liquid sample supply section 940 are arranged on the side of the sample inlet 902i of the flow channel mounting section 902. In other words, the liquid sample supply section 940 is a supply section that supplies a fluid sample 9013 containing multiple biological cells to the flow channel mounting section 902 upstream of the focused region FS in the flow channel mounting section 902.

[0057] <Passage Detection Unit> The cell sorter unit 900 of this embodiment includes a passage detection unit 922 between the sample inlet 902i of the flow path mounting unit 902 and the focal point FS, which detects the passage and flow velocity of cells 906. The passage detection unit 922 can also be referred to as a counter 922. The counter 922 is composed of electrostatic, optical, acoustic, and other sensors. The timing of primary light irradiation and secondary light collection is controlled by the control unit 613 using the time tp and time Δtp of the predicted focal point FS, based on the monitoring results of the counter 922, when the re-cells 906 pass through. The focal point FS may also be referred to as the focusing region FS.

[0058] <Focus> The flow channel mounting section 902 is positioned between a pair of objective lenses 606 and 609 such that the fluid sample 9013 passing through it passes through a region where the focal points of the pair of objective lenses 606 and 609 overlap. Coherent light, including an excitation light pulse train 6011 and a probe light pulse train 6021, is focused through the objective lens 903 and irradiated as a primary light pulse onto the cells 906 flowing within the flow channel mounting section 902. Secondary light emitted from the cells 906 is approximately parallelized by the objective lens 609 and guided toward the filter 610 as collimated light. The secondary light includes a transmitted light component that has passed through the cells 906 and a scattered light component that has been scattered by the cells 906. The process of detecting a Raman signal from the secondary light after passing through the filter 610 is the same as in the SRS microscope 600, reconstruction unit 614, and determination device 400 of the first embodiment.

[0059] <Preparative section> As shown in Figure 5, the SRS microscope 670 of this embodiment further includes a sorting unit 950 that sorts the sample 906 downstream of the focal point FS of the flow channel mounting unit 902 based on the determination result of the determination unit 130.

[0060] The dispensing unit 950 includes a droplet forming unit 905 for discharging droplets 913 containing cells 906 from the sample outlet 902e of the flow channel mounting unit 902. The droplet forming unit 905 is composed of an excitation means such as a piezoelectric element that vibrates the flow channel mounting unit 902. The droplets 913 are composed of cells, buffer solution, and sheath solution. The concentration of cells in the cell suspension 9011 is pre-adjusted so that each droplet 913 contains one cell, and the flow velocity of the cell suspension 9011 and sheath solution 9012 in the flow channel mounting unit 902, as well as the excitation operation of the droplet forming unit 905, are controlled by the control unit 613. The droplet forming unit 905 is configured so that multiple droplets 913 are discharged at intervals from the discharge outlet 902e of the flow channel mounting unit 902.

[0061] The sorting unit 950 includes a charging plate 908 for charging the droplets 913, and a charging control unit 907 for controlling the amount of charge and the polarity of the charge applied to the droplets 913 by the charging plate 908. The charging control unit 907 controls the amount of charge and the polarity of the charge based on the determination result from the cell 916 determination device 100, which determines the cell corresponding to the droplet 913 that has reached the charging plate 908.

[0062] The sorting unit 950 is equipped with an electrode pair 909 that applies an electrostatic field Fdc in a direction intersecting the discharge direction near the outlet 902e of the droplet 913 in order to sort droplets 913 that have a predetermined charge. Due to the electrostatic field Fdc, the droplets 913 that have a predetermined charge are collected in predetermined collection containers 9101, 9102, and 9103, respectively. In this embodiment, droplets 913 that have been determined to be living cells or dead cells are collected in collection containers 9101 and 9103, respectively, while droplets 913 that have been determined to be in the boundary region are not charged and travel straight as neutral droplets 913, and are collected in collection container 9102.

[0063] The determination system 4000 sorts the cell suspension 9011 based on the control method 40000 shown in Figure 6.

[0064] <Start supplying the fluid sample to the flow path> The control method 40000 includes a starting step S100 followed by a step S410 in which the supply of the fluid sample 9013 to the flow path mounting section 902 is started.

[0065] <Obtain the predicted transit times of cells in a fluid sample as they pass through the focus and sorting area.> The control method 40000 includes a step S420 following the supply start step S410, in which the predicted passage time of the cells 906 in the fluid sample 9013 as they pass through the focus FS and the sorting unit 950 is obtained. The predicted passage time as they pass through the sorting unit 950 is obtained by acquiring the predicted passage times as they pass through the droplet formation unit 905, the charging plate 908, and the electrode pair 909, respectively.

[0066] <Irradiate with primary light at the predicted focal point transit time and perform spectral imaging.> The control method 40000 includes a step S420 for acquiring predicted passage times, followed by a step S430 for irradiating primary light at the predicted passage time of the focal point FS and performing spectral imaging. The predicted passage times for passing through the separation unit 950 are obtained as well as the predicted passage times for passing through the droplet formation unit 905, the charging plate 908, and the electrode pair 909, respectively.

[0067] <Obtain Raman spectroscopic images (Psrs) for the spectrally imaged sample.> The control method 40000 includes a step S110 to acquire Raman spectroscopic images Psrs of the spectrally imaged sample 906, following the spectroscopic imaging step S430.

[0068] <Generates spectral difference information I(p / f) from Raman spectroscopic images Psrs, emphasizing the Raman signal corresponding to proteins relative to the Raman signal corresponding to lipids.> The control method 40000 includes a step S120 following the spectral image acquisition step S110, which generates spectral difference information I(p / f) from the Raman spectral image Psrs, in which the Raman signal corresponding to protein is emphasized relative to the Raman signal corresponding to lipid.

[0069] <Obtain cell identification information (Ics) for coordinate values ​​in Raman spectroscopy images> The control method 40000 includes a step S130 to acquire cell identification information Ics for coordinate values ​​in a Raman spectroscopic image, following a spectral difference information generation step S120.

[0070] <Determine the state of cells based on spectral difference information I(p / f) and cell identification information Ics> The control method 40000 includes a cell identification information acquisition step S130 followed by a step S140 in which the state of the cell is determined based on the spectral difference information I(p / f) and the cell identification information Ics. Each of steps S110, S120, S130, and S140 corresponds to the control method 10000 of the determination system 1000 according to the first embodiment.

[0071] <Changes the ejection trajectory of droplets after passing through the dispensing section.> The control method 40000 includes a cell state determination step S140 followed by a step S440 to change the ejection trajectory of the droplet 913 that has passed through the dispensing unit 950, and the ejection trajectory changing step S440 is followed by a termination step S150.

[0072] This embodiment describes the operation of separating living and dead cells using a cell sorter device, but the cells to be separated are not limited to living and dead cells. Furthermore, although the function of separating cells has been mainly described, it may also be used as a flow cytometer to analyze and statistically display the spectra of individual cells. [Explanation of Symbols]

[0073] 100, 400 judgment device 110 Acquisition Department 120 Generation part 150 Cell region identification section 130 Judgment section

Claims

1. A determination device comprising: an acquisition unit that acquires a Raman spectral image captured by an imaging device that captures stimulated Raman scattered light from biological cells irradiated with coherent primary light; a generation unit that generates spectral difference information that emphasizes the Raman scattered light signal corresponding to one of the different wavenumber shift bands of the Raman spectral image with respect to the other wavenumber shift band; a cell region identification unit that acquires cell identification information that identifies the cell region in the imaging field of the Raman spectral image; and a determination unit that determines the state of the cell based on the spectral difference information and the cell identification information.

2. The determination device according to claim 1, wherein the different wavenumber shift bands correspond to a wavenumber shift band corresponding to the protein and a wavenumber shift band corresponding to the lipid, and the different wavenumber shift bands do not overlap with each other.

3. The wavenumber shift band corresponding to the aforementioned lipids is 2800 cm⁻¹. －１ More than 2900cm －１ The wavenumber shift band corresponding to the protein is 2920 cm², which is included in the following wavenumber shift band. －１ More than 2940cm －１ The determination device according to claim 2, which is included in the following wavenumber shift band.

4. The determination device according to claim 1, wherein the Raman spectroscopic image includes information about coordinate values ​​in the imaging field, information about wavenumber shift values ​​included in the wavenumber shift band, and information about the intensity of the Raman scattered light signal corresponding to the wavenumber shift value.

5. The determination device according to claim 1, wherein the spectral difference information includes information relating to coordinate values ​​in the imaging field and difference value information corresponding to the difference value of the other Raman scattered light signal with respect to the one Raman scattered light signal.

6. The determination device according to claim 1, wherein the state of the cell includes at least one of the activity level, life or death, cause of death, or pharmacological effect of the cell.

7. The determination device according to claim 1, wherein the cell identification information includes information relating to coordinate values ​​corresponding to a cell region in the Raman spectroscopic image and information relating to a cell identification code corresponding to the cell region.

8. The determination device according to claim 1, wherein the determination unit determines the state of the cell based on the comparison result between a predetermined reference value and the spectral difference information.

9. The determination device according to claim 8, further comprising a first storage unit for storing the predetermined reference value.

10. The determination device according to claim 1, comprising the imaging device.

11. The determination device according to claim 10, wherein the acquisition unit acquires the Raman spectral image by reading the Raman spectral image from a second storage unit that stores a plurality of Raman spectral images captured by the imaging device.

12. The determination device according to claim 11, wherein the acquisition means acquires the Raman spectral image from the storage unit 2 via a network.

13. The determination device according to claim 11, wherein the second storage unit constitutes the imaging device.

14. The determination device according to claim 9 or 10, comprising: an imaging device, a placement section on which a sample containing the biological cells is placed; a pair of objective lenses facing each other on either side of the placement section such that at least a portion of the focal point overlaps with the sample; an illumination optical system that irradiates the sample with coherent primary light through one of the pair of objective lenses; and a detection optical system that detects a portion of the secondary light emitted from the sample by a nonlinear optical effect through the other of the pair of objective lenses.

15. The determination device according to claim 1 or 2, comprising an excitation light source that emits an excitation light pulse train and a probe light source that emits a probe light pulse train.

16. The determination device according to claim 15, wherein the irradiation optical system comprises an adjustment unit for adjusting the time difference between one of the excitation light pulse train and the probe light pulse train and the other; a sweeping unit for sweeping the wavelength of the emitted coherent primary light; and a multiplexing unit for optically coupling to one of the objective lenses and combining the excitation light pulse train and the probe light pulse train.

17. The determination device according to claim 16, further comprising: a scanning unit that changes the relative position of the focal point with respect to the sample; and a control unit that coordinately controls the scanning unit, the sweeping unit, the excitation light source, and the probe light source.

18. The determination device according to claim 17, comprising: an optical filter that selectively transmits the wavelength component of the excitation light or the probe light from the secondary light collected through the other objective lens; a focusing lens that focuses the transmitted light that has passed through the optical filter; and a detection unit that detects a portion of the secondary light guided through the optical filter and the focusing lens.

19. The determination device according to claim 18, further comprising a reconstruction unit that reconstructs an image based on the position of the focal point and the detection signal output from the detection unit, and generates the Raman spectral image.

20. The determination device according to claim 14, wherein the mounting portion is configured, at least in part, as a light-transmitting channel so that the fluid sample passes through the focal point.

21. The determination device according to claim 20, further comprising a dispensing unit that dispenses the sample based on the determination result of the determination unit downstream of the focal point in the flow path.

22. The determination device according to claim 21, wherein the dispensing unit includes a droplet forming unit that forms a plurality of droplets of the fluid at intervals from at least a portion of the fluid.

23. The determination device according to claim 22, further comprising a supply unit that supplies the fluid containing a plurality of biological cells to the flow channel upstream of the focusing region in the flow channel.

24. A control method for a determination device comprising: an acquisition unit for acquiring a Raman spectroscopic image; a generation unit for generating spectral difference information by emphasizing the Raman signal of a first component with respect to the Raman signal of a second component; a cell region identification unit for identifying cell regions; and a determination unit for determining the state of cells contained in a sample. The process of acquiring the Raman spectroscopic image of the sample obtained by spectral imaging using the acquisition unit, Using the generation unit, a step is made to generate spectral difference information from the Raman spectral image in which the Raman signal corresponding to the protein is emphasized relative to the Raman signal corresponding to the lipid contained in the sample. A step of obtaining cell identification information by identifying cell regions in the imaging field of the Raman spectroscopic image using the cell region identification unit, A control method for a determination device comprising the step of determining the state of the cell based on the spectral difference information and the cell identification information using the determination unit.

25. A control method for a determination device according to claim 24, wherein the determination unit is operated to determine the state of the cell based on the result of comparing a predetermined reference value with the spectral difference information.

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