JP2015166763A - Microscope device, and analysis method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a microscope device that can classify an object with high precision and make an undyed analysis.SOLUTION: A microscope device includes: a light source which irradiates an object with light; image forming means of forming an image of the object on a primary image plane; imaging means including a detector on which luminous flux from the image is dispersed and made incident through image-capturing and which acquires a signal based upon the incident luminous flux and moving means of moving an image-capturing position in a predetermined direction; control means of controlling a timing of image-capturing; control means of controlling the moving means; means of generating first data having one-dimensional space information and high-resolution wavelength information at the image-capturing position based upon a signal, and also generating second data having two-dimensional space information and one-dimensional wavelength information from the first data at each image-capturing position; means of creating a spectral image for each wavelength from the second data; means of acquiring a spectral radiance, a spectral luminance, and a spectral reflectivity or spectral transmittance of each pixel of the spectral image; and means of analyzing the spectral image by a predetermined analyzing method.

Description

  The present invention relates to a microscope apparatus and an analysis method, and more particularly to a microscope apparatus capable of acquiring spectral information of an observation object and an analysis method using the microscope apparatus.

  In general, as a microscope apparatus for observing an object, for example, the object is enlarged using visible light and light in a wavelength region in the vicinity thereof, and a person visually recognizes details of the object and a minute object. It is known to be able to.

  In such a microscope apparatus, transmitted light from an object obtained by irradiating light from a light source to an object to be observed (hereinafter, “object to be observed” is simply referred to as “object”), Observe the reflected light or radiated light (hereinafter referred to as “transmitted light, reflected light or radiated light from the object” as appropriate, “light flux from the object”) directly, The image of the light beam from the object is observed with the imaging device, the object is observed, and processing such as classification and discrimination of the object (hereinafter referred to as "classification and discrimination, etc." Is simply referred to as “classification processing” as appropriate).

However, the basis of the classification processing of the object is that only monochrome or RGB composite information can be obtained as color information (spectral information) in both cases of direct visual observation and visual inspection of an image. Absent. For this reason, it has been pointed out as a problem that classification processing is difficult for approximate color information, and the accuracy of classification processing is limited.

  In addition, when observing an object with poor color information (for example, a living cell, a tissue section, a crystal, etc.), the microscope apparatus usually stains the object to classify the structure and properties of the object. It is necessary to perform such processing.

However, when such treatments as staining are performed, the state of the object (for example, living cells, tissue slices, crystals, etc.) deteriorates and changes in properties, so that the state of the object in real life (for example, biological activity) It has been pointed out as a problem that it is impossible to observe over time a living state cell that maintains the condition or a tissue constructed by a living cell.

  Note that the prior art that the applicant of the present application knows at the time of filing a patent application is not an invention related to a known literature invention, and therefore there is no prior art document information to be described in the present specification.

  The present invention has been made in view of the various problems of the conventional techniques as described above. The object of the present invention is to acquire high wavelength resolution spectral information in a wide range of the object, An object of the present invention is to provide a microscope apparatus that can classify and discriminate objects with high accuracy.

  In addition, an object of the present invention is to provide a microscope apparatus capable of observing an object (for example, a living cell, a tissue slice, a crystal, etc.) over time without causing state deterioration or property change. To do.

  Another object of the present invention is to provide an analysis method for analyzing an object without staining.

  In order to achieve the above object, a microscope apparatus according to the present invention includes a light source that irradiates light on an object and an imaging optical that forms an image of the object on a primary image plane by a light beam incident through an objective lens. Means and two-dimensional imaging in which a light beam from the image incident through a lens is incident by being dispersed in a direction orthogonal to a predetermined direction by a dispersion optical element and a signal based on the incident light beam is acquired by photographing An imaging means comprising a detector and a moving means for moving an imaging position imaged by the two-dimensional imaging detector in the predetermined direction; and a first timing for controlling the imaging timing of the two-dimensional imaging detector. Based on the control means, the second control means for controlling the movement of the moving means, and the signal, two-dimensional spectroscopic data having one-dimensional spatial information and one-dimensional wavelength information at the photographing position is created. , Spectral data creation means for creating three-dimensional spectral data having two-dimensional spatial information and one-dimensional wavelength information from the two-dimensional spectral data at the photographing position, and an image for creating a spectral image for each wavelength from the three-dimensional spectral data Creating means; acquisition means for acquiring spectral radiance, spectral brightness and spectral reflectance or spectral transmittance in each pixel of the spectral image; and analysis processing means for analyzing the spectral image by a predetermined analysis method. It is what I did.

  The one-dimensional wavelength information is a wavelength range from ultraviolet to infrared (for example, 200 nm to 13 μm) including visible light visible to humans, with a wavelength resolution of 0.1 nm to 100 nm. High-wavelength-resolved spectroscopic information (hyperspectral data) that splits into several hundred bands was used.

  In addition, a microscope apparatus according to the present invention includes a light source that irradiates light on an object, and a microscope unit that includes an imaging optical unit that forms an image of the object on a primary image plane by a light beam incident through an objective lens. A two-dimensional imaging detector that obtains a signal based on the incident light flux by dispersing the light flux from the image incident through the lens by photographing in a direction orthogonal to a predetermined direction by the dispersion optical element. And a moving means for moving a photographing position photographed by the two-dimensional imaging detector in the predetermined direction, and an imaging means detachably disposed on the microscope unit, and the two-dimensional imaging detector First control means for controlling the timing of photographing, second control means for controlling movement of the moving means, and one-dimensional spatial information and one-dimensional wavelength information at the photographing position based on the signal. 2D spectroscopy A spectral data generating means for generating three-dimensional spectral data having two-dimensional spatial information and one-dimensional wavelength information from the two-dimensional spectral data at each photographing position, and a wavelength from the three-dimensional spectral data. An image creating means for creating a spectral image for each pixel, an obtaining means for obtaining spectral radiance, spectral luminance and spectral reflectance or spectral transmittance at each pixel of the spectral image, and analyzing the spectral image by a predetermined analysis method Analysis processing means for processing.

  In the microscope apparatus according to the present invention, in the above-described microscope apparatus, in the imaging unit, the two-dimensional imaging detector can be attached and detached together with an optical element including the lens and the dispersion optical element. By exchanging the three-dimensional imaging detector and the optical element, it is possible to change the wavelength range capable of photographing, wavelength resolution, and the number of spatial pixels.

  The analysis method according to the present invention is to analyze the object without staining using the above-described microscope apparatus.

  In the microscope apparatus according to the present invention, in the above-described microscope apparatus, the moving unit is a moving member on which a configuration subsequent to the lens is placed.

  The microscope apparatus according to the present invention includes a light source that irradiates light on an object, an imaging optical unit that forms an image of the object on a primary image plane with a light beam incident through an objective lens, and photographing. Imaging with a two-dimensional imaging detector for acquiring a light beam from the image incident through the lens after being dispersed in a direction orthogonal to a predetermined direction by a dispersion optical element and acquiring a signal based on the incident light beam And two-dimensional spectroscopic data having one-dimensional spatial information and one-dimensional wavelength information at the photographing position based on the signal, control means for controlling photographing timing of the two-dimensional imaging detector, and the signal. In addition, spectral data creating means for creating three-dimensional spectral data having two-dimensional spatial information and one-dimensional wavelength information from the two-dimensional spectral data at each photographing position, and a wave from the three-dimensional spectral data An image creating means for creating a spectral image for each pixel, an obtaining means for obtaining spectral radiance, spectral luminance and spectral reflectance or spectral transmittance at each pixel of the spectral image, and analyzing the spectral image by a predetermined analysis method Analysis processing means for processing, and the imaging means captures the image while moving the photographing position in the predetermined direction by changing the positional relationship with the object in the predetermined direction. It is what I did.

  According to the present invention, a microscope apparatus includes a light source that irradiates light on an object, and a microscope unit that includes an imaging optical unit that forms an image of the object on a primary image plane with a light beam incident through an objective lens; A two-dimensional imaging detector that obtains a signal based on the incident light flux by dispersing the light flux from the image incident through the lens by photographing in a direction orthogonal to a predetermined direction by the dispersion optical element. Imaging means that is detachably disposed in the microscope unit, control means for controlling the timing of imaging of the two-dimensional imaging detector, and one-dimensional spatial information at the imaging position based on the signal, In addition to creating two-dimensional spectral data having one-dimensional wavelength information, three-dimensional spectral data having two-dimensional spatial information and one-dimensional wavelength information from two-dimensional spectral data at each photographing position Data creation means, image creation means for creating a spectral image for each wavelength from the three-dimensional spectral data, and acquisition means for obtaining spectral radiance, spectral luminance and spectral reflectance or spectral transmittance at each pixel of the spectral image And an analysis processing means for analyzing the spectral image by a predetermined analysis method, and the imaging means changes the positional relationship with the object in the predetermined direction, thereby changing the shooting position to the predetermined position. The image is taken while moving in the direction of.

  Since the present invention is configured as described above, the present invention has an excellent effect that it is possible to classify and discriminate objects with high accuracy, which cannot be classified by the prior art.

  In addition, since the present invention is configured as described above, it is possible to observe an object such as a living cell, a tissue section, a crystal, etc. over time without causing deterioration of the state or property change. There is an effect.

  Moreover, since this invention is comprised as demonstrated above, there exists the outstanding effect that a target object can be analyzed without dyeing | staining.

FIG. 1A is an explanatory diagram of a schematic configuration of a microscope apparatus according to the present invention, and FIG. 1B is a view taken in the direction of arrow A in FIG. FIG. 2 is a block diagram illustrating the control unit of the microscope apparatus according to the present invention. FIG. 3 is a flowchart showing a processing routine of imaging processing in the microscope apparatus. FIG. 4 is a flowchart showing a processing routine of analysis processing in the microscope apparatus. FIG. 5 is a flowchart showing the processing routine of the calibration process. 6 (a) is an RGB image of a nematode photographed by the microscope apparatus according to the present invention, and FIG. 6 (b) is a transmission spectrum of the photographed image obtained by the microscope apparatus according to the present invention. FIG. 6C is a spectrum analysis image acquired by the microscope apparatus according to the present invention. FIG. 7 (a) is an RGB image of alga bodies photographed by the microscope apparatus according to the present invention, and FIG. 7 (b) is a transmission spectrum of the photographed image acquired by the microscope apparatus according to the present invention. FIG. 7C is a spectrum analysis image acquired by the microscope apparatus according to the present invention. FIG. 8 is a schematic configuration explanatory diagram of a modified example of the imaging unit in the microscope apparatus according to the present invention. FIG. 9 is a schematic configuration explanatory diagram of a modified example of the imaging unit in the microscope apparatus according to the present invention. FIG. 10 is a schematic configuration explanatory diagram of a modified example of the imaging unit in the microscope apparatus according to the present invention. FIG. 11A is an explanatory diagram of a pair of achromatic prisms arranged in the initial state, and FIG. 11B is a view taken in the direction of arrow B in FIG. (C) is explanatory drawing of a pair of achromatic prism of the state which functions as a parallel plane board, and FIG.11 (d) is C arrow line view of FIG.11 (c). 12 (a) and 12 (b) are schematic configuration explanatory views showing a modification of the microscope apparatus according to the present invention.

Hereinafter, an example of an embodiment of a microscope apparatus and an analysis method according to the present invention will be described in detail with reference to the accompanying drawings.

FIG. 1 (a) shows a schematic configuration explanatory diagram of a microscope apparatus according to the present invention, and FIG. 1 (b) shows a view as seen from the arrow A in FIG. 1 (a). FIG. 2 is an explanatory diagram of the block configuration of the control unit.

  The microscope apparatus 10 shown in FIG. 1 has a variable magnification optical system (not shown) that changes the diameter of a substantially parallel light beam incident through an objective lens (not shown) and emits it again as a substantially parallel light beam. ), And an image forming optical unit 12 that forms an image of a light beam from the object 200 on a primary image plane via a variable power optical system (not shown), and an image is formed on the primary image plane by the image forming optical unit 12. The imaging unit 14 that performs imaging of a predetermined region of the primary image plane including the image 210 that is formed, and the imaging by the imaging unit 14 are controlled, and the zooming optical system (not shown) in the imaging optical unit 12 is controlled. And a control unit 16 that adjusts the magnification of the image formed on the primary image plane.

In the microscope apparatus 10, a transmission type light source unit 18 that irradiates light that passes through the object 200 and an epi-illumination type light source unit 18 that irradiates light that is reflected by the object 200 are provided. A halogen lamp, a xenon lamp, a mercury lamp, an LED lamp, a laser, or the like is used. In FIG. 1, the case where a transmissive light source unit is used is described.

More specifically, the imaging unit 14 includes an objective lens 22 that receives a light beam from an image 210 formed on the primary image plane, a precision linear motion stage 24 that moves in the Y-axis direction in an XYZ orthogonal coordinate system, and an objective. A slit plate 26 disposed so that a slit opening 26a extending in the Z-axis direction is positioned on the image plane of the lens 22, a collimating lens 28 that converts the light beam that has passed through the slit opening 26a into parallel light, and The parallel light from the collimator lens 28 is detected on the image plane of the dispersion optical element 30 for dispersing in the Y-axis direction, the imaging lens 32 for imaging the light beam emitted from the dispersion optical element 30, and the imaging lens 32. And a two-dimensional imaging detection unit 34 disposed so that the unit 34a is located.

  In the precision linear motion stage 24, a slit plate 26, a collimating lens 28, a dispersion optical element 30, an imaging lens 32, and a two-dimensional imaging detection unit 34 are fixedly disposed.

  For this reason, the slit plate 26, the collimating lens 28, the dispersion optical element 30, the imaging lens 32, and the two-dimensional imaging detection unit 34 are integrated with the Y axis as the precision linear motion stage 24 moves in the Y axis direction. Will move in the direction.

The precision linear motion stage 24 can use a conventionally known technique, and will not be described in detail.

The slit plate 26 is disposed so that the light beam from the objective lens 22 passes through a slit opening 26a that extends in the Z-axis direction.

  Further, the dispersion optical element 30 can use, for example, a diffraction grating, a prism, or a grism. The grism is a direct-view diffraction grating that combines a transmission diffraction grating and a prism.

The diffraction grating, prism, or grism is disposed so as to disperse the incident light beam in the Y-axis direction perpendicular to the Z-axis direction that is the extension direction of the slit opening 26a.

The objective lens 22, the slit plate 26, the collimating lens 28, the dispersion optical element 30, and the imaging lens 32 are configured to be exchangeable as appropriate.

  The two-dimensional imaging detector 34 is an area sensor in which the detection unit 34a is arranged in parallel with the Z axis and the Y axis (that is, parallel to the primary image plane and parallel to the YZ plane).

  Further, the two-dimensional imaging detector 34 has a spatial pixel number of 1 to 29 million pixels, an obtainable wavelength range of 200 nm to 13 μm, a wavelength resolution of 0.1 nm to 100 nm, and an observation purpose or object. It is possible to change according to the type.

  That is, in the imaging unit 14, the two-dimensional imaging detector 34 having a different wavelength range, wavelength resolution, number of spatial pixels, and the like is replaced, and the objective lens 22, slit plate 26, collimating lens 28, dispersion optical element 30 and imaging lens By appropriately replacing 32, it is possible to change the wavelength range, wavelength resolution, number of spatial pixels, and the like that can be captured by the imaging unit 14.

For this reason, in the microscope apparatus 10, the wavelength region, wavelength resolution, and number of spatial pixels in the acquired spectral image can be changed.

  The control unit 16 is connected to the precision linear motion stage 24 and the two-dimensional imaging device 34, and is configured by, for example, a microcomputer or a general-purpose personal computer. Further, the control unit 16 is connected to the imaging optical unit 12 and controls a variable power optical system (not shown), and is connected to the light source unit 18 to control ON / OFF of the light source.

The control unit 16 moves the Y-axis direction of the imaging control unit 40 that controls the timing at which the two-dimensional imaging detector 34 acquires an electrical signal (that is, the timing for imaging) and the precision linear motion stage 24. It is created by a movement control unit 42 that controls the direction, amount of movement, and timing of movement, a spectral data creation unit 44 that creates spectral data based on an electrical signal from the two-dimensional imaging detector 34, and a spectral data creation unit 44. Based on the obtained spectral data, an image creating unit 46 that creates a spectral image, an image processing unit 48 that processes a spectral image for each wavelength acquired by photographing the object 200, and a spectral processed by the image processing unit 48. And an analysis processing unit 56 that performs image analysis processing.

  More specifically, when the information indicating that the precision linear motion stage 24 is moved is output from the movement control unit 42, the imaging control unit 40 performs imaging in the two-dimensional imaging detector 34 (that is, the electric power in the two-dimensional imaging detector). Signal is acquired), and information indicating that photographing has been performed is output to the movement control unit 42.

Further, the imaging control unit 40 determines whether or not the precision linear motion stage 24 has been moved until the imaging position in the predetermined imaging area including the image 210 reaches the imaging end position, and from the movement control unit 42 to the imaging end position. When information indicating that the camera has moved is output, the shooting process is terminated after shooting.

  The movement control unit 42 moves the precision linear motion stage 24 from the one end 210c in the Y-axis direction of the image 210 to the Y-axis direction within a predetermined imaging region including the other end 210d, and the slit of the slit opening 26a. It is sequentially moved at a predetermined interval according to the width. Thereby, the photographing position in the predetermined photographing region is moved in the Y-axis direction.

  More specifically, the movement control unit 42 moves the precise linear motion stage 24 so that the photographing position of the two-dimensional imaging detector 34 becomes the photographing start position when the operator instructs the start of photographing, and the precise linear motion is performed. Information indicating that the stage 24 has been moved is output to the imaging control unit 40.

  Then, when the information indicating that photographing has been performed is output from the photographing control unit 40, the movement control unit 42 moves the precision linear motion stage 24 at a predetermined interval corresponding to the slit width of the slit opening 26a. Information indicating that the linear motion stage 24 has been moved is output to the imaging control unit 40.

Further, when the movement control unit 42 moves the precision linear motion stage 24 until the shooting position of the two-dimensional imaging detector 34 reaches the shooting end position, the movement control unit 42 outputs information indicating that the movement has been moved to the shooting end position to the shooting control unit 40. .

  The spectral data creation unit 44 creates and creates two-dimensional spectral data having one-dimensional spatial information and one-dimensional wavelength information based on the electrical signal output from the two-dimensional imaging detector 34. The dimensional spectroscopic data is output and stored in the storage unit 50 provided in the control unit 16.

Further, the spectral data creation unit 44 uses the two-dimensional spectral data stored in the storage unit 50 to complete the three-dimensional spectroscopy having two-dimensional spatial information and one-dimensional wavelength information when imaging at each imaging position is completed. Data is generated, and the generated three-dimensional spectral data is output to the storage unit 50 and stored.

  The image creation unit 46 creates a spectral image for each wavelength based on the three-dimensional spectral data created by the spectral data creation unit 44.

In this image creation unit 46, each of the three-dimensional spectroscopic data spectrally separated into several hundred bands in a predetermined wavelength range of 200 nm to 13 μm and a predetermined wavelength resolution of 0.1 nm to 100 nm. A spectral image for each band (each wavelength) is acquired.

The image processing unit 48 includes a calibration processing unit 52 that performs dark noise correction processing, inter-pixel sensitivity deviation correction processing, luminance calibration processing, and light source correction processing on a spectral image for each wavelength acquired by photographing the object 200. Spectral luminance (cd / m ² · nm) is calculated from the spectral radiance (W / sr · m ² · nm) calculated during the luminance calibration process, and a sensitivity correction coefficient, luminance calibration coefficient, And a calculation unit 54 for calculating reflectance or spectral transmittance.

More specifically, the calibration processing unit 52 performs dark noise correction (dark correction) processing for removing noise caused by dark current in the two-dimensional imaging detector 34. In addition, in order to correct the sensitivity deviation for each pixel of the two-dimensional imaging detector 34, the sensitivity deviation correction process between pixels is performed using the sensitivity correction coefficient on the spectral image that has been subjected to the dark noise correction process. Further, the luminance calibration processing is performed using the luminance calibration coefficient on the spectral image that has been subjected to the inter-pixel sensitivity deviation correction processing. Furthermore, the illumination unevenness of the light source light in the space in the spectral image subjected to the dark noise correction process is corrected, and the light source correction process for acquiring the spectral reflectance or the spectral transmittance of the object 200 is performed.

Here, the dark noise correction data used in the dark noise correction process is not light-blocked without placing the target object 200 in the shooting region before observing the target object 200, that is, before shooting the target object 200. This is spectral image data for each wavelength created based on three-dimensional spectral data acquired by photographing in a state. Note that the shooting processing procedure at this time is the same as the shooting processing described later.

  In addition, the sensitivity correction coefficient is such that the spatial distribution of the radiance is equalized by an integrating sphere or the like before the object 200 is observed, that is, before the object 200 is imaged, without placing the object 200 in the imaging region. 3D spectroscopic data acquired by photographing the light source light (hereinafter referred to as “uniform uniform light source”). Note that the shooting processing procedure at this time is the same as the shooting processing described later.

That is, the sensitivity correction coefficient is output from a specified pixel (can be a single pixel or an average of a plurality of pixels) as a reference value in a spectral image for each wavelength of uniform standard light source imaging data, and the reference value is output from each pixel. The correction coefficient for each pixel is calculated by dividing by the value, and is created as three-dimensional spectral data having a correction count value for each pixel of the spectral image for each wavelength. Note that the calculation of the sensitivity correction coefficient is performed by the calculation unit 54 and output to the storage unit 50 for storage.

  Further, the luminance calibration coefficient is a light source light having a spectral radiance value that is not placed on the imaging region before the object 200 is observed, that is, before the object 200 is imaged. Hereinafter, this is one-dimensional data created based on three-dimensional spectral data acquired by photographing "spectral radiance standard light source". Note that the shooting processing procedure at this time is the same as the shooting processing described later.

That is, the luminance calibration coefficient is one-dimensional data corresponding to each wavelength, and the spectral radiance value valued for each wavelength of the spectral radiance standard light source is obtained for each wavelength obtained by photographing the spectral radiance standard light source. A luminance calibration coefficient having a conversion coefficient for each wavelength is created by dividing by the output value of a designated pixel (which may be a single pixel or an average of a plurality of pixels) in the spectral image. Note that the calculation of the luminance calibration coefficient is performed by the calculation unit 54 and output to the storage unit 50 for storage.

  Further, the light source data is applied to the reflection reference such as a standard white plate without placing the object 200 in the imaging region before observing the object 200, that is, before photographing the object 200. It is created as three-dimensional spectroscopic data acquired by photographing the reflected light obtained in this way. Note that the shooting processing procedure at this time is the same as the shooting processing described later.

Further, the spectral reflectance or spectral transmittance R (λ) of each pixel in the spectral image for each wavelength is expressed by the following equation.
R (λ) = C (λ) / E (λ)
R (λ): Spectral reflectance or spectral transmittance for each pixel of the spectral image C (λ): Output value of each pixel of the spectral image for each wavelength acquired by photographing the object 200 E (λ): Light source Output value of pixel corresponding to C (λ) of spectral image for each wavelength of data

The light source data and the spectral reflectance or the spectral transmittance are calculated by the calculation unit 54 and output to the storage unit 50 for storage.

The calculation unit 54 calculates the spectral luminance L (λ) (cd / m ² · nm) from the spectral radiance Le (λ) (W / sr · m ² · nm) in the spectral image for each wavelength processed in the calibration processing unit 52. nm).

Specifically, it is calculated by the following formula.
L (λ) = Km × Le (λ) × V (λ)
L (λ): Spectral luminance (cd / m ² · nm) for each pixel of the spectral image
Le (λ): Spectral radiance (W / sr · m ² · nm) for each pixel of the spectral image
V (λ): CIE standard spectral luminous efficiency Km: Maximum luminous efficacy (683 lm · W ⁻¹ )

  As for the method for calculating the spectral luminance from such spectral radiance, conventionally known techniques (for example, Japanese Society for Color Science (ed.), “New Color Science Handbook (Third Edition)”, University of Tokyo Press, pp. 51-53, 2011.), detailed description thereof will be omitted.

In addition, for example, data used for spectral analysis is generally spectral radiance or spectral reflectance (or spectral transmittance), and spectral luminance is used only in specific situations such as color relations. The process of calculating the luminance is executed when an instruction from the operator is given.

  The analysis processing unit 56 uses, for example, spectral radiance, spectral luminance, spectral reflectance, or spectral transmittance at each pixel of the spectral image for each wavelength acquired by photographing the object 200 processed by the image processing unit 48. Then, analysis processing is performed based on the set data analysis method.

In the analysis processing unit 56, various analysis methods can be used. For example, in the spectral data analysis method, the K-mean method, ISODATA method, spectral angle mapper (Support Angle Mapper), support vector machine (Support) Vector Machine), Support Vector Regression Method, Cross-Correlation Method, Linear Unmixing Method, Principal Components Analysis (Minimum PCA), Principal Components Analysis (PCA) Minimum Noise Fraction (MNF), matched filter method (Matched Filter), restriction Nergie minimum method (Constrained Energy Minimization), Orthogonal subspace projection method decision boundary (Decision Boundary), Feature extraction Battacha distance method (B-Distance), Cluster analysis Discriminant analysis (Discriminant Analysis), Linear multiple regression analysis (uls) : MLR), principal component regression analysis (Principal Components Regression: PCR), classical least square method, PLS regression analysis (Partial Last Squares Regression: PLSR), Fourier transform regression analysis N der quasi evaluation index: NDVI), normalized differential reflectance index (NDSI), normalized soil index (NDSI), normalized water index (including NDSI), normalized water index (NMD) Analysis (Normalized Difference Soil Moisture Index: NDSMI), Pixel Purity Index (PPI) processing, equal area normalization (Equal Area Normalization), band mapping method (Band Mapping method), SMACCS edge method Fitting), decision tree method (Decision Tree Classifier), shortest distance classification method (Minimum Distance Classifier), maximum likelihood classification method (Maximum Likelihood Classifier), Fisher Linear level The Euclidean shortest distance method (MED), the shape difference method (SD), the distance / correlation method (DC), the standard normal variate (SNV), and the like can be used.

  In the above configuration, when the object 200 is observed with the microscope apparatus 10 according to the present invention, first, the two-dimensional imaging detector 34 uses the one end 210a and the other end 210b in the Z-axis direction of the image 210. In such a state that can be photographed, the operator gives an instruction to start photographing.

When the operator gives an instruction to start shooting, the control unit 16 starts shooting processing.

  Here, the flowchart of FIG. 3 shows the detailed processing contents of the photographing process in the microscope apparatus 10 according to the present invention. In this photographing process, first, the photographing position by the two-dimensional imaging detector 34 starts photographing. The precision linear motion stage 24 is moved so as to coincide with the position (step S302).

  This shooting start position is set in advance, and is, for example, one end in the Y-axis direction in a predetermined shooting region including the image 210.

That is, in the process of step S302, the movement controller 42 moves the precision linear motion stage 24 to a position where the imaging position coincides with the imaging start position by the two-dimensional imaging detector 34, and the precision linear motion stage 24 is Information indicating that it has moved is output to the imaging control unit 40.

  Next, the imaging control unit 40 executes processing for imaging in the two-dimensional imaging detector 34 (step S304).

  That is, in the process of step S304, the photographing control unit 40 performs photographing (acquisition of electric signals) in the two-dimensional imaging detector 34 and outputs information indicating that photographing has been performed to the movement control unit 42.

  Specifically, for example, when a light beam from the other end 210d in the Y-axis direction of the image 210 is incident on the two-dimensional imaging detector 34 by photographing, the two-dimensional imaging detector 34 determines the light intensity of the incident light. The distribution is converted into an electrical signal, and this electrical signal is output to the spectral data creation unit 44.

Thereafter, the spectral data creating unit 44 creates two-dimensional spectral data having one-dimensional spatial information and one-dimensional wavelength information based on the electrical signal output from the two-dimensional imaging detector 34. The created two-dimensional spectroscopic data is output to and stored in the storage unit 50.

  When the two-dimensional imaging detector 34 performs photographing, the movement control unit 42 moves the precision linear motion stage 24 at a predetermined interval (step S306).

  That is, in the process of step S306, the movement control unit 42 moves the precision linear motion stage 24 at a predetermined interval and outputs information to the imaging control unit 40 that the precision linear motion stage 24 has moved. To do.

At this time, when the precise linear motion stage 24 is moved until the photographing position of the two-dimensional imaging detector 34 reaches the photographing end position, the movement control unit 42 outputs information indicating that it has moved to the photographing end position. The Rukoto.

  Thereafter, the imaging control unit 40 determines whether or not the precise linear motion stage 24 has moved until the imaging position of the two-dimensional imaging detector 34 reaches the imaging end position (step S308).

  Note that this photographing end position is set in advance, and is, for example, the other end in the Y-axis direction in a predetermined photographing region.

  That is, in the determination process of step S308, the imaging control unit 40 determines whether or not information indicating that the movement control unit 42 has moved to the imaging end position has been output.

  That is, in the determination process in step S308, if the information indicating that the movement control unit 42 has moved to the shooting end position is not output, the shooting position of the two-dimensional imaging detector 34 is set to the shooting end in the shooting control unit 40. It is determined that the precision linear motion stage 24 has not moved until the position is reached. On the other hand, when information indicating that the movement control unit 42 has moved to the photographing end position is output, the precise linear motion stage 24 is used until the photographing position of the two-dimensional imaging detector 34 reaches the photographing end position in the photographing control unit 40. Is determined to have moved.

  If it is determined in the determination process of step S308 that the imaging linear movement stage 24 has not moved until the imaging position of the two-dimensional imaging detector 34 reaches the imaging end position, the process returns to the process of step S304, and the process of step S304. Perform the following processing.

On the other hand, if it is determined in step S308 that the photographing linear movement stage 24 has moved until the photographing position of the two-dimensional imaging detector 34 reaches the photographing end position, the two-dimensional imaging detector 34 is obtained at the photographing end position. Then, photographing (acquisition of electrical signals) is performed (step S310), and this photographing process is terminated.

When the photographing process is finished, the analysis process is started next.

  Here, the flowchart of FIG. 4 shows the detailed processing contents of the analysis processing in the microscope apparatus 10 according to the present invention. In this analysis processing, first, the spectral data creation unit 44 acquires it at each photographing position. 3D spectroscopic data having two-dimensional spatial information and one-dimensional wavelength information indicating information in a predetermined imaging region by integrating the two-dimensional spectroscopic data having the one-dimensional spatial information and the one-dimensional wavelength information. Is created (step S402).

Next, the image creation unit 46 creates a spectral image for each wavelength in the created three-dimensional spectral data (step S404), and performs a calibration process for the created spectral image for each wavelength (step S406).

  Here, FIG. 5 shows a flowchart showing the detailed processing contents of the calibration processing. In this calibration processing, first, dark noise correction processing is performed (step S502).

  That is, in the process of step S502, dark noise correction processing is performed using dark noise correction data created in advance and stored in the storage unit 50.

Specifically, in the calibration processing unit 52, the dark noise correction data of the corresponding wavelength with respect to the output value in each pixel of the spectral image for each wavelength acquired by photographing the object 200 created in the process of step S404. The output value of the corresponding pixel in is subtracted.

  Next, an inter-pixel sensitivity deviation correction process is performed on the spectral image for each wavelength subjected to the dark noise correction process in this way (step S504).

In other words, in the process of step S504, the calibration processing unit 52 performs sensitivity correction created in advance and stored in the storage unit 50 for each pixel of the spectral image for each wavelength subjected to dark noise correction processing. Multiply by a coefficient.

  Thereafter, a luminance calibration process is performed on the spectral image for each wavelength subjected to the inter-pixel sensitivity deviation correction process (step S506).

That is, in the process of step S506, the calibration processing unit 42 creates and stores in advance in the storage unit 50 the pixel output value of the spectral image for each wavelength subjected to the inter-pixel sensitivity deviation correction process. Multiply by the luminance calibration factor. In addition, what was obtained by multiplying the luminance calibration coefficient indicates the spectral radiance at each pixel of the spectral image for each wavelength.

  Then, the calculation unit 54 calculates the spectral luminance at each pixel of the spectral image for each wavelength from the acquired spectral radiance at each pixel of the spectral image for each wavelength (step S508), and the analysis processing at step S408. Proceed to processing.

  Since the spectral luminance is used only in a specific case as described above, for example, when there is no instruction to calculate the spectral luminance from the operator, the processing in step S508 may be omitted. .

In the process of step S408, the light source correction process (step S408) is performed, and each pixel of the spectral image for each wavelength subjected to the dark noise correction process acquired in the process of step S502 in the calibration processing unit 52. The output value of the corresponding pixel in the spectral image of the wavelength corresponding to the light source data is divided from the output value of the light source data, and the light source correction process is performed, so that the spectral for each pixel in the spectral image for each wavelength of the object 200 is obtained. Obtain reflectance or spectral transmittance.

Next, using the spectral radiance and spectral luminance and the spectral reflectance or spectral transmittance in the spectral image for each wavelength acquired by photographing the object 200, the analysis processing unit 56 sets, for example, the user. The analysis image analyzed by the predetermined analysis method is displayed on a display unit (not shown) (step S412), and the analysis process is terminated.

Then, the operator refers to the analysis image data displayed on the display unit (not shown) and classifies or discriminates the object.

Here, the observation result of observing nematodes and algae as the object 200 using the microscope apparatus 10 according to the present invention will be described.

  First, nematodes were grown on NGM plates in which Escherichia coli (OP50) was cultured until they became adults by performing morphological culture at an interval of 4 days at a constant temperature of 20 ° C.

  Then, the nematode thus obtained was collected, and the collected nematode was anesthetized, and placed on the preparation in an unfixed and unstained state while alive.

  Then, the nematode mounted on this preparation was observed in the microscope apparatus 10 using the transmitted light of the halogen light source.

  The observation results are shown in FIGS. 6 (a), (b) and (c).

  As an analysis method in the analysis process, spectrum data was analyzed by a supervised maximum likelihood method, classified into tissues and visualized.

  Specifically, FIG. 6A shows an RGB image of a nematode photographed by the microscope apparatus 10, and FIG. 6B shows a transmission of the photographed image acquired by the microscope apparatus 10. A spectrum is shown, and FIG. 6C shows a spectrum analysis image acquired by a microscope apparatus.

  In FIG. 6B, the spectral transmittance R (λ) is calculated for each pixel of the spectral image, a representative location is selected for each nematode organ, and the average spectral transmittance of each region (ROI) is calculated. It is displayed as a graph.

In FIG. 6C, each ROI selected in FIG. 6C is assigned to each class as a teacher location, and the occurrence probability (likelihood) for each living class is determined based on the spectral information of each spatial pixel. It was obtained from the following formula and classified into each class by the maximum likelihood method, and organ classification of nematodes that remained unstained was performed.

In addition,
Is the likelihood of class c _i (selection range), and
Is the variance-covariance determinant of class c _i , and
Is the transpose of the deviation, and
Is the variance-covariance inverse of class c _i , and
Is a deviation matrix, and n is the number of bands.

  Moreover, about the algae, the germination body of the zoospore of two types of algae was cultured in the beaker containing a culture solution, and the growth alga body was used.

  The cultured algae are collected, and the collected algae are placed on the preparation in an unfixed and unstained state, and placed on the preparation in the microscope apparatus 10 using the transmitted light of the halogen light source. The placed alga bodies were observed.

  The observation results are shown in FIGS. 7 (a), (b) and (c).

  As an analysis method in the analysis process, the inclination of the feature point was analyzed for the spectrum data, and the algae and bacteria were classified and visualized.

  Specifically, FIG. 7A shows an RGB image of algal bodies photographed by the microscope apparatus 10, and FIG. 7B shows transmission of the photographed image acquired by the microscope apparatus 10. A spectrum is shown, and FIG. 7C shows a spectrum analysis image acquired by a microscope apparatus.

  In FIG. 7B, the spectral transmittance R (λ) is calculated for each pixel of the spectral image, and the spectral transmittance graph of the selected portion is displayed.

FIG. 7 (c) estimates the characteristic wavelength at which the difference in the transmission spectrum of algae and bacteria appears from the spectral transmission graph of FIG. 7 (b), and calculates the normalized spectral reflectance index from the estimated wavelength by the following formula. Calculation and classification analysis of algae and bacteria by threshold.
NDSI = (R (1) -R (2) / (R (1) + R (2))
NDSI: normalized spectral reflection index R (1), R (2): spectral transmittance of characteristic wavelength

  As described above, the microscope apparatus 10 according to the present invention includes the slit plate 26, the collimator lens 28, the dispersion optical element 30, the imaging lens 32, and the two-dimensional imaging detector in the imaging unit 14 after the objective lens 22. 34 is provided so as to acquire one-dimensional spatial information extended in the Z-axis direction and one-dimensional wavelength information extended in the Y-axis direction.

  Then, the slit plate 26, the collimating lens 28, the dispersion optical element 30, the imaging lens 32, and the two-dimensional imaging detector 34 are disposed on the precision linear motion stage 24 that is movably disposed in the Y-axis direction. The shooting position is changed in the Y-axis direction.

  Thereby, in the microscope apparatus 10 according to the present invention, it is possible to photograph all the predetermined photographing areas including the image 210 on the primary image plane on which the image 210 of the object 200 is formed without moving the object 200. It becomes possible.

For this reason, according to the microscope apparatus 10 of the present invention, it is not necessary to perform processing such as moving the target object 200 when observing the target object 200. For example, the microscope apparatus 10 is sensitive to physical stimulation (for example, vibration). Even when a simple object is observed, the object 200 can be observed without giving a physical stimulus.

  In addition, the microscope apparatus 10 according to the present invention performs a calibration process and the like, and performs spectral radiance and spectral brightness of each pixel and spectral reflectance or spectral transmittance in a spectral image for each wavelength acquired by photographing the object 200. The image analyzed by a predetermined analysis method is displayed on a display unit (not shown).

  Therefore, the microscope apparatus 10 according to the present invention can classify and discriminate different organs, different cells or different crystals with high accuracy.

  Furthermore, in the microscope apparatus 10 according to the present invention, an analysis image analyzed using the spectral radiance or the like in the spectral image can be acquired, so that the cells are not stained, in addition to the stained cells. Unstained cells can be observed.

  That is, with the microscope apparatus 10 according to the present invention, for example, it becomes possible to observe the living cells without staining them.

Therefore, according to the microscope apparatus 10 according to the present invention, the object 200 can be observed over time without causing state deterioration or property change.

  The embodiment described above may be modified as shown in the following (1) to (7).

  (1) In the above-described embodiment, the slit plate 26, the collimating lens 28, the dispersion optical element 30, the imaging lens 32, and the two-dimensional imaging detector 34 are on the precision linear motion stage 24 that can move in the Y-axis direction. Of course, the arrangement is not limited to this.

  That is, the configuration after the slit plate 26 is integrally formed by a configuration based on a technology capable of moving control on a straight line, for example, an actuator such as a fluid type (hydraulic pressure, pneumatic pressure), electromagnetic type, ultrasonic type, piezo type. It is good also as a structure which moves to an axial direction.

(2) In the above-described embodiment, the configuration after the slit plate 26 is arranged on the precision linear motion stage 24 movable in the Y-axis direction. However, the present invention is not limited to this. Of course, the imaging unit 14 may have the following three configurations.

(I) Configuration in which objective lens 22 is arranged on a precision linear motion stage (II) Configuration using galvanometer mirror (III) Configuration using a pair of achromatic prisms

Hereinafter, the above (I) to (III) will be described.

(I) Configuration in which the objective lens 22 is disposed on the precision linear motion stage FIG. 8 is a schematic configuration explanatory diagram of a modification of the imaging unit in the microscope apparatus according to the present invention.

  The imaging unit 104 shown in FIG. 8 includes an objective lens 112 that receives a light beam from an image 210, a precision linear motion stage 114 that is provided with the objective lens 112 and moves in the Y-axis direction, and an image plane of the objective lens 12. , A slit plate 26 disposed so that a slit opening 26a extending in the Z-axis direction is located, a collimating lens 28 that collimates the light beam that has passed through the slit opening 26a, and the collimating lens 28 The dispersive optical element 30 that disperses the parallel light in the Y-axis direction, the image forming lens 32 that forms an image of the light beam emitted from the dispersive optical element 30, and the detection unit 34a are positioned on the image plane of the image forming lens 32. And a two-dimensional imaging detection unit 34 arranged as described above.

The slit plate 26, the collimating lens 28, the dispersion optical element 30, the imaging lens 32, and the two-dimensional imaging detector 34 are fixedly disposed.

  Further, the movement control unit 42 moves the precision linear motion stage 114 in the Y-axis direction within the predetermined imaging region including the one end 210c in the Y-axis direction of the image 210 and the other end 210d in the Y-axis direction. 26a is sequentially moved at a predetermined interval corresponding to the width in the Y-axis direction.

  More specifically, the movement control unit 42 moves the precise linear motion stage 114 so that the photographing position of the two-dimensional imaging detector 34 becomes the photographing start position when the operator gives an instruction to start photographing. Information indicating that the moving stage 114 has moved is output to the imaging control unit 40.

  When the information indicating that the photographing has been performed is output from the photographing control unit 40, the movement control unit 42 moves the precision linear motion stage 114 at a predetermined interval corresponding to the width of the slit opening 26a in the Y-axis direction. The information indicating that the precision linear motion stage 114 has been moved is output to the imaging control unit 40.

Further, when the movement control unit 42 moves the precision linear motion stage 114 until the shooting position of the two-dimensional imaging detector 34 reaches the shooting end position, the movement control unit 42 outputs information indicating that the movement to the shooting end position is performed to the shooting control unit 40. .

Then, the movement controller 42 moves the precision linear motion stage 114 to move the imaging position, and the imaging controller 40 performs imaging and creates spectral data based on the electrical signal from the two-dimensional imaging detector 34. In the unit 44, two-dimensional spectroscopic data having one-dimensional spatial information and one-dimensional wavelength information is created.

(II) Configuration Using Galvano Mirror FIG. 9 shows a schematic configuration explanatory diagram of a modified example of the imaging unit in the microscope apparatus according to the present invention.

  The imaging unit 124 illustrated in FIG. 9 includes an F-θ lens 132 that receives a light beam from the image 210, a galvano mirror 134 provided at the rear stage of the F-θ lens 132, and a light beam reflected by the galvano mirror 134. The imaging lens 136 that forms an image, the slit plate 26 that is disposed so that the slit opening 26a extending in the Z-axis direction on the image plane of the imaging lens 136 is positioned, and the slit opening 26a. A collimator lens 28 that converts the light beam into parallel light; a dispersion optical element 30 that disperses the parallel light from the collimator lens 28 in the X-axis direction; and an imaging lens 32 that forms an image of the light beam emitted from the dispersion optical element 30; The two-dimensional imaging detection unit 34 is arranged so that the detection unit 34 a is positioned on the image plane of the imaging lens 32.

  Note that the F-θ lens 134, the imaging lens 136, the slit plate 26, the collimating lens 28, the dispersion optical element 30, the imaging lens 32, and the two-dimensional imaging detector 34 are fixedly disposed.

  The galvanometer mirror 134 reflects the light substantially collimated by the F-θ lens 132 on the reflecting surface and enters the imaging lens 136, and the reflecting surface rotates around the Z axis. It is arranged.

  Then, the movement control unit 42 controls the rotation angle and rotation direction of the reflecting surface. Note that such a rotation angle and a rotation direction are set before photographing processing.

  In the following description, “turning the galvanometer mirror 134” means “turning the reflecting surface of the galvanometer mirror 134”.

That is, the galvanometer mirror 134 rotates the reflection surface by rotating, and changes the reflection angle of the light collimated by the F-θ lens 132.

  In addition, the movement control unit 42 galvanometer mirror 134 so that the imaging position moves in the Y-axis direction within a predetermined imaging region including the other end 210d from one end 210c in the Y-axis direction of the image 210. Are sequentially rotated around the Z axis at a predetermined rotation angle corresponding to the width of the slit opening 26a in the Y axis direction.

  More specifically, when the start of shooting is instructed by the operator, the movement control unit 42 rotates the galvano mirror 134 so that the shooting position of the two-dimensional imaging detector 34 becomes the shooting start position. Information about the rotation is output to the imaging control unit 40.

  Then, when the information indicating that the photographing has been performed is output from the photographing control unit 40, the movement control unit 42 rotates the galvano mirror 134 at a predetermined rotation angle corresponding to the width of the slit opening 26a in the Y-axis direction. The information indicating that the galvano mirror 134 is rotated is output to the imaging control unit 40.

In addition, when the galvano mirror 134 is rotated until the photographing position of the two-dimensional imaging detector 34 reaches the photographing end position, the movement control unit 42 outputs information indicating that the two-dimensional imaging detector 34 has moved to the photographing end position to the photographing control unit 40.

Then, while the movement control unit 42 controls the rotation of the galvano mirror 134 to move the imaging position, the imaging control unit 40 performs imaging, and based on the electrical signal from the two-dimensional imaging detector 34, spectral data is obtained. The creating unit 44 creates two-dimensional spectral data having one-dimensional spatial information and one-dimensional wavelength information.

(III) Configuration Using a Pair of Achromatic Prisms FIG. 10 shows a schematic configuration explanatory diagram of a modified example of the imaging unit in the microscope apparatus according to the present invention.

  The imaging unit 144 shown in FIG. 10 is extended in the Z-axis direction on the image plane of the objective lens 22 and a pair of achromatic prisms 146 disposed in front of the objective lens 22 on which the light beam from the image 210 is incident. The slit plate 26 disposed so that the slit opening 26a is positioned, the collimating lens 28 that makes the light beam that has passed through the slit opening 26a parallel light, and the parallel light from the collimating lens 28 in the Y-axis direction. A dispersive optical element 30 that disperses, an image forming lens 32 that forms an image of a light beam emitted from the dispersive optical element 30, and a two-dimensional arrangement in which the detector 34a is positioned on the image plane of the image forming lens 32. And an imaging detection unit 34.

  The objective lens 22, the slit plate 26, the collimating lens 28, the dispersion optical element 30, the imaging lens 32, and the two-dimensional imaging detector 34 are fixedly disposed.

  Further, the pair of achromatic prisms 146 are constituted by achromatic prisms 146-1 and 146-2, and the achromatic prisms 146-1 and 146-2 are aligned in the X-axis direction with the same apex angle direction. Arranged. In the following description, the state of the pair of achromatic prisms shown in FIGS. 11A and 11B is referred to as “initial state”.

  In addition, the pair of achromatic prisms 146 are rotated by the same angle in the opposite directions from the initial state with the achromatic prisms 146-1 and 146-2 being controlled by the movement control unit 42, respectively.

  In addition, in the pair of achromatic prisms 146, the achromatic prisms 146-1 and 146-2 rotate in directions opposite to each other so that the apex angle direction is in the opposite direction and the Z axis direction is horizontal (FIG. 11 ( c) (Refer to (d)), it functions as a plane parallel plate.

  FIG. 11C shows a state in which the achromatic prism 146-1 in FIG. 11A is rotated 90 ° in the direction of arrow I and the achromatic prism 146-2 is rotated 90 ° in the direction of arrow II. It is.

  That is, the achromatic prisms 146-1 and 146-2 are respectively rotated in the opposite directions around the coincident central axes O. For example, the achromatic prism 146-1 is moved in the direction of the arrow I. When rotating, the achromatic prism 146-2 rotates in the direction of arrow II (see FIG. 13A). At this time, the achromatic prisms 146-1 and 146-2 are rotatable in the range of 0 ° to 180 °.

The pair of achromatic prisms 146 having such a configuration makes it possible to change the position of the light beam incident on the two-dimensional imaging detector 34 disposed downstream of the pair of achromatic prisms 146. As a result, the shooting position of shooting is moved in the Y-axis direction.

  Further, the movement control unit 42 is an achromatic prism so that the photographing position moves in the Y-axis direction within a predetermined photographing region including the other end 210d from one end 210c in the Y-axis direction of the image 210. 146-1 and 146-2 are sequentially rotated around the X axis at respective predetermined rotation angles according to the width of the slit opening 26a in the Y axis direction.

  More specifically, the movement control unit 42 sets the achromatic prisms 146-1 and 146-2 so that the imaging position of the one-dimensional imaging detector 236 becomes the imaging start position when the operator gives an instruction to start imaging. Information indicating that the achromatic prisms 146-1 and 146-2 are rotated is output to the imaging control unit 40 in the opposite directions.

  When the information indicating that the photographing has been performed is output from the photographing control unit 40, the movement control unit 42 moves the achromatic prisms 146-1 and 146-2 according to the width of the slit opening 26a in the Y-axis direction. Information indicating that the achromatic prisms 146-1 and 146-2 are rotated is output to the imaging control unit 40 by rotating at predetermined rotation angles.

Further, when the achromatic prisms 146-1 and 146-2 are rotated until the photographing position of the two-dimensional imaging detector 34 reaches the photographing end position, the movement control unit 42 photographs information indicating that the two-dimensional imaging detector 34 has moved to the photographing end position. Output to the control unit 40.

  Then, while the movement control unit 42 rotates the achromatic prisms 146-1 and 146-2 to move the imaging position, the imaging control unit 40 performs imaging and converts the electrical signal from the two-dimensional imaging detector 34 into an electric signal. Based on this, the spectral data creating unit 44 creates two-dimensional spectral data having one-dimensional spatial information and one-dimensional wavelength information.

  (3) In the above-described embodiment, the calibration process is performed after the spectral image for each wavelength acquired by photographing the object 200 is created. However, the present invention is not limited to this. Yes, the dark noise correction process and the light source correction process in the calibration process may be performed after an instruction from the operator, for example.

  (4) In the above-described embodiment, the microscope apparatus 10 is configured by the imaging optical unit 12, the light source unit 18, and the imaging unit 14, but it is needless to say that the present invention is not limited to this. As a microscope apparatus according to the present invention, a microscope unit having an imaging optical unit for forming an image of an object on a primary image plane and a light source for irradiating the object with light, specifically, a conventionally known optical unit. It is good also as a structure arrange | positioned in the lens-barrel part of a microscope apparatus detachably via attachments, such as C mount.

  (5) In the above-described embodiment, the precision linear motion stage 24 provided in the imaging unit 14 in the microscope apparatus 10 is used to move the calibration after the objective lens 22 in the Y-axis direction so that the imaging position is set to Y. Although it is configured to move in the axial direction, it is of course not limited to this. In the photographing unit 14, the objective lens 22, the slit plate 26, the collimator lens 28, the dispersion optical element 30, the imaging lens 32, and the like The two-dimensional imaging detector 34 may be fixedly arranged, and the photographing position may be moved in the Y-axis direction by moving the positional relationship with the object 200 in the Y-axis direction.

  That is, by fixing either one of the imaging unit 14 or the target object 200 and moving it in the other Y-axis direction, the imaging position is moved in the Y-axis direction.

  Specifically, when moving the microscope apparatus 10, for example, in the imaging apparatus 14, the objective lens 22, the slit plate 26, the collimating lens 28, the dispersion optical element 30, the imaging lens 32, and the two-dimensional imaging detector 34 are used. Is fixedly arranged, and the imaging unit 14 is arranged on the Y axis by moving means that is provided separately from the imaging unit 14 and controlled by a control unit different from the control unit 16 with respect to the fixed object 200. It is also possible to move in the direction (refer to FIG. 12A). Note that the control unit that controls the moving unit and the control unit 16 of the imaging unit 14 are connected to this difference and moved. And the timing of shooting are controlled.

  When moving the object 200, the objective lens 22, the slit plate 26, the collimating lens 28, the dispersion optical element 30, the imaging lens 32, and the two-dimensional imaging detector 34 are fixedly arranged in the imaging device 14. The object 200 is arranged on a stage movable in the Y-axis direction, and the object 200 is moved in the Y-axis direction by the stage controlled by the control unit with respect to the fixed imaging unit 14. (Refer to FIG. 12B). At this time, the control unit that controls the moving unit and the control unit 16 of the imaging unit 14 are connected, and the timing of movement and the timing of imaging are controlled.

  (6) In the above-described embodiment, the wavelength is changed by exchanging hardware configurations such as the objective lens 22, the slit plate 26, the collimating lens 28, the dispersion optical element 30, the imaging lens 32, and the two-dimensional imaging detector 34. The area, wavelength resolution, and number of spatial pixels are changed, but this is not limited to this. Using the functions of the shooting software, reading externally defined files or changing parameters to the worker May specify not only the wavelength range, wavelength resolution, and number of spatial pixels, but also the imaging speed, exposure time, and gain.

  (7) You may make it combine suitably the embodiment shown above and the modification shown in said (1) thru | or (6).

  The present invention is suitable for use as a microscope apparatus for observing cells and tissues.

DESCRIPTION OF SYMBOLS 10 Microscope apparatus 12 Imaging optical part 14,104,124,144 Imaging part 16 Control part 22,112 Objective lens 24,114 Precision linear motion stage 26 Slit board 28 Collimating lens 30 Dispersion optical element 32, 136 Imaging lens 34 2 Dimensional imaging detector 40 Imaging control unit 42 Movement control unit 44 Spectral data creation unit 46 Image creation unit 48 Analysis unit 50 Storage unit 132 F-θ lens 134 Galvano mirror 146 A pair of achromatic prisms 146-1 and 146-2 Prism 200 Object 210 Image

Claims (7)

A light source that irradiates the object with light;
Imaging optical means for forming an image of the object on a primary image plane by a light beam incident through an objective lens;
A two-dimensional imaging detector that obtains a signal based on the incident light flux, the light flux from the image incident through the lens dispersed by the dispersion optical element in a direction orthogonal to a predetermined direction by the photographing by photographing. An imaging means comprising: a moving means for moving an imaging position photographed by the two-dimensional imaging detector in the predetermined direction;
First control means for controlling shooting timing of the two-dimensional imaging detector;
Second control means for controlling movement of the moving means;
Based on the signal, two-dimensional spectral data having one-dimensional spatial information and one-dimensional wavelength information at the photographing position is created, and two-dimensional spatial information and one-dimensional data are obtained from the two-dimensional spectral data at each photographing position. Spectral data creating means for creating three-dimensional spectral data having wavelength information;
Image creating means for creating a spectral image for each wavelength from the three-dimensional spectral data;
Obtaining means for obtaining spectral radiance, spectral luminance and spectral reflectance or spectral transmittance in each pixel of the spectral image;
An analysis processing means for analyzing the spectral image by a predetermined analysis method. A light source that irradiates light on the object, and a microscope unit that includes an imaging optical unit that forms an image of the object on a primary image plane by a light beam incident through the objective lens;
A two-dimensional imaging detector that obtains a signal based on the incident light flux, the light flux from the image incident through the lens dispersed by the dispersion optical element in a direction orthogonal to a predetermined direction by the photographing by photographing. A moving means for moving a photographing position photographed by the two-dimensional imaging detector in the predetermined direction, and an imaging means detachably disposed on the microscope unit;
First control means for controlling shooting timing of the two-dimensional imaging detector;
Second control means for controlling movement of the moving means;
Based on the signal, two-dimensional spectral data having one-dimensional spatial information and one-dimensional wavelength information at the photographing position is created, and two-dimensional spatial information and one-dimensional data are obtained from the two-dimensional spectral data at each photographing position. Spectral data creating means for creating three-dimensional spectral data having wavelength information;
Image creating means for creating a spectral image for each wavelength from the three-dimensional spectral data;
Obtaining means for obtaining spectral radiance, spectral luminance and spectral reflectance or spectral transmittance in each pixel of the spectral image;
An analysis processing means for analyzing the spectral image by a predetermined analysis method. The microscope apparatus according to claim 1 or 2,
In the imaging means, the two-dimensional imaging detector is configured to be detachable together with the optical element including the lens and the dispersion optical element, and photographing is performed by exchanging the two-dimensional imaging detector and the optical element. A microscope apparatus characterized by being capable of changing a possible wavelength range, wavelength resolution, and number of spatial pixels.   The analysis method which analyzes the said target object unstained using the microscope apparatus of any one of Claim 1 or 2. The microscope apparatus according to claim 1 or 2,
The moving device is a moving member on which a configuration subsequent to the lens is mounted. A light source that irradiates the object with light;
Imaging optical means for forming an image of the object on a primary image plane by a light beam incident through an objective lens;
A two-dimensional imaging detector that captures a light beam from the image incident through a lens by being dispersed in a direction orthogonal to a predetermined direction by a dispersion optical element and acquires a signal based on the incident light beam by photographing. Imaging means provided,
Control means for controlling the shooting timing of the two-dimensional imaging detector;
Based on the signal, two-dimensional spectral data having one-dimensional spatial information and one-dimensional wavelength information at the photographing position is created, and two-dimensional spatial information and one-dimensional data are obtained from the two-dimensional spectral data at each photographing position. Spectral data creating means for creating three-dimensional spectral data having wavelength information;
Image creating means for creating a spectral image for each wavelength from the three-dimensional spectral data;
Obtaining means for obtaining spectral radiance, spectral luminance and spectral reflectance or spectral transmittance in each pixel of the spectral image;
Analysis processing means for analyzing the spectral image by a predetermined analysis method,
The imaging apparatus captures the image while moving a photographing position in the predetermined direction by changing a positional relationship with the object in the predetermined direction. A light source that irradiates light on the object, and a microscope unit that includes an imaging optical unit that forms an image of the object on a primary image plane by a light beam incident through the objective lens;
A two-dimensional imaging detector that captures a light beam from the image incident through a lens by being dispersed in a direction orthogonal to a predetermined direction by a dispersion optical element and acquires a signal based on the incident light beam by photographing. An imaging means detachably disposed in the microscope unit;
Control means for controlling the shooting timing of the two-dimensional imaging detector;
Based on the signal, two-dimensional spectral data having one-dimensional spatial information and one-dimensional wavelength information at the photographing position is created, and two-dimensional spatial information and one-dimensional data are obtained from the two-dimensional spectral data at each photographing position. Spectral data creating means for creating three-dimensional spectral data having wavelength information;
Image creating means for creating a spectral image for each wavelength from the three-dimensional spectral data;
Obtaining means for obtaining spectral radiance, spectral luminance and spectral reflectance or spectral transmittance in each pixel of the spectral image;
Analysis processing means for analyzing the spectral image by a predetermined analysis method,
The imaging apparatus captures the image while moving a photographing position in the predetermined direction by changing a positional relationship with the object in the predetermined direction.