Microscope device, spectrometer, and microscope system

Abstract

This microscope device comprises: an opening (31) that has a first slit and a second slit through which a plurality of beams, which come out from an observation target and are respectively caused by a plurality of irradiation beams emitted to the observation target and having different wavelengths, pass; a dispersion element that disperses the wavelength of each of the plurality of beams that have passed through the opening (31); and an imaging sensor (32) that receives the plurality of beams the wavelengths of which are dispersed by the dispersion element. The imaging sensor (32) receives the plurality of wavelength-dispersed beams so that a 0th-order beam passing through the second slit and a 1st-order beam passing through the first slit do not overlap each other.

Description

[Technical field]

[0001] The present disclosure relates to a microscope apparatus, a spectrometer, and a microscope system. [Background technology]

[0002] Conventionally, in relation to technologies for acquiring imaging data of an object to be observed, there are fluorescent observation methods used, for example, for acquiring pathological images and imaging cytometry. In addition, various imaging diagnostic methods using fluorescent staining have been proposed as methods excellent in quantitativeness and polychromaticity.

[0003] Fluorescence observation techniques are advantageous in that they are easier to multiplex than color staining, and detailed diagnostic information can be obtained. In fluorescence imaging other than pathological diagnosis, an increase in the number of colors makes it possible to simultaneously examine various antigens expressed in a sample. In general fluorescence imaging, excitation light is irradiated at the absorption wavelength (excitation wavelength) of the dye, and the dye spectrum emitted by this is selectively captured by a bandpass filter. When there are multiple colors, the absorption wavelength (excitation wavelength) varies depending on the dye, so a method of imaging by switching filters for each dye is used. However, since the absorption and emission spectra of the dyes are broad and overlap, when staining with multiple colors, multiple dyes are excited by one excitation wavelength. Furthermore, the fluorescence of adjacent dyes leaks into the bandpass filter, causing color mixing (fluorescence overlap).

[0004] On the other hand, a method is known in which the wavelength of the excitation light and the wavelength of the fluorescence to be detected are switched in a time-division manner to capture images. However, this method has a problem in that the capture time increases linearly as the number of colors increases. As an observation method that takes the above circumstances into consideration, there is a fluorescence observation method that uses multiple excitation lights and multiple slits. With this method, multiple excitation lights can be irradiated at once, and data on the fluorescence due to all excitations can be obtained with a single scan. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 4452850 [Non-patent literature]

[0006] [Non-Patent Document 1] Edward C. Stack, "Multiplexed immunohistochemistry, imaging, and quantitation: A review, with an assessment of Tyramide signal amplification, multispectral imaging and multiplex analysis", Methods 70 (2014) 46-58 Summary of the Invention [Problem to be solved by the invention]

[0007] However, the conventional fluorescence observation method using multiple excitation lights and multiple slits described above has a problem in that, for example, in the imaging section, the first-order light passing through one slit overlaps with the zeroth-order light passing through another slit, making it difficult to obtain accurate imaging data.

[0008] Therefore, the present disclosure has been made in consideration of the above circumstances, and proposes a microscope device, a spectrometer, and a microscope system that are capable of obtaining accurate imaging data in an imaging section when a plurality of illumination lights with different wavelengths are irradiated onto an object to be observed, and each of the plurality of lights from the object to be observed is passed through a plurality of slits and then wavelength dispersed. [Means for solving the problem]

[0009] In order to solve the above problems, a microscope apparatus according to one embodiment of the present disclosure includes an opening having a first slit and a second slit for passing a plurality of light beams from an observation object resulting from a plurality of irradiation light beams having different wavelengths irradiated onto the observation object, a dispersion element for wavelength-dispersing each of the plurality of light beams passing through the opening, and an image sensor for receiving the plurality of light beams wavelength-dispersed by the dispersion element. The image sensor receives the plurality of wavelength-dispersed light beams such that a zeroth-order light beam passing through the second slit and a first-order light beam passing through the first slit do not overlap with each other. [Brief description of the drawings]

[0010] [Figure 1] 1 is a schematic block diagram of a fluorescence observation apparatus according to an embodiment. [Diagram 2] FIG. 2 is a diagram showing an example of an optical system in a fluorescence observation device according to an embodiment. [Diagram 3] FIG. 2 is an explanatory diagram of light dispersion by a diffraction grating. [Figure 4] FIG. 2 is a schematic diagram of a pathological specimen which is an observation object in the embodiment. [Diagram 5] 5 is a schematic diagram showing a state of line illumination irradiating an observation object in the embodiment. FIG. [Figure 6] 3 is an enlarged view of the spectral imaging unit 30 in FIG. 2. [Figure 7] 11 is a graph showing the positions of the 0th order light and 1st order light of slit #1 and the 0th order light and 1st order light of slit #2 in an imaging section in an embodiment. [Figure 8] 4 is a graph showing a first example in the embodiment. [Figure 9] 3 is a schematic diagram showing a field of view of image pickup data captured by an image pickup unit in the embodiment. FIG. [Figure 10] 11 is a graph showing a second example in the embodiment. [Figure 11] 11 is a graph showing a simulation example of diffraction efficiency in the embodiment. [Figure 12] 11 is a graph showing a third example in the embodiment. [Figure 13] 13 is a graph showing a fourth example in the embodiment. [Figure 14] 13 is a graph showing a fifth example in the embodiment. [Figure 15] 13 is a graph showing a sixth example in the embodiment. [Figure 16] 13 is a graph showing a seventh example in the embodiment. [Figure 17] 4 is a graph for explaining limitations on the wavelength range to be observed in the embodiment. [Figure 18] 6 is a graph for explaining data correction relating to secondary light in the embodiment. [Figure 19] 6 is a graph for explaining a specific example of data correction relating to secondary light in the embodiment. [Figure 20] 6 is a graph for explaining data correction regarding −1st order light in the embodiment. [Figure 21] 11 is a graph for explaining a specific example of data correction in the embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, an embodiment of the present disclosure will be described in detail with reference to the drawings. In this embodiment, a fluorescence observation device that observes fluorescence from an observation object will be described as an example of a microscope device.

[0012] First, the configuration of a fluorescence observation device 100 according to an embodiment will be described with reference to Fig. 1 and Fig. 2. Fig. 1 is a schematic block diagram of the fluorescence observation device 100 according to an embodiment. Fig. 2 is a diagram showing an example of an optical system in the fluorescence observation device 100 according to an embodiment.

[0013] The fluorescence observation device 100 of this embodiment includes an observation unit 1. The observation unit 1 includes an excitation section 10, a stage 20, and a spectral imaging section 30 (hereinafter also referred to as a spectroscope). The excitation section 10 (illumination section) irradiates an observation object with a plurality of irradiation lights having different wavelengths. The excitation section 10 irradiates, for example, a pathology specimen (pathology sample) that is an observation object with a plurality of line lights having different wavelengths that are arranged parallel to different axes.

[0014] Here, "different axes and parallel" refers to multiple line illuminations that are different axes and parallel. Different axes refers to not being on the same axis, and the distance between the axes is not particularly limited. Parallel is not limited to parallel in the strict sense, but also includes a state of being approximately parallel. For example, deviation from the parallel state due to distortion from an optical system such as a lens or manufacturing tolerances is acceptable, and in such cases, the state is still considered to be parallel.

[0015] The stage 20 is a platform for supporting the pathological specimen. The spectroscopic imaging unit 30 acquires a fluorescence spectrum (spectroscopic data) of the pathological specimen excited in a line shape.

[0016] The fluorescence observation device 100 further includes a processing unit 2. The processing unit 2 typically forms an image of a pathological specimen (hereinafter also referred to as sample S) based on the fluorescence spectrum of the pathological specimen acquired by the observation unit 1, or outputs a distribution of the fluorescence spectrum. The image referred to here refers to the component ratio of the pigments constituting the spectrum, autofluorescence derived from the sample, etc., the waveform converted into RGB (red-green-blue) color, the luminance distribution of a specific wavelength band, etc.

[0017] The excitation unit 10 and the spectroscopic imaging unit 30 are connected to the stage 20 via an observation optical system 40, such as an objective lens 44. The observation optical system 40 has a function of tracking the optimal focus by a focus mechanism 60. A non-fluorescence observation unit 70 for performing dark-field observation, bright-field observation, etc. may be connected to the observation optical system 40.

[0018] Furthermore, the fluorescence observation device 100 may be connected to a control unit 80 that controls the excitation unit 10, the spectroscopic imaging unit 30, the scanning mechanism 50, the focus mechanism 60, the non-fluorescence observation unit 70, and the like.

[0019] The excitation unit 10 includes a plurality of light sources L1, L2, ... capable of outputting light of a plurality of excitation wavelengths Ex1, Ex2, .... The plurality of light sources are typically constituted by light-emitting diodes (LEDs), laser diodes (LDs), mercury lamps, etc., and each light is converted into line illumination and irradiated onto the sample S on the stage 20.

[0020] Here, FIG. 4 is a schematic diagram of a pathological specimen, which is an observation object in the embodiment. FIG. 5 is a schematic diagram showing the state of line illumination irradiated to the observation object in the embodiment. The sample S is typically composed of a slide including an observation object Sa, such as a tissue slice, as shown in FIG. 4, but may be other than that. The observation object Sa is, for example, a biological sample such as nucleic acid, cell, protein, bacteria, or virus. The sample S (observation object Sa) is stained with multiple fluorescent dyes. The observation unit 1 observes the sample S by enlarging it to a desired magnification. When the portion A in FIG. 4 is enlarged, the illumination area is arranged with multiple line illuminations (two (Ex1, Ex2) in the illustrated example) as shown in FIG. 5. Then, the imaging areas R1, R2 of the spectral imaging unit 30 are arranged so as to overlap with each illumination area. The two line illuminations Ex1, Ex2 are irradiated in parallel in the Z-axis direction, and are arranged at a predetermined distance (Δy) apart in the Y-axis direction.

[0021] The spectral imaging unit 30 also includes an opening 31, an image sensor 32, a prism 33, a mirror 34, and a diffraction grating 35. The opening 31 shown in Fig. 2 has a number of slits that pass through a number of light beams from the observation object resulting from a number of irradiation beams. The photographing areas R1 and R2 correspond to the slits of the opening 31. That is, the number of slits of the spectral imaging unit 30 is the same as that of the line illumination.

[0022] The wavelengths constituting the first line illumination Ex1 and the wavelengths constituting the second line illumination Ex2 are different from each other. Line-shaped fluorescence excited by these line illuminations Ex1 and Ex2 is observed by the spectral imaging unit 30 via the observation optical system 40.

[0023] The imaging element 32 (imaging section) captures (receives) a plurality of light beams (fluorescence, etc.) that have been wavelength-dispersed by a diffraction grating 35 (dispersion element). The imaging element 32 employs, for example, a two-dimensional imager such as a CCD (Charge Coupled Device) or a CMOS (Complementary Metal Oxide Semiconductor).

[0024] The spectral imaging section 30 acquires fluorescence spectral data (x, λ) from each of the line illuminators Ex1 and Ex2, using the pixel array in one direction (e.g., the vertical direction) of the image sensor 32 as a wavelength channel. The acquired spectral data (x, λ) is recorded in the processing unit 2 in a state where it is linked to the excitation wavelength from which each piece of spectral data was excited.

[0025] The processing unit 2 (correction section) processes the image data captured by the image sensor 32. For example, the processing unit 2 is realized by hardware elements used in a computer, such as a central processing unit (CPU), random access memory (RAM), and read only memory (ROM), and a necessary program (software). Instead of or in addition to the CPU, a programmable logic device (PLD) such as a field programmable gate array (FPGA), a digital signal processor (DSP), or an application specific integrated circuit (ASIC), etc. may be used.

[0026] The processing unit 2 has a storage unit 21. The storage unit 21 stores spectroscopic data representing a correlation between the wavelengths of the multiple line illuminations Ex1 and Ex2 and the fluorescence received by the image sensor 32. A storage device such as a non-volatile semiconductor memory or a hard disk drive is used for the storage unit 21, and a standard spectrum of the autofluorescence of the sample S and a standard spectrum of a dye alone that stains the sample S are stored in advance. The spectroscopic data (x, λ) received by the image sensor 32 is stored in the storage unit 21. In this embodiment, the storage unit that stores the autofluorescence of the sample S and the standard spectrum of the dye alone and the storage unit that stores the spectroscopic data (measurement spectrum) of the sample S acquired by the image sensor 32 are configured in the common storage unit 21, but are not limited to this and may be configured in separate storage units. In addition, the processing unit 2 can correct the optical information obtained from the light that has passed through the first slit based on the optical information obtained from the light that has passed through the second slit in the optical information obtained from each of the multiple wavelength-dispersed lights (details will be described later).

[0027] As shown in FIG. 2, a dichroic mirror 42 and a bandpass filter 45 are inserted in the optical path to prevent the excitation light (Ex1, Ex2) from reaching the image sensor 32.

[0028] The diffraction grating 35 is disposed on the optical path between the opening 31 and the image sensor 32. The diffraction grating 35 is an example of a wavelength dispersion element that wavelength-disperses the multiple light beams that have passed through the multiple slits of the opening 31. The diffraction grating 35 causes wavelength dispersion due to the coherence of light. In general, a diffraction grating has an advantage in that the wavelength dependency of the wavelength dispersion is almost constant, and it is easy to design the wavelength dispersion by the grating pitch. In other words, by using the diffraction grating 35, it is easy to design what kind of wavelength dispersion the light should have when it is incident on the image sensor 32.

[0029] Moreover, the diffraction grating 35 is, for example, a blazed diffraction grating in which the cross-sectional shape of the groove is sawtooth. By using a blazed diffraction grating, the amount of light can be concentrated on a specific order of light. For example, since the wavelength component of the second-order light can be suppressed, even if the first-order light of a certain slit overlaps with the second-order light of another slit, the influence of color mixing can be suppressed to a small extent, and the spectrum of each slit can be correctly acquired. Also, for example, even if the first-order light of a certain slit overlaps with the -1st order light of another slit #2, the blazed diffraction grating can suppress the -1st order light to a small extent relative to the first order light, so the influence is minor.

[0030] Here, Fig. 3 is an explanatory diagram of light dispersion by a diffraction grating. When a diffraction grating is used, for example, -3rd order light to 3rd order light are generated for incident light (wavelengths λ1 to λ2). Problems with the conventional technology will be described in detail below.

[0031] In fluorescence observation techniques using multiple excitation lights and multiple slits, it is necessary to separate the light using a spectroscope and observe using 1st order light and -1st order light. However, in reality, unsegregated 0th order light and undesired higher order light (2nd order light and higher) are generated. Furthermore, because each light that passes through multiple slits is separated, the diffracted light obtained from one slit overlaps with the diffracted light obtained from other slits in the imaging section, making it impossible to obtain a correct spectrum. This has been an issue that has not been apparent in the past, as most spectroscopes were configured with one slit per dispersing element.

[0032] For example, consider the case where there are two slits. Ideally, only the images of the two dispersed 1st order light (or -1st order light) would be projected onto the imager (imaging element), but in reality, 0th order light and higher order light are generated, so only the ideal 1st order light image is not necessarily obtained. For example, the 0th order light from slit #1 may overlap with the 1st order light (or -1st order light) from slit #2, or the higher order light (e.g. 2nd order light) from slit #1 may overlap with the 1st order light (or -1st order light) from slit #2. If there is such overlap, the spectrum of the 1st order light (or -1st order light) to be observed cannot be measured correctly.

[0033] In this embodiment, a method will be described that can obtain accurate (more accurate than conventional) imaging data in the imaging section by avoiding or reducing such overlap.

[0034] Returning to Figs. 1 and 2, the observation unit 1 further includes a scanning mechanism 50. The scanning mechanism 50 changes the position on the observation object where the illumination light is irradiated over time. For example, the scanning mechanism 50 can scan the stage 20 with the multiple line illuminations Ex1 and Ex2 in the Y-axis direction, that is, in the arrangement direction of the line illuminations Ex1 and Ex2. By driving the scanning mechanism 50, the image sensor 32 continuously captures images of multiple wavelength-dispersed lights in the scanning direction over time, and the processing unit 2 processes the image data captured by the image sensor 32 to obtain two-dimensional data or three-dimensional data on the observation object.

[0035] Specifically, by using the scanning mechanism 50, dye spectra (fluorescence spectra) excited by different excitation wavelengths spatially separated by Δy on the sample S (object of observation Sa) can be continuously recorded in the Y-axis direction.

[0036] In the examples so far, the line illumination as excitation light is configured with two lines, but the present invention is not limited to this and may be configured with three, four, five or more lines. Also, each line illumination may include a plurality of excitation wavelengths selected so as to minimize deterioration of color separation performance.

[0037] Next, the observation unit 1 will be described in detail with reference to Fig. 2. The excitation section 10 has a plurality of (four in this example) excitation light sources L1, L2, L3, and L4. Each of the excitation light sources L1 to L4 is composed of a laser light source that outputs laser light having a wavelength of 405 nm, 488 nm, 561 nm, and 645 nm, respectively.

[0038] The excitation section 10 further includes a plurality of collimator lenses 11, a laser line filter 12, dichroic mirrors 13a, 13b, and 13c, a homogenizer 14, a condenser lens 15, and an entrance slit 16 so as to correspond to the respective excitation light sources L1 to L4.

[0039] The laser light emitted from the excitation light source L1 and the laser light emitted from the excitation light source L3 are each collimated by a collimator lens 11, then transmitted through a laser line filter 12 for cutting the base of each wavelength band, and made coaxial by a dichroic mirror 13a. The two coaxial laser lights are further beam-shaped by a homogenizer 14 such as a fly-eye lens and a condenser lens 15 to become the line illumination Ex1.

[0040] The laser light emitted from the excitation light source L2 and the laser light emitted from the excitation light source L4 are also coaxialized by the dichroic mirrors 13b and 13c, and are converted into line illumination to become line illumination Ex2 having a different axis from the line illumination Ex1. The line illuminations Ex1 and Ex2 form different-axis line illuminations (primary images) separated by Δy in an entrance slit 16 (slit conjugate) having a plurality of slit portions through which each of the line illuminations can pass. Note that, in this embodiment, an example in which four lasers are two coaxial and two different-axis will be described, but other than this, two lasers may be configured as two different-axis configurations, or four lasers may be configured as four different-axis configurations.

[0041] This primary image is irradiated onto the sample S on the stage 20 via the observation optical system 40. The observation optical system 40 has a condenser lens 41, dichroic mirrors 42 and 43, an objective lens 44, a bandpass filter 45, and a condenser lens 46 (imaging lens). The line illuminations Ex1 and Ex2 are collimated by the condenser lens 41 paired with the objective lens 44, reflected by the dichroic mirrors 42 and 43, transmitted through the objective lens 44, and irradiated onto the sample S.

[0042] Illumination is formed on the surface S of the sample as shown in Fig. 5. Fluorescence excited by these illuminations is collected by the objective lens 44, reflected by the dichroic mirror 43, transmitted through the dichroic mirror 42 and a bandpass filter 45 that cuts the excitation light, collected again by the condenser lens 46, and enters the spectral imaging unit 30.

[0043] The aperture 31 is disposed at the focal point of the condenser lens 46, and has slits in the same number as the number of excitation lines. The fluorescence spectrum originating from the two excitation lines that passed through the aperture 31 is reflected by the prism 33 and the mirror 34, then wavelength-separated by the diffraction grating 35, and then reflected by the mirror 34 again to enter the image sensor 32.

[0044] The stage 20 and the scanning mechanism 50 constitute an XY stage, and the sample S is moved in the X-axis direction and the Y-axis direction to acquire a fluorescent image of the sample S. In WSI (Whole Slide Imaging), the sample S is scanned in the Y-axis direction, then moved in the X-axis direction, and then scanned again in the Y-axis direction, and this operation is repeated.

[0045] The non-fluorescence observation section 70 is composed of a light source 71, a dichroic mirror 43, an objective lens 44, a condenser lens 72, an image pickup element 73, etc. In the non-fluorescence observation system, an observation system using dark-field illumination is shown in FIG.

[0046] The light source 71 is disposed below the stage 20, and irradiates the sample S on the stage 20 with illumination light from the opposite side to the line illuminations Ex1 and Ex2. In the case of dark-field illumination, the light source 71 illuminates from outside the NA (numerical aperture) of the objective lens 44, and the light diffracted by the sample S (dark-field image) is captured by the image sensor 73 via the objective lens 44, the dichroic mirror 43, and the condenser lens 72. By using dark-field illumination, even seemingly transparent samples such as fluorescently stained samples can be observed with contrast.

[0047] The dark-field image may be observed simultaneously with the fluorescence and used for real-time focusing. In this case, the illumination wavelength may be selected so as not to affect the fluorescence observation. The non-fluorescence observation unit 70 is not limited to an observation system that acquires a dark-field image, but may be configured with an observation system that can acquire non-fluorescence images such as bright-field images, phase-contrast images, phase images, and in-line hologram images. For example, various observation methods such as the Schlieren method, phase-contrast method, polarized observation method, and epi-illumination method can be adopted as a method for acquiring a non-fluorescence image. The position of the illumination light source is not limited to below the stage, but may be above the stage or around the objective lens. In addition to a method of performing focus control in real time, other methods such as a pre-focus map method in which focus coordinates (Z coordinates) are recorded in advance may be adopted.

[0048] The fluorescence spectrum acquired by the image sensor 32 is output to the processing unit 2. In addition to the storage unit 21, the processing unit 2 includes a data calibration unit 22 and an image formation unit 23. The data calibration unit 22 calibrates the spectroscopic data stored in the storage unit 21. The image formation unit 23 forms a fluorescence image of the sample S based on the spectroscopic data and the interval Δy between the multiple line illuminations Ex1 and Ex2.

[0049] The display unit 3 displays various information created by the processing unit 2. The display unit 3 may be configured, for example, as a monitor integrally attached to the processing unit 2, or may be a display device connected to the processing unit 2. The display unit 3 includes, for example, a display element such as a liquid crystal device or an organic EL device, and a touch sensor, and is configured as a UI (User Interface) that displays input settings of shooting conditions, shot images, etc.

[0050] The structure of the observation unit 1 for obtaining accurate imaging data with the imaging element 32 will be described in detail below. The observation unit 1 is configured such that, for the multiple wavelength-dispersed lights, the zeroth-order light of any light does not overlap with the first-order light of other lights at the imaging element 32. Note that, for the multiple wavelength-dispersed lights, it is preferable that the wavelength band of the first-order light of at least any of the lights includes part or all of 400 to 800 nm. This is because the wavelength of fluorescence mainly used in fluorescent observation of living organisms is 400 to 800 nm. However, the wavelength band is not limited to this.

[0051] FIG. 6 is an enlarged view of the spectral imaging section 30 in FIG. 2. Here, an example is shown in which an Offner type is adopted as the spectral imaging section 30. FIG. 7 is a graph showing the positions of the zeroth order light and the first order light of slit #1 (first slit) and the zeroth order light and the first order light of slit #2 (second slit) in the imaging section (image sensor 32) in the embodiment. The horizontal axis is the Y coordinate position in the image sensor 32, and the vertical axis is the light intensity (I). For example, the zeroth order light of the light that has passed through slit #1 is referred to as the "zeroth order light of slit #1", and the same applies to other lights.

[0052] Mirror 34 is a concave mirror having an inner reflective surface with a radius of curvature R. Diffraction grating 35 is a reflective diffraction grating (convex mirror) having an outer reflective surface with a radius of curvature R / 2. There are two slits in aperture 31, slit #1 and slit #2. The distance between slit #1 and slit #2 is L.

[0053] For the zero-order light of slit #1, the y coordinate position on the imaging element 32 is defined as y1. For the zero-order light of slit #2, the y coordinate position on the imaging element 32 is defined as y2.

[0054] For the first-order light of slit #1, the y-coordinate position in the image sensor 32 is set to y11 to y12, and the wavelength band is set to λ min1 ~λ max1 For the first-order light of slit #2, the y-coordinate position in the image sensor 32 is set to y21 to y22, and the wavelength band is set to λ min2 ~λ max2 In addition, the diffraction order is defined as m.

[0055] In this case, y11, y12, y21, and y22 are expressed as in the following formulas (1) to (4).

number

[0056] Moreover, when considering the first-order light, since the diffraction order m=1, y11, y12, y21, and y22 are expressed by the following formulas (5) to (8).

number

[0057] 8 is a graph showing a first example of the embodiment. The first example is an example in which the observation unit 1 is configured so that the zeroth order light of slit #2 is positioned between the zeroth order light and the first order light of slit #1 in the image sensor 32. In addition, L=y2-y1.

[0058] The condition for this is as shown in the following equation (9).

number

[0059] Subtracting y1 from each term in equation (9) gives the following equation (10), which is also the condition for the slit spacing L.

number

[0060] Thus, according to the first example, for slits #1 and #2, the zero-order light of one and the first-order light of the other do not overlap at the image sensor 32, so accurate imaging data can be obtained at the image sensor 32. In other words, more accurate (less noisy) diffraction data of the first-order light can be obtained.

[0061] 9 is a schematic diagram showing the field of view of image data captured by the imaging section (image sensor 32) in the embodiment. In this embodiment, the slit interval is set to be equal to or less than the diameter of the field of view of image data captured by image sensor 32, which is defined by objective lens 44 and condenser lens 46. Moreover, the first example is a configuration that can make the slit interval between slits #1 and #2 the narrowest, compared with the second to seventh examples below. The imaging performance is better near the center of the field of view, and by making the slit interval narrower, the imaging performance can be further improved.

[0062] In particular, fluorescent materials with fluorescence peaks of around 400 nm to 800 nm (for example, fluorescent dye Indo-1 fluoresces around 400 nm, and APC Alexa Fluor 750 fluoresces around 800 nm) are often used in fluorescent observation of living organisms. Therefore, by making the wavelength band of the primary light of at least one of the multiple wavelength-dispersed lights include part or all of the wavelength band of 400 to 800 nm, the accuracy of fluorescent observation of living organisms can be improved.

[0063] Although the configuration using 1st order light has been illustrated, the same applies to the case where -1st order light is used.

[0064] Next, a second example will be described. Fig. 10 is a graph showing a second example in the embodiment. The second example is an example in which the observation unit 1 is configured so that the zero-order light of slit #2 is located closer to the second-order light side than the first-order light of slit #1 in the image sensor 32.

[0065] The condition for this is as shown in the following formula (11).

number

[0066] Furthermore, by subtracting y1 from each term in equation (11) and assuming that the diameter of the observation field of view is D, the condition for the slit interval L (=y2-y1) is given by the following equation (12).

number

[0067] Thus, according to the second example, for slits #1 and #2, the zero-order light of one and the first-order light of the other do not overlap at the image sensor 32, so accurate imaging data can be obtained at the image sensor 32. In other words, more accurate (less noisy) diffraction data of the first-order light can be obtained.

[0068] As described above, if a blazed diffraction grating is used as the diffraction grating 35, the amount of light can be concentrated on a specific order of light. For example, the wavelength components of the second-order light can be suppressed. Therefore, for example, the second-order light of slit #1 partially overlaps with the first-order light of slit #2, but the effect is minor.

[0069] Here, Fig. 11 is a graph showing a simulation example of the diffraction efficiency in the embodiment. It can be seen that the diffraction efficiency of the first-order light is high, and the diffraction efficiency of the second-order light and the diffraction efficiency of the third-order light are low. Note that the diffraction efficiency of the second-order light is somewhat high on the short wavelength side, but as shown in Fig. 10, the portion where the second-order light of slit #1 overlaps with the first-order light of slit #2 is on the long wavelength side, so the effect is small, and an accurate spectrum can be obtained with little effect of color mixing. In addition, when a blazed diffraction grating is used, the diffraction efficiency of -1st order light to -3rd order light can be made negligibly small.

[0070] In addition, the second example has a configuration that allows the slit spacing between slits #1 and #2 to be narrower, although not as much as the first example, compared to the third to seventh examples below, and can achieve high imaging performance.

[0071] The third to seventh examples will be described below, but as the mathematical expressions are similar to those in the first and second examples, the description will be omitted.

[0072] A third example will be described. Fig. 12 is a graph showing a third example in the embodiment. In comparison with the second example, the third example is an example in which the observation unit 1 is configured so that the second order light of slit #1 does not overlap with the first order light of slit #2, but the third order light of slit #1 overlaps with it.

[0073] Generally, the higher the order of diffracted light, the lower the diffraction efficiency, so by overlapping the 1st order light from slit #2 with the 3rd order light from slit #1 instead of the 2nd order light, the accuracy of the imaging data can be further improved. The slit interval may be determined within a range that ensures sufficient imaging performance.

[0074] Next, a fourth example will be described. Fig. 13 is a graph showing a fourth example in the embodiment. The graph itself is similar to Fig. 10. Here, a method of suppressing the influence of color mixing of higher-order light between slits by data correction is considered. For example, the wavelengths and intensities of overlapping higher-order light are evaluated (calculated) in advance for the first-order light of slit #1 (area A) and the first-order light of slit #2 (area B). By correcting the data of the mixed color portion based on this evaluation, it becomes possible to obtain a correct spectrum even if there is color mixing.

[0075] Although color mixing between multiple slits has been described here, overlapping of secondary light and primary light may occur for one slit. This can be dealt with by evaluating the overlapping parts in advance and correcting the data as in the above-mentioned method, or by using an optical filter to limit the wavelength band of the fluorescence from the observation object that reaches the image sensor 32. These methods may also be combined.

[0076] Next, a fifth example will be described. Fig. 14 is a graph showing a fifth example in an embodiment. Although the case where there are two slits has been described so far, there may be three or more slits. The example in Fig. 14 shows a case where there are three slits in the first example. The first order light of slit #1 is placed between the zeroth order light of slit #2 and the zeroth order light of slit #3.

[0077] The intervals between the three first-order beams do not necessarily have to be the same, but can be set to be different. Therefore, the slit intervals do not have to be constant, but can be different.

[0078] Thus, according to the fifth example, even when there are three slits, any zero-order light does not overlap with other first-order lights, so accurate imaging data can be obtained by the imaging element 32. In other words, more accurate (less noisy) diffraction data of the first-order light can be obtained.

[0079] Next, a sixth example will be described. Fig. 15 is a graph showing a sixth example in an embodiment. The example in Fig. 15 shows a case where the number of slits in the first example is four. The first order light of slit #2 is placed between the zeroth order light of slit #3 and the zeroth order light of slit #4.

[0080] Thus, according to the sixth example, even when there are four slits, any zero-order light does not overlap with other first-order lights, so accurate imaging data can be obtained by the imaging element 32. In other words, more accurate (less noisy) diffraction data of first-order light can be obtained. Note that the same can be achieved when there are five or more slits.

[0081] Next, a seventh example will be described. Fig. 16 is a graph showing a seventh example in the embodiment. In the example of Fig. 16, the zeroth order light of slit #2 is arranged in the second order light region of slit #1, and the zeroth order light of slit #3 is arranged in the second order light region of slit #2.

[0082] The intervals between the three first-order beams do not necessarily have to be the same, but can be set to be different. Therefore, the slit intervals do not have to be constant, but can be different.

[0083] Thus, according to the seventh example, even when there are three slits, any zero-order light does not overlap with other first-order lights, so accurate imaging data can be obtained by the imaging element 32. In other words, more accurate (less noisy) diffraction data of the first-order light can be obtained. Note that the same can be achieved when there are four or more slits.

[0084] 17 is a graph for explaining the limitation of the wavelength range to be observed in the embodiment. The observation unit 1 may further include an optical filter that passes only light in a predetermined wavelength range of the obtained fluorescence.

[0085] In some cases, the actual fluorescence from the object of observation is spread over a wide band. Therefore, the wavelength range of the observation can be limited by using an optical filter or adjusting the sensitivity of the light receiving element. Specifically, the following (A) to (C) can be considered.

[0086] (A) An optical filter is placed in a portion of the emission spectrum to limit the wavelength. (B) The observation wavelength is restricted by utilizing the sensitivity characteristics of the light receiving element. (C) Combine (A) and (B).

[0087] With these, for example, as shown in FIG. 17, the fluorescence in area B1 can be restricted and acquired as data for area B2.

[0088] Next, Fig. 18 is a graph for explaining data correction related to secondary light in the embodiment. The horizontal axis is wavelength, and the vertical axis is light intensity. The processing unit 2 can estimate the amount of secondary light generated from the amount of primary light for the same light.

[0089] By acquiring the diffraction characteristics of the diffraction grating 35 in advance, it is possible to estimate the amount of second-order light generated from the amount of first-order light from slit #1, for example. In other words, for example, when the first-order light from slit #2 overlaps with the second-order light from slit #1, the signal of the first-order light from slit #2 can be acquired more accurately by data processing.

[0090] Specifically, the efficiency of secondary light is different from that of primary light, but the same waveform appears in a wavelength region twice as large. Therefore, by evaluating the occurrence rate of secondary light relative to primary light in advance, it is possible to calculate how much secondary light is generated in actual measurements. In the following formula (13), the left side indicates the secondary light intensity, the first term on the right side indicates the primary light intensity, and the second term on the right side indicates the secondary light occurrence rate.

number

[0091] In this way, the secondary light can be calculated (estimated) with high accuracy from the measurement results of the primary light, thereby enabling highly accurate data correction.

[0092] Next, Fig. 19 is a graph for explaining a specific example of data correction related to secondary light in the embodiment. An example of a method for evaluating the secondary light appearance rate will be explained. For example, a filter that blocks light in the secondary light region to be evaluated is placed in a light source having broad emission wavelength characteristics such as a halogen lamp. This light is introduced individually into the slit of the spectroscope. In this way, the ratio of the secondary light to the primary light can be evaluated, and the appearance rate of the secondary light can be evaluated.

[0093] In Fig. 19(a), C1 indicates the original light source spectrum, C2 indicates the filtered light source spectrum, and C3 indicates the secondary light appearance rate evaluation area. In Fig. 19(b), C4 indicates the filtered light source spectrum, and C5 indicates the filtered secondary light of the light source. The ratio of C4 to C5 is the appearance rate of the secondary light.

[0094] In addition to this method, there is also a method of obtaining the appearance rate of secondary light for each wavelength by using a method of continuously extracting monochromatic wavelengths from a high-luminance white light source.

[0095] Next, Fig. 20 is a graph for explaining data correction regarding -1st order light in an embodiment. Although the efficiency of -1st order light is different from that of 1st order light, the same waveform z appears symmetrically with respect to the 0th order light. By evaluating the occurrence rate of -1st order light with respect to 1st order light in advance, it is possible to calculate how much -1st order light is generated in actual measurement. In the following formula (14), the left side indicates the -1st order light intensity, the first term on the right side indicates the 1st order light intensity, and the second term on the right side indicates the occurrence rate of -1st order light.

number

[0096] An example of a method for evaluating the occurrence rate of -1st order light relative to 1st order light will be described. When broadband light such as from a halogen lamp is introduced into one slit, the spectrum of the dispersed 1st order light appears in the data acquired from the image sensor 32. At the same time, -1st order light is generated. This information makes it possible to evaluate the occurrence ratio of -1st order light relative to 1st order light. This is evaluated for each slit.

[0097] In addition to this method, there is also a method of obtaining the occurrence rate of −1st order light for each wavelength by continuously extracting monochromatic wavelengths from a high-brightness white light source.

[0098] Next, FIG. 21 is a graph for explaining a specific example of data correction in the embodiment. Here, data correction in the case of FIG. 12 will be explained. D is the light intensity in the D region, which is actual measurement data. I1 is the first-order light intensity of slit #1. I2 is the first-order light intensity of slit #2. β -1 is the −1st order light appearance rate of slit #2, and is a correction coefficient that is evaluated in advance.

[0099] Also, I Eis the light intensity in the E region, which is actual measurement data. α3 is the third-order light appearance rate of slit #1, which is a correction coefficient evaluated in advance. I1 and I2 can be calculated using the following formulas (15) and (16).

number

[0100] In addition, β -1 Regarding the pre-evaluation of α3, it is possible to apply the evaluation method of the 2nd order light described in FIG. 18 or the evaluation method of −1st order light described in FIG.

[0101] In this manner, according to the observation unit 1 of this embodiment, for multiple wavelength-dispersed light beams, the imaging element 32 is configured so that the zeroth light of any light does not overlap with the first order light of other light beams, thereby making it possible to obtain accurate imaging data at the imaging element 32.

[0102] For example, in a conventional line-scan type fluorescence imaging device, a fluorescence observation method using multiple excitation lights and multiple slits had a problem in that the light from the multiple slits mixed with each other, making it impossible to obtain accurate spectral data. In contrast, by using the method of this embodiment, it is possible to obtain more accurate data with reduced color mixing.

[0103] Conventionally, one spectrometer could only disperse light for one line (or point). Therefore, in order to simultaneously obtain spectroscopic data for multiple lines (or points), the only option was to place multiple spectrometers. This was a complicated system, and it also placed significant constraints on the physical layout. In reality, it is very difficult to configure an observation system such as a microscope with multiple spectrometers.

[0104] In contrast, according to this embodiment, accurate spectroscopy of multiple lines (or points) is possible with one spectroscope (diffraction grating 35, image sensor 32, etc.). This simplifies the device, enables a compact layout, and enables simultaneous spectroscopy of multiple lines. In terms of fluorescence observation, it used to take a lot of time to scan the entire surface with one line and take images repeatedly for the number of excitation wavelengths, but if different excitation wavelengths are used for each of the multiple lines, it is possible to complete the imaging of multiple color excitations with a single scan. In other words, it is possible to significantly shorten the observation time.

[0105] In addition, in this embodiment, specifically, for example, as shown in FIG. 9, the observation unit 1 is configured so that the zeroth order light of the light that has passed through one slit is positioned between the zeroth order light and the first order light of the light that has passed through the other slit, thereby achieving the above-mentioned effect.

[0106] Furthermore, for example, the above-mentioned effects can be achieved by configuring the observation unit 1 so that the zero-order light of the light that has passed through one slit is positioned closer to the second-order light side than the first-order light of the light that has passed through the other slit, as shown in Figure 10.

[0107] In addition, by correcting the imaging data obtained from any one of the multiple wavelength-dispersed lights based on the imaging data obtained from the other lights, the accuracy of the imaging data can be further improved.

[0108] Furthermore, if an optical filter that passes only light in a predetermined wavelength range from the obtained fluorescent light is further used, the accuracy of the imaging data can be further improved.

[0109] Furthermore, by making the wavelength band of the primary light of at least one of the multiple wavelength-dispersed lights include part or all of 400 to 800 nm, it is more effective when the observation object is a living organism.

[0110] Furthermore, by using a diffraction grating as a dispersive element, it is possible to easily design what wavelength dispersion the light incident on the imaging element 32 will have.

[0111] Furthermore, by using a blazed diffraction grating as the diffraction grating 35, the amount of light can be concentrated on a specific order of light, and even if there is a region where higher order light overlaps with the first order light, the effect can be kept small.

[0112] Furthermore, by setting the slit interval of the opening 31 to be equal to or smaller than the diameter of the visual field of the imaging data, imaging data using a plurality of excitation lights can be obtained accurately.

[0113] Furthermore, by having the scanning mechanism 50, it is possible to obtain two-dimensional or three-dimensional data about the object to be observed.

[0114] Furthermore, a fluorescence observation system (microscope system) is configured by including the fluorescence observation device 100 and a program used for processing the imaging data. This program is a program for causing an information processing device to execute a correction process for processing the imaging data.

[0115] This program is provided in a state stored in a recording medium such as a magnetic disk, an optical disk, a magneto-optical disk, or a flash memory, and is used by being downloaded to a computer or the like connected to the fluorescence observation device 100.

[0116] This program may also be downloaded to an external computer or the like via a network such as the Internet for use.

[0117] The present technology can also be configured as follows. (1) an opening having a first slit and a second slit for passing a plurality of light beams from an observation object, the light beams being caused by a plurality of irradiation light beams having different wavelengths irradiated onto the observation object; a dispersion element that performs wavelength dispersion on each of the plurality of light beams that have passed through the opening; an image sensor that receives the plurality of light beams that have been wavelength-dispersed by the dispersion element; The imaging element receives the plurality of wavelength-dispersed light beams such that zero-order light passing through the second slit and first-order light passing through the first slit do not overlap. (2) The microscope apparatus according to (1), further comprising an illumination unit that illuminates the object to be observed with a plurality of illumination lights having different wavelengths. (3) The microscope apparatus according to (1), further comprising a correction unit that processes image data captured by the image sensor. (4) The microscope apparatus according to (1), wherein the imaging element receives light such that a zeroth order light of the light that has passed through the second slit is positioned between a zeroth order light of the light that has passed through the first slit and a first order light of the light that has passed through the first slit. (5) The microscope apparatus according to (1), wherein the imaging element receives light such that the zeroth order light of the light that has passed through the second slit is located on the second order light side relative to the first order light of the light that has passed through the first slit. (6) The microscope device described in (3), wherein the correction unit corrects the optical information obtained from the light that has passed through the first slit based on the optical information obtained from the light that has passed through the second slit, in the optical information obtained from each of the multiple wavelength-dispersed light beams. (7) The microscope apparatus according to (1), further comprising an optical filter that passes only light within a predetermined wavelength range from the plurality of light beams from the object to be observed. (8) The microscope apparatus according to (1), wherein a wavelength band of a first-order light of at least any of the plurality of wavelength-dispersed light beams includes a part or all of 400 to 800 nm. (9) The microscope apparatus according to (1), wherein the dispersive element is a diffraction grating that causes wavelength dispersion due to optical coherence. (10) The microscope apparatus according to (9), wherein the diffraction grating is a blazed diffraction grating in which the cross-sectional shape of the grooves is sawtooth. (11) The imaging element receives a plurality of fluorescent lights obtained when the object to be observed is irradiated with a plurality of excitation lights having different wavelengths. (12) The microscope apparatus according to (1), wherein the slit spacing of the opening is equal to or smaller than the field diameter of the imaging data captured by the imaging element, as determined by the objective lens and the imaging lens. (13) a scanning mechanism for changing a position on the object to be observed where the irradiation light is irradiated over time, The microscope device described in (3) above, wherein by driving the scanning mechanism, the image sensor continuously captures the wavelength-dispersed multiple light beams in a scanning direction over time, and the correction unit processes the image data captured by the image sensor to obtain two-dimensional data or three-dimensional data about the object to be observed. (14) The microscope apparatus according to (2), wherein the irradiation unit irradiates excitation light, which is the plurality of irradiation lights having different wavelengths, in a line shape. (15) A spectroscope in a microscope apparatus, an opening having a first slit and a second slit for passing a plurality of light beams from an observation object, the light beams being caused by a plurality of irradiation light beams having different wavelengths irradiated onto the observation object; a dispersion element that performs wavelength dispersion on each of the plurality of light beams that have passed through the opening; an image sensor that receives the plurality of light beams that have been wavelength-dispersed by the dispersion element; The imaging element receives the plurality of wavelength-dispersed light beams such that zero-order light passing through the second slit and first-order light passing through the first slit do not overlap. (16) A microscope system including the microscope device according to any one of (1) to (14) and a program used to process imaging data captured by the microscope device, The program is a program for causing an information processing device to execute a correction process for processing the imaging data.

[0118] Although the embodiments of the present disclosure have been described above, the technical scope of the present disclosure is not limited to the above-described embodiments, and various modifications are possible without departing from the gist of the present disclosure.

[0119] 6 shows an example in which an Offner type is used as the spectral imaging unit 30, and the radius of curvature of the outer reflecting surface of the diffraction grating 35 is set to half the radius of curvature of the inner reflecting surface of the mirror 34, but the present invention is not limited to this. Depending on the design, the ratio of the radii of curvature may be other than 2:1.

[0120] Furthermore, the effects of each embodiment described in this specification are merely examples and are not limiting, and other effects may also be provided. [Explanation of symbols]

[0121] 1...observation unit, 2...processing unit, 3...display unit, 10...excitation unit, 11...collimator lens, 12...laser line filter, 13...dichroic mirror, 14...homogenizer, 15...condenser lens, 16...entrance slit, 20...stage, 21...storage unit, 22...data calibration unit, 23...image formation unit, 30...spectral imaging unit, 31...aperture, 32...image sensor, 33...prism, 34...mirror, 35...diffraction grating, 40...observation optical system, 41...condenser lens, 42...dichroic mirror, 43...dichroic mirror, 44...objective lens, 45...bandpass filter, 46...condenser lens, 50...scanning mechanism, 60...focus mechanism, 70...non-fluorescence observation unit, 80...control unit

Claims

1. an opening having a first slit and a second slit for passing a plurality of light beams from the observation object, the light beams being caused by a plurality of irradiation light beams having different wavelengths and irradiated onto the observation object; a dispersion element that performs wavelength dispersion on each of the plurality of light beams that have passed through the opening; an image sensor that receives the plurality of light beams that have been wavelength-dispersed by the dispersion element; The imaging element receives the zeroth order light and the first order light of the light that has passed through the second slit and the zeroth order light and the first order light of the light that has passed through the first slit such that the zeroth order light and the first order light of the light that has passed through the second slit do not overlap with the zeroth order light and the first order light of the light that has passed through the first slit, a microscope device.

2. The microscope apparatus of claim 1, wherein the imaging element receives zeroth-order and first-order light of the light that has passed through the second slit and zeroth-order, first-order and second-order light of the light that has passed through the first slit, such that zeroth-order light of the light that has passed through the second slit overlaps with second-order light of the light that has passed through the first slit, for the multiple wavelength-dispersed light beams.

3. The microscope apparatus of claim 1, wherein the imaging element receives zeroth-order and first-order light of the light that has passed through the second slit and zeroth-order, first-order and second-order light of the light that has passed through the first slit, such that secondary light of the light that has passed through the first slit does not overlap with primary light of the light that has passed through the second slit, for the multiple wavelength-dispersed light beams.

4. The microscope apparatus of claim 3, wherein the imaging element receives, for the multiple wavelength-dispersed light beams, zeroth-order and first-order light that have passed through the second slit and zeroth-order, first-order, second-order and third-order light that have passed through the first slit such that third-order light that has passed through the first slit overlaps with first-order light that has passed through the second slit.

5. The microscope apparatus of claim 4, wherein the imaging element receives, for the multiple wavelength-dispersed light beams, zeroth order light, first order light and -1st order light that has passed through the second slit and zeroth order light, first order light, second order light and third order light that has passed through the first slit such that the first order light of the light that has passed through the first slit overlaps with the -1st order light of the light that has passed through the second slit.

6. The opening has a third slit that passes the light from the observation object, 2. The microscope device of claim 1, wherein the imaging element receives the zeroth order light and the first order light of the light that has passed through the second slit, the zeroth order light and the first order light of the light that has passed through the first slit, and the zeroth order light and the first order light of the light that has passed through the third slit, such that the zeroth order light and the first order light of the light that has passed through the second slit, the zeroth order light and the first order light of the light that has passed through the first slit, and the zeroth order light and the first order light of the light that has passed through the third slit do not overlap with each other.

7. The opening has a fourth slit that passes the light from the observation object, 7. The microscope apparatus of claim 6, wherein the imaging element receives, for the plurality of wavelength-dispersed light beams, the zeroth-order light and the first-order light of the light that has passed through the second slit, the zeroth-order light and the first-order light of the light that has passed through the first slit, the zeroth-order light and the first-order light of the light that has passed through the third slit, and the zeroth-order light and the first-order light of the light that has passed through the fourth slit, such that the zeroth-order light and the first-order light of the light that has passed through the second slit, the zeroth-order light and the first-order light of the light that has passed through the first slit, the zeroth-order light and the first-order light of the light that has passed through the third slit, and the zeroth-order light and the first-order light of the light that has passed through the fourth slit do not overlap.

8. The microscope apparatus of claim 6, wherein the imaging element receives zeroth-order light, first-order light and second-order light of the light that has passed through the second slit, zeroth-order light, first-order light and second-order light of the light that has passed through the first slit, and zeroth-order light and first-order light of the light that has passed through the third slit, such that, for the multiple wavelength-dispersed light beams, the zeroth-order light of the light that has passed through the second slit overlaps with the second order light of the light that has passed through the first slit, and the zeroth-order light of the light that has passed through the third slit overlaps with the second order light of the light that has passed through the second slit.

9. The microscope apparatus according to claim 1 , further comprising an illumination unit that illuminates the object to be observed with a plurality of illumination lights having different wavelengths.

10. The microscope apparatus according to claim 1 , further comprising a correction unit that processes image data captured by the image sensor.

11. The microscope device according to claim 10, wherein the correction unit corrects optical information obtained from the light that has passed through the first slit based on optical information obtained from the light that has passed through the second slit, in the optical information obtained from each of the plurality of wavelength-dispersed light beams.

12. The microscope apparatus according to claim 1 , further comprising an optical filter that passes only light in a predetermined wavelength range from the plurality of light beams from the observation object.

13. 2. The microscope apparatus according to claim 1, wherein a wavelength band of primary light of at least any of the plurality of wavelength-dispersed light beams includes part or all of 400 to 800 nm.

14. 2. The microscope apparatus according to claim 1, wherein the dispersive element is a diffraction grating that generates wavelength dispersion due to optical coherence.

15. A spectroscope in a microscope apparatus, an opening having a first slit and a second slit for passing a plurality of light beams from the observation object, the light beams being caused by a plurality of irradiation light beams having different wavelengths and irradiated onto the observation object; a dispersion element that performs wavelength dispersion on each of the plurality of light beams that have passed through the opening; an image sensor that receives the plurality of light beams that have been wavelength-dispersed by the dispersion element; The imaging element is a spectroscope that receives the zeroth order light and the first order light of the light that has passed through the second slit and the zeroth order light and the first order light of the light that has passed through the first slit, such that the zeroth order light and the first order light of the light that has passed through the second slit do not overlap with the zeroth order light and the first order light of the light that has passed through the first slit, for the multiple wavelength-dispersed light beams.

16. 15. A microscope system including the microscope apparatus according to claim 1 and a program used to process image data captured by the microscope apparatus, The program is a program for causing an information processing device to execute a correction process for processing the imaging data.

Images