Patent Publication Number: US-11037294-B2

Title: Image processing device, image processing method, and computer-readable recording medium

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
     This application is a continuation of International Application No. PCT/JP2017/000624, filed on Jan. 11, 2017, the entire contents of which are incorporated herein by reference. 
    
    
     BACKGROUND 
     1. Technical Field 
     The present disclosure relates to an image processing device, an image processing method, and a computer-readable recording medium to process a stained-specimen image that captures a stained specimen. 
     2. Related Art 
     Spectral transmittance is one of the physical quantities representing the physical property unique to an object. Spectral transmittance is the physical quantity that represents the ratio of transmitted light to incident light at each wavelength, and it is information unique to an object and its values are not changed due to extrinsic influences unlike color information such as RGB values that depend on changes in illumination light. For this reason, the spectral transmittance is used in various fields as information for reproducing the color of the object itself. For example, in the fields of pathological diagnosis using a living tissue specimen, particularly a pathological specimen, the spectral transmittance is used as an example of the spectral characteristic value for analysis of an image that captures the specimen. 
     As one of the pathological diagnosis, tissue diagnosis is known, which is to collect tissue in a lesion area and observe it with a microscope so as to diagnose a disease or examine how much the lesion is expanding. This tissue diagnosis is also called biopsy, and it is popular that a block specimen obtained after extraction of an organ or a pathological specimen obtained after biopsy using a needle is sliced in a thickness of approximately several micrometers and then it is enlarged and observed by using a microscope so as to obtain various findings. Especially, transmission observation using an optical microscope is one of the most popular observation methods because of relatively inexpensive equipment, easy handling, and conventionality in practice. In this case, a sliced specimen hardly causes light absorption and scattering and it is almost colorless and transparent; therefore, it is typical to conduct staining using a dye prior to observation. 
     Various staining techniques are proposed, and there are more than 100 types in total; particularly for pathological specimens, hematoxylin-eosin staining (hereafter, referred to as “HE staining”) using two dyes, hematoxylin in blue-violet and eosin in red, is typically used. 
     Hematoxylin is a natural substance collected from plants, and it does not have staining properties. However, its oxidized material, hematin, is a basophilic dye, and it binds with a negatively charged substance. As deoxyribonucleic acid (DNA) included in a cell nucleus is negatively charged due to the phosphate group included as a component, it binds with hematin so as to be stained in blue-violet. As described above, it is not hematoxylin but hematin, its oxidized material, that has staining properties; however, as it is typical to use hematoxylin as the name of the stain, it is hereafter used. 
     Meanwhile, eosin is an acidophilic dye, and it binds with a positively charged substance. The pH environment has an effect on whether an amino acid or a protein is charged positively or negatively, and under the acidic environment, it is more likely to be positively charged. For this reason, acetic acid is sometimes added to an eosin solution for use. A protein included in a cytoplasm binds with eosin so as to be stained in red to pink. 
     In a specimen (hereafter, referred to as stained specimen) after HE staining, cell nuclei, bone tissue, and the like, are stained in blue-violet, cytoplasm, connective tissue, red blood cells, and the like, are stained in red so that they may be easily visually recognized. As a result, an observer is capable of determining the size, the positional relationship, and the like, of elements included in tissue, such as cell nuclei, and is capable of morphologically judging the state of a stained specimen. 
     Observation of a stained specimen is conducted by not only observer&#39;s eyes but also multiband capturing of the stained specimen and presentation on the display screen of an external device. In the case of display on the display screen, for example, a process is performed to estimate the spectral transmittance at each point on a stained specimen based on a captured multiband image, or a process is performed to estimate the quantity of dye with which the stained specimen is stained based on the estimated spectral transmittance, and a display image which is an RGB image of the stained specimen for display is composed. 
     Techniques for estimating the spectral transmittance at each point on the stained specimen based on a multiband image of the stained specimen include, for example, an estimation technique using analysis of principal component and an estimation technique using Wiener estimation. Wiener estimation is widely known as one of the linear filtering techniques to estimate original signals from observed signals having noise superimposed thereon, and it is a technique to minimize errors in consideration of statistical properties of the observation target and properties of noise (observed noise). As signals from a camera contain some noise, Wiener estimation is extremely useful as a technique to estimate original signals. 
     An explanation is given below of a conventional method of composing a display image from a multiband image of a stained specimen. 
     First, a multiband image of the stained specimen is captured. For example, 16 bandpass filters are rotated by a filter wheel and switched so that a multiband image is captured in sequential lighting. Thus, a multiband image having pixel values in 16 bands at each point on the stained specimen is obtained. Although a dye is supposed to be distributed in three dimensions within the stained specimen, which is the target to be observed, it is difficult to directly capture it as a three-dimensional image with the normal transmission observation system, and it is observed as a two-dimensional image that is obtained by projecting the illumination light, passed through the stained specimen, onto the imaging element of the camera. Therefore, each point mentioned here refers to a point on the stained specimen, corresponding to each pixel of the imaging element projected. 
     Here, with regard to any point (pixel) x of a captured multiband image, the relation between the pixel value g(x,b) in the band b and the spectral transmittance t(x,λ) at the corresponding point on the stained specimen satisfies the following Equation (1) based on the response system of the camera.
 
 g ( x,b )=∫ λ   f ( b ,λ) s (λ) e (λ) t ( x ,λ) dλ+n ( b )  (1)
 
     In Equation (1), λ denotes the wavelength, f(b,λ) denotes the spectral transmittance of the b-th bandpass filter, s(λ) denotes the spectral sensitivity property of the camera, e(λ) denotes the spectral radiant property of illumination, and n(b) denotes observed noise in the band b. As b is a serial number for identifying a band, and it is here an integer number that satisfies 1≤b≤16. The actual calculation uses the following Equation (2) in which Equation (1) is discretized in a wavelength direction.
 
 G ( x )= FSET ( x )+ N   (2)
 
     In Equation (2), when the number of sample points in the wavelength direction is D and the number of bands is B (here, B=16), G(x) is the matrix in the row B and the column 1 corresponding to the pixel value g(x,b) at the point x. Similarly, T(x) is the matrix in the row D and the column 1 corresponding to t(x,λ), and F is the matrix in the row B and the column D corresponding to f(b,λ). Furthermore, S is the diagonal matrix in the row D and the column D, and the diagonal element corresponds to s(λ). Similarly, E is the diagonal matrix in the row D and the column D, and the diagonal element corresponds to e(λ). N is the matrix in the row B and the column 1 corresponding to n(b). In Equation (2), as equations regarding multiple bands are summarized by using a matrix, the variable b representing a band is not described. Moreover, the integrate regarding the wavelength λ is replaced with the product of matrices. 
     Here, to simplify description, the matrix H defined by the following Equation (3) is introduced. The matrix H is also called a system matrix.
 
 H=FSE   (3)
 
     Therefore, Equation (2) is replaced with the following Equation (4).
 
 G ( x )= HT ( x )+ N   (4)
 
     Then, by using Wiener estimation, the spectral transmittance at each point on the stained specimen is estimated based on a captured multiband image. The estimated value of the spectral transmittance (hereafter, referred to as spectral transmittance data) T^(x) is calculatable by using the following Equation (5). Here, T^ indicates that the symbol “^ (hat)” indicating an estimated value is attached to the upper section of T.
 
 {circumflex over (T)} ( x )= WG ( x )  (5)
 
     In Equation (5), W is represented by using the following Equation (6), and it is called “Wiener estimation matrix” or “estimation operator used for Wiener estimation”.
 
 W=R   SS   H   T ( HR   SS   H   T   +R   NN ) −1   (6)
 
     In Equation (6), R SS  is the matrix in the row D and the column D, and it represents the autocorrelation matrix of the spectral transmittance of the stained specimen. Furthermore, R NN  is the matrix in the row B and the column B, and it represents the autocorrelation matrix of noise of the camera used for capturing. Furthermore, with regard to any matrix X, the matrix X T  represents the transposed matrix of the matrix X, and the matrix X −1  represents the inverse matrix of the matrix X. The matrices F, S, E, i.e., the spectral transmittance of the bandpass filter, the spectral sensitivity property of the camera, and the spectral radiant property of the illumination, constituting the system matrix H, the matrix R SS , and the matrix R NN  are previously acquired. 
     As described above, after the spectral transmittance data T^(x) is estimated, the quantity of dye at the corresponding point (hereafter, referred to as sample point) on the stained specimen is estimated based on the spectral transmittance data T^(x). Here, the target dyes to be estimated are three types, i.e., hematoxylin, eosin with which a cytoplasm is stained, eosin with which a red blood cell is stained, or the inherent pigment of a red blood cell that is not stained, and they are abbreviated as H dye, E dye, and R dye, respectively. Strictly speaking, red blood cells have the inherent color although it is not stained, and after HE staining, the inherent color of red blood cells and the color of eosin changed in the process of staining are observed in an overlapped manner. 
     Therefore, in a precise sense, the combination of them is referred to as the R dye. 
     It is generally known that, with regard to a light transmissive substance, the Lambert-beer law represented by the following Equation (7) holds for the relation between the intensity I 0 (λ) of incident light at each wavelength λ and the intensity I(λ) of output light. 
     
       
         
           
             
               
                 
                   
                     
                       I 
                       ⁡ 
                       
                         ( 
                         λ 
                         ) 
                       
                     
                     
                       
                         I 
                         0 
                       
                       ⁡ 
                       
                         ( 
                         λ 
                         ) 
                       
                     
                   
                   = 
                   
                     e 
                     
                       
                         - 
                         
                           k 
                           ⁡ 
                           
                             ( 
                             λ 
                             ) 
                           
                         
                       
                       ⁢ 
                       d 
                     
                   
                 
               
               
                 
                   ( 
                   7 
                   ) 
                 
               
             
           
         
       
     
     In Equation (7), k(λ) denotes a value unique to a substance, determined depending on a wavelength, and d denotes the thickness of a substance. Here, as the left side of Equation (7) means the spectral transmittance t(λ), Equation (7) is replaceable with the following Equation (8).
 
 t (λ)= e   −k(λ)d   (8)
 
     Furthermore, the spectral absorbance a(λ) is represented by the following Equation (9).
 
 a (λ)= k (λ) d   (9)
 
     Thus, Equation (8) is replaceable with the following Equation (10).
 
 t (λ)= e   −a(λ)   (10)
 
     When the stained specimen after HE staining have been stained with three types of dyes, the H dye, the E dye, and the R dye, the following Equation (11) holds according to the Lambert-Beer law with regard to each wavelength λ. 
     
       
         
           
             
               
                 
                   
                     
                       I 
                       ⁡ 
                       
                         ( 
                         λ 
                         ) 
                       
                     
                     
                       
                         I 
                         0 
                       
                       ⁡ 
                       
                         ( 
                         λ 
                         ) 
                       
                     
                   
                   = 
                   
                     e 
                     
                       - 
                       
                         ( 
                         
                           
                             
                               
                                 k 
                                 H 
                               
                               ⁡ 
                               
                                 ( 
                                 λ 
                                 ) 
                               
                             
                             ⁢ 
                             
                               d 
                               H 
                             
                           
                           + 
                           
                             
                               
                                 k 
                                 E 
                               
                               ⁡ 
                               
                                 ( 
                                 λ 
                                 ) 
                               
                             
                             ⁢ 
                             
                               d 
                               E 
                             
                           
                           + 
                           
                             
                               
                                 k 
                                 R 
                               
                               ⁡ 
                               
                                 ( 
                                 λ 
                                 ) 
                               
                             
                             ⁢ 
                             
                               d 
                               R 
                             
                           
                         
                         ) 
                       
                     
                   
                 
               
               
                 
                   ( 
                   11 
                   ) 
                 
               
             
           
         
       
     
     In Equation (11), k H (λ), k E (λ), and k R (λ) represent k(λ) that corresponds to the H dye, the E dye, and the R dye, respectively, and they are, for example, the dye spectra (hereafter, referred to as “reference dye spectra”) of the respective dyes with which the stained specimen is stained. Furthermore, d H , d E , and d R  represent the virtual thickness of the H dye, the E dye, and the R dye at each sample point corresponding to each pixel location of a multiband image. Naturally, dye is dispersedly present in a stained specimen, and the idea of thickness is not precise; however, it is an index for a relative quantity of dye, indicating how much dye is present, as compared to a case where it is assumed that a stained specimen is stained with a single dye. That is, it can be said that d H , d E , and d R  represent the quantity of dye with regard to the H dye, the E dye, and the R dye, respectively. Furthermore, k H (λ), k E (λ), and k R (λ) may be easily obtained according to the Lambert-Beer law by preparing stained specimens that are individually stained by using the H dye, the E dye, and the R dye, respectively, and measuring the spectral transmittance thereof with a spectroscope. 
     Here, when the spectral transmittance at the position x is t(x,λ) and the spectral absorbance is a(x,λ), Equation (9) is replaceable with the following Equation (12).
 
 a ( x ,λ)= k   H (λ) d   H   +k   E (λ) d   E   +k   R (λ) d   R   (12)
 
     When the estimated spectral transmittance at the wavelength λ for the spectral transmittance data T^(x) estimated by using Equation (5) is t^(x,λ) and the estimated spectral absorbance is a^(x,λ), Equation (12) is replaceable with the following Equation (13). Here, t^ indicates that the symbol “^” is attached to the upper section of t, and a^ indicates that the symbol “^” is attached to the upper section of a.
 
 â ( x ,λ)= k   H (λ) d   H   +k   E (λ) d   E   +k   R (λ) d   R   (13)
 
     In Equation (13), there are three unknown variables, d H , d E , and d R ; therefore, they may be solved when simultaneous equations are produced with Equation (13) with regard to at least three different wavelengths λ. To further increase the accuracy, simultaneous equations may be produced with Equation (13) with regard to four or more different wavelengths λ and multiple regression analysis may be conducted. For example, when simultaneous equations are produced with Equation (13) with regard to three wavelengths λ1, λ2, λ3, matrix representation is possible as in the following Equation (14). 
     
       
         
           
             
               
                 
                   
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                     ⁢ 
                     
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                   ( 
                   14 
                   ) 
                 
               
             
           
         
       
     
     Here, Equation (14) is replaceable with the following Equation (15).
 
 Â ( x )= Kd ( x )  (15)
 
     In Equation (15), when the number of sample points in the wavelength direction is D, A^(x) is the matrix in the row D and the column 1 corresponding to a^(x,λ), K is the matrix in the row D and the column 3 corresponding to k(λ), and d(x) is the matrix in the row 3 and the column 1 corresponding to d H , d E , and d R  at the point x. Here, A^ indicates that the symbol “^” is attached to the upper section of A. 
     Furthermore, according to Equation (15), the dye quantities d H , d E , d R  are calculated by using the least squares method. The least squares method is a method for determining d(x) such that the sum of squares of an error is minimized in a single regression analysis equation, and it is calculable with the following Equation (16).
 
 {circumflex over (d)} ( x )=( K   T   K ) −1   K   T   Â ( x )  (16)
 
     In Equation (16), d^(x) is the estimated quantity of dye. When the estimated dye quantities d H ^, d E ^, d R ^ are substituted into Equation (12), the restored spectral absorbance a˜(x,λ) is obtained with the following Equation (17). Here, a˜ indicates that the symbol “˜ (tilde)” is attached to the upper section of a.
 
 ã ( x ,λ)= k   H (λ) {circumflex over (d)}   H   +k   E (λ) {circumflex over (d)}   E   +k   R (λ) {circumflex over (d)}   R   (17)
 
     Thus, the estimated error e(λ) for dye-quantity estimation is obtained based on the estimated spectral absorbance a^(x,λ) and the restored spectral absorbance a˜(x,λ) by using the following Equation (18).
 
 e (λ)= â ( x ,λ)− ã ( x ,λ)  (18)
 
     Hereafter, e(λ) is described as a residual spectrum. By using Equations (17), (18), the estimated spectral absorbance a^(x,λ) is also representable with the following Equation (19).
 
 â ( x ,λ)= k   H (λ) {circumflex over (d)}   H   +k   E (λ) {circumflex over (d)}   E   +k   R (λ) {circumflex over (d)}   R   +e (λ)  (19)
 
     After the dye quantities d H ^, d E ^, d R ^ are obtained, they are corrected so that changes in the quantity of dye in the stained specimen may be simulated. Here, the dye quantities d H ^, d E ^ for staining according to a staining method are corrected. The quantity of dye d R ^, which is the inherent color of a red blood cell, is not corrected. That is, the corrected dye quantities d H ^*, d E ^* are obtained by using appropriate coefficients α H , α E  according to the following Equations (20), (21).
 
 {circumflex over (d)}   H   •   =a   H   {circumflex over (d)}   H   (20)
 
 {circumflex over (d)}   E   •   =a   E   {circumflex over (d)}   E   (21)
 
     By substituting the corrected dye quantities d H ^*, d E ^* into Equation (17), a new restored spectral absorbance a˜*(x,λ) is obtained by using the following Equation (22).
 
 ã   • ( x ,λ)= k   H (λ) {circumflex over (d)}   H   •   +k   E (λ) {circumflex over (d)}   E   •   +k   R (λ) {circumflex over (d)}   R   (22)
 
     Furthermore, when the residual spectrum e(λ) is included, the new estimated spectral absorbance a^*(x,λ) is obtained by using the following Equation (23).
 
 â   • ( x ,λ)= k   H (λ) {circumflex over (d)}   H   *+k   E (λ) {circumflex over (d)}   E   *+k   R (λ) {circumflex over (d)}   R   +e (λ)  (23)
 
     By substituting the new restored spectral absorbance α˜*(x,λ) or the new estimated spectral absorbance α^*(x,λ) into Equation (10), the new spectral transmittance t*(x,λ) is obtained by using the following Equation (24).
 
 t *(λ)= e   −a*(λ)   (24)
 
     In Equation (24), the spectral absorbance a*(x,λ) means either the new restored spectral absorbance α˜*(x,λ) or the new estimated spectral absorbance α^*(x,λ). 
     By substituting Equation (24) into Equation (1), the new pixel value g*(x,b) is obtained by using the following Equation (25). In this case, calculation may be based on zero observed noise n(b).
 
 g *( x,b )=∫ λ   f ( b ,λ) s (λ) e (λ) t *( x ,λ) dλ   (25)
 
     Here, Equation (25) is replaced with the following Equation (26).
 
 G *( x )= HT *( x )  (26)
 
     In Equation (26), G*(x) is the matrix in the row B and the column 1 corresponding to g*(x,b), and T*(x) is the matrix in the row D and the column 1 corresponding to t*(x,b). Thus, it is possible to compose the pixel value G*(x) of the stained specimen while the quantity of dye is virtually changed. The above procedure allows virtual adjustment on the quantity of dye in the stained specimen. 
     The Lambert-Beer law is a formulation of attenuation of light transmitted through a translucent object based on the assumption that there is no refraction or scattering; however, refraction and scattering may occur in the actual stained specimen. Therefore, if attenuation of light due to a stained specimen is modeled by using only the Lambert-Beer law, errors occur due to this modeling. Unfortunately, it is extremely difficult to configure a model including refraction and scattering inside a living body specimen, and it is unexecutable in operation. Therefore, addition of a residual spectrum that is a modeling error including effects of refraction and scattering may prevent unnatural color fluctuation due to a physical model. 
     Nowadays, there is a disclosed technology of using the above-described Equations (1) to (26) to calculate, from a multiband image of the stained specimen, a single stain image representing the stained state due to the target single staining dye out of staining dyes with which a stained specimen is stained (for example, see Japanese Patent No. 5490568). The staining dyes used are, for example, coloring due to a DAB response (hereafter, referred to as DAB dye) using an EGFR antibody that recognizes an EGFR receptor as well as the above-described H dye and E dye. 
     Furthermore, according to the technology disclosed in Japanese Patent No. 5490568, a single stain image representing the stained state due to the H dye (hereafter, referred to as H dye-quantity image) is processed with a threshold to extract negative cells, and a single stain image representing the stained state due to the DAB dye (hereafter, referred to as DAB dye-quantity image) is processed with a threshold to extract positive cells, whereby a positive/negative determination may be conducted. Here, an example of the method for extracting cells is a method with which Otsu&#39;s method, or the like, is applied to the histogram of pixel values (quantity of dye) of a single stain image to automatically calculate a threshold for binarization so as to extract cells. 
     SUMMARY 
       FIG. 18  to  FIG. 22  are diagrams that illustrate a conventional problem. Specifically,  FIG. 18  is a diagram that illustrates a multiband image (stained-specimen image)  100 . Here, in  FIG. 18 , the darkest part is a positive cell PC that is visualized with the H dye and the DAB dye. Furthermore, the next darkest part is a negative cell NC that is visualized with the H dye.  FIG. 19  is a diagram that illustrates a DAB dye-quantity image  200  calculated from the multiband image  100  illustrated in  FIG. 18 . In the illustration of  FIG. 19 , a part closer to white has a larger quantity of DAB dye.  FIG. 20  is a diagram that illustrates an H dye-quantity image  300  calculated from the multiband image  100  illustrated in  FIG. 18 . In the illustration of  FIG. 20 , a part closer to white has a larger quantity of H dye.  FIG. 21  is a diagram that illustrates the histogram of pixel values (quantity of dye) of the H dye-quantity image  300 . In  FIG. 21 , the horizontal axis indicates a quantity of dye, and the vertical axis indicates a frequency. (a) of  FIG. 21  illustrates the histogram of the entire H dye-quantity image  300 . (b) of  FIG. 21  is the histogram of pixel values (quantity of dye) in only a partial range (0.01 to 0.1), extracted from the histogram illustrated in (a) of  FIG. 21 . (c) of  FIG. 21  is the histogram of pixel values (quantity of dye) in only a partial range (0.1 to 2), extracted from the histogram illustrated in (a) of  FIG. 21 .  FIG. 22  is a diagram that illustrates an image  400  after Otsu&#39;s method, or the like, is applied to the histogram illustrated in  FIG. 21  to automatically calculate a threshold for binarization and an H stained area ArH′ (part illustrated in white) due to the H dye is extracted. 
     When there are a small number of bands in a multiband image, little information causes a decrease in the accuracy of a calculated single stain image. For example, when cell nucleus immunostain for visualizing the positive cell PC with the DAB dye and cell nucleus counterstain for visualizing the negative cell NC with the H dye are applied to the stained specimen, the H dye tends to be excessively detected on an area of the positive cell PC of the H dye-quantity image  300  ( FIG. 18  to  FIG. 20 ). That is, artifacts occur in a range of a high quantity of dye in the histogram of pixel values (quantity of dye) of the H dye-quantity image  300  ( FIG. 21 ). Therefore, when a threshold is automatically calculated by applying Otsu&#39;s method, or the like, to the histogram of pixel values of the H dye-quantity image  300 , artifacts present in the histogram make it difficult to calculate a desirable threshold. As a result, as it is understood from the comparison between the multiband image  100  illustrated in  FIG. 18  or the DAB dye-quantity image  200  illustrated in  FIG. 19  and the H stained area ArH′ illustrated in  FIG. 22 , there is a problem in that the positive cell PC is sometimes extracted as the negative cell NC and it is difficult to extract cells in a desirable manner. 
     In some embodiments, an image processing device includes a processor including hardware. The processor is configured to: calculate each single stain image representing a stained state due to each stain from a stained-specimen image that has captured a specimen to which at least two types of stains are applied; sequentially perform, on all the at least two types of stains, an extraction process to extract a stained area due to a target stain from the single stain image of the target stain, starting from a stain having high specificity with regard to a target site; sequentially perform a correction process, on all at least one type of second stain except for a first stain having the highest specificity among the at least two types of stains, to correct the single stain image by excluding stained areas of all stains having higher specificity than the target stain from the single stain image of the target stain, starting from the stain having the high specificity; extract the stained area due to the target stain from a corrected single stain image that is obtained after the correction process is performed on the single stain image of the target stain, to extract the stained area due to the second stain; and analyze an image based on the stained area. 
     In some embodiments, an image processing method includes: calculating each single stain image representing a stained state due to each stain from a stained-specimen image that has captured a specimen to which at least two types of stains are applied; sequentially performing, on all the at least two types of stains, an extraction process to extract a stained area due to a target stain from the single stain image of the target stain, starting from a stain having high specificity with regard to a target site; sequentially performing a correction process, on all at least one type of second stain except for a first stain having the highest specificity among the at least two types of stains, to correct the single stain image by excluding stained areas of all stains having higher specificity than the target stain from the single stain image of the target stain, starting from the stain having the high specificity; extracting the stained area due to the target stain from a corrected single stain image that is obtained after the correction process is performed on the single stain image of the target stain, to extract the stained area due to the second stain; and analyzing an image based on the stained area. 
     In some embodiments, a non-transitory computer-readable recording medium recording an image processing program causing an image processing device to implement the image processing method. 
     The above and other features, advantages and technical and industrial significance of this disclosure will be better understood by reading the following detailed description of presently preferred embodiments of the disclosure, when considered in connection with the accompanying drawings. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a block diagram that illustrates a configuration of an imaging system according to a first embodiment; 
         FIG. 2  is a diagram that schematically illustrates a configuration of an imaging device illustrated in  FIG. 1 ; 
         FIG. 3  is a diagram that illustrates an example of the spectral sensitivity property of an RGB camera illustrated in  FIG. 2 ; 
         FIG. 4  is a diagram that illustrates an example of the spectral property of a first filter illustrated in  FIG. 2 ; 
         FIG. 5  is a diagram that illustrates an example of the spectral property of a second filter illustrated in  FIG. 2 ; 
         FIG. 6  is a flowchart that illustrates operation of the imaging system illustrated in  FIG. 1 ; 
         FIG. 7  is a flowchart that illustrates the procedure at Step S 2  illustrated in  FIG. 6 ; 
         FIG. 8  is a flowchart that illustrates the procedure of a single-stain image calculation step S 2 A illustrated in  FIG. 7 ; 
         FIG. 9  is a diagram that illustrates an example of the histogram of pixel values of a DAB dye-quantity image calculated at a stained-area extraction step S 2 C illustrated in  FIG. 7 ; 
         FIG. 10  is a diagram that illustrates an example of an image of DAB stained areas extracted at the stained-area extraction step S 2 C illustrated in  FIG. 7 ; 
         FIG. 11  is a diagram that illustrates an example of an H corrected dye-quantity image calculated at a single-stain image correction step S 2 D illustrated in  FIG. 7 ; 
         FIG. 12  is a diagram that illustrates an example of the histogram of pixel values of an H corrected dye-quantity image calculated at a stained-area extraction step S 2 E illustrated in  FIG. 7 ; 
         FIG. 13  is a diagram that illustrates an example of an image of H stained areas extracted at the stained-area extraction step S 2 E illustrated in  FIG. 7 ; 
         FIG. 14  is a flowchart that illustrates an image processing method according to a second embodiment; 
         FIG. 15  is a diagram that illustrates an example of a multiband image (stained-specimen image) of the stained specimen to which three types of stains, cell nucleus immunostain, cytoplasmic immunostain, and cell nucleus counterstain, have been applied; 
         FIG. 16  is a diagram that illustrates an example of a second corrected dye-quantity image that is calculated at a single-stain image correction step S 2 J illustrated in  FIG. 14 ; 
         FIG. 17  is a diagram that illustrates an example of a third corrected dye-quantity image that is calculated at a single-stain image correction step S 2 L illustrated in  FIG. 14 ; 
         FIG. 18  is a diagram that illustrates a conventional problem; 
         FIG. 19  is a diagram that illustrates a conventional problem; 
         FIG. 20  is a diagram that illustrates a conventional problem; 
         FIG. 21  is a diagram that illustrates a conventional problem; and 
         FIG. 22  is a diagram that illustrates a conventional problem. 
     
    
    
     DETAILED DESCRIPTION 
     With reference to drawings, an explanation is given below of an aspect (hereafter, referred to as “embodiment”) for implementing the disclosure. Furthermore, the disclosure is not limited to the embodiment described below. Moreover, in description of drawings, the same components are attached with the same reference numeral. 
     First Embodiment 
     Schematic Configuration of an Imaging System 
       FIG. 1  is a block diagram that illustrates a configuration of an imaging system  1  according to the first embodiment. 
     The imaging system  1  is a system that captures a stained specimen to which at least two types of stains have been applied and that processes a stained-specimen image during capturing. 
     Here, at least two types of stains applied to a stained specimen may be, for example, cell nucleus immunostain using Ki-67, ER, PgR, or the like, as an antibody, cell membrane immunostain using HER2, or the like, as an antibody, cytoplasmic immunostain using serotonin, or the like, as an antibody, cell nucleus counterstain using hematoxylin (H) as a dye, or cytoplasmic counterstain using eosin (E) as a dye. 
     Furthermore, as illustrated in  FIG. 1 , the imaging system  1  includes an imaging device  2  and an image processing device  3 . 
     Configuration of the Imaging Device 
       FIG. 2  is a diagram that schematically illustrates a configuration of the imaging device  2 . 
     The imaging device  2  is a device that acquires a multiband image (an equivalence of the stained-specimen image according to the disclosure) of a stained specimen S to which at least two types of stains have been applied. As illustrated in  FIG. 2 , the imaging device  2  includes a stage  21 , an illumination unit  22 , a tube lens  23 , an RGB camera  24 , and a filter unit  25 . 
     The stage  21  is a section on which the stained specimen S is placed, and it is configured to move under the control of the image processing device  3  so as to change the observed area of the stained specimen S. 
     The illumination unit  22  emits illumination light to the stained specimen S placed on the stage  21  under the control of the image processing device  3 . 
     The tube lens  23  focuses transmitted light, which has been emitted to the stained specimen S and passed through the stained specimen S, to the RGB camera  24 . 
       FIG. 3  is a diagram that illustrates an example of the spectral sensitivity property of the RGB camera  24 . 
     The RGB camera  24  includes an imaging element such as CCD (Charge Coupled Device) or CMOS (Complementary Metal Oxide Semiconductor), and it captures transmitted light having passed through the stained specimen S under the control of the image processing device  3 . For example, the RGB camera  24  has spectral sensitivity properties of respective R (red), G (green), and B (blue) bands illustrated in  FIG. 3 . 
       FIG. 4  is a diagram that illustrates an example of the spectral property of a first filter  252 .  FIG. 5  is a diagram that illustrates an example of the spectral property of a second filter  253 . 
     The filter unit  25  is disposed on the optical path leading from the tube lens  23  to the RGB camera  24 , and it limits the wavelength band of light focused on the RGB camera  24  to a predetermined range. The filter unit  25  includes: a filter wheel  251  that is rotatable under the control of the image processing device  3 ; and the first and the second filters  252 ,  253  provided on the filter wheel  251  and having different spectral properties (e.g., the spectral properties in  FIG. 4 ,  FIG. 5 ) to divide a transparent wavelength region of each R, G, B band into two. 
     Furthermore, the imaging device  2  acquires a multiband image of the stained specimen S under the control of the image processing device  3  as described below. 
     First, the imaging device  2  locates the first filter  252  on the optical path leading from the illumination unit  22  to the RGB camera  24  and emits illumination light from the illumination unit  22  to the stained specimen S. Then, the RGB camera  24  captures (first capturing) the transmitted light, which has been transmitted through the stained specimen S and passed through the first filter  252  and the tube lens  23 . 
     Then, the imaging device  2  locates the second filter  253  on the optical path leading from the illumination unit  22  to the RGB camera  24  and conducts second capturing in the same manner as the first capturing. 
     Thus, different images in three bands are acquired during the first and the second capturings so that multiband images in the total of six bands are acquired. 
     Here, the number of filters provided in the filter unit  25  may be not only two but also equal to or more than three to acquire images in more bands. Furthermore, the imaging device  2  may be configured not to include the filter unit  25  so as to acquire only RGB images by using the RGB camera  24 . Furthermore, instead of the filter unit  25 , it is possible to adopt a liquid crystal tunable filter or an acousto-optical tunable filter that is capable of changing the spectral property. Moreover, multiband images may be acquired by switching lights having different spectral properties and emitting them to the stained specimen S. 
     Configuration of the Image Processing Device 
     The image processing device  3  is a device that processes multiband images of the stained specimen S acquired by the imaging device  2 . As illustrated in  FIG. 1 , the image processing device  3  includes an image acquiring unit  31 , a control unit  32 , a storage unit  33 , an input unit  34 , a display unit  35 , and a calculating unit  36 . 
     The image acquiring unit  31  is configured as appropriate in accordance with the configuration of the imaging system  1 . For example, when the imaging device  2  is connected to the image processing device  3 , the image acquiring unit  31  is configured by using an interface that loads multiband images (image data) output from the imaging device  2 . Furthermore, when a server is installed to store multiband images acquired by the imaging device  2 , the image acquiring unit  31  is configured by using a communication device, or the like, connected to the server so as to conduct data communications with the server and acquire multiband images. Alternatively, the image acquiring unit  31  may be configured by using a reader to which a portable recording medium is removably attached and which reads multiband images stored in the recording medium. 
     The control unit  32  is configured by using a CPU (Central Processing Unit), or the like. The control unit  32  includes an image-acquisition controller  321  that controls operations of the image acquiring unit  31  and the imaging device  2  to acquire multiband images. Furthermore, the control unit  32  controls operations of the image acquiring unit  31  and the imaging device  2  on the basis of input signals input from the input unit  34 , multiband images input from the image acquiring unit  31 , and programs and data stored in the storage unit  33 . 
     The storage unit  33  is configured by using various IC memories, e.g., ROM (Read Only Memory) such as flash memory that is updatable and recordable, or RAM (Random Access Memory), a hard disk that is built in or connected via a data communication terminal, or an information storage device such as CD-ROM, and an information writing/reading device for the information storage device, or the like. As illustrated in  FIG. 1 , the storage unit  33  includes: a program storage unit  331  that stores an image processing program; an image-data storage unit  332  that stores multiband images acquired by the image acquiring unit  31  and image data, various parameters, and the like, used when the image processing program is executed; and a specificity-information storage unit  333  that stores specificity information illustrated in, for example, Table 1 described below. 
     The specificity information is information used for setting a processing rank during an extraction process and a correction process, described later, by the calculating unit  36 , and it is information in which stains are sequentially ranked, starting from the stain having the highest specificity (the highest binding power) with regard to the target site, which is targeted for staining. Specifically, in the specificity information, as illustrated in Table 1, cell nucleus immunostain ranks at the top, and cell membrane immunostain, cytoplasmic immunostain, cell nucleus counterstain, and cytoplasmic counterstain are set to be lower sequentially in rank. Here, the following Table 1 is an example, and the rank is changed depending on an individually produced antibody. For example, it is determined, depending on the produced antibody, that serotonin ranks first and Ki-67 ranks third. 
     
       
         
           
               
               
             
               
                 TABLE 1 
               
               
                   
               
               
                 Rank 
                 Types of stains (antibody or dye) 
               
               
                   
               
             
            
               
                 1 
                 Cell nucleus immunostain (Ki-67, ER, PgR) 
               
               
                 2 
                 cell membrane immunostain (HER2) 
               
               
                 3 
                 cytoplasmic immunostain (serotonin) 
               
               
                 4 
                 cell nucleus counterstain (H) 
               
               
                 5 
                 cytoplasmic counterstain (E) 
               
               
                   
               
            
           
         
       
     
     Hereafter, among at least two types of stains applied to the stained specimen S, the stain that ranks at the top (the highest specificity) is described as a first stain, and the other stains as second stains. 
     The input unit  34  is configured by using various types of input devices such as keyboard, mouse, touch panel, or various switches, and it outputs input signals corresponding to operation input to the control unit  32 . For example, the input unit  34  takes input of the type of stain applied to the stained specimen S in accordance with a user operation. Then, the control unit  32  adds, as metadata, information indicating the input type of stain to a multiband image stored in the image-data storage unit  332 . 
     The display unit  35  is implemented by using a display device such as LCD (Liquid Crystal Display), EL (Electro Luminescence) display, or CRT (Cathode Ray Tube) display, and it displays various types of screens based on display signals input from the control unit  32 . 
     The calculating unit  36  is configured by using a CPU, or the like. The calculating unit  36  reads a multiband image stored in the image-data storage unit  332  and recognizes the type of stain applied to the stained specimen S based on the metadata added to the multiband image. Then, the calculating unit  36  executes image processing on the multiband image in accordance with each recognized stain. As illustrated in  FIG. 1 , the calculating unit  36  includes a single-stain image calculating unit  361 , a processing-rank setting unit  365 , a stained-area extracting unit  366 , a single-stain image correcting unit  367 , and an analyzing unit  368 . 
     The single-stain image calculating unit  361  calculates single stain images each representing a stained state due to each stain from the multiband image. As illustrated in  FIG. 1 , the single-stain image calculating unit  361  includes a spectral-transmittance estimating unit  362 , a dye-quantity estimating unit  363 , and a calculating unit  364 . 
     The spectral-transmittance estimating unit  362  estimates the spectral transmittance of each pixel from the multiband image. 
     The dye-quantity estimating unit  363  estimates the quantity of dye in each pixel with regard to each stain by using the spectral transmittance estimated by the spectral-transmittance estimating unit  362 . 
     The calculating unit  364  calculates a single stain image of each of the stains based on the quantity of dye in each pixel with regard to each stain, estimated by the dye-quantity estimating unit  363 . 
     The processing-rank setting unit  365  sets the processing rank of each stain applied to the stained specimen S during an extraction process of the stained-area extracting unit  366  and a correction process of the single-stain image correcting unit  367  based on the specificity information stored in the specificity-information storage unit  333 . 
     The stained-area extracting unit  366  performs a process to extract a stained area due to the target stain from a single stain image of the target stain with every stain applied to the stained specimen S in accordance with the processing rank set by the processing-rank setting unit  365 . Furthermore, to extract a stained area due to the second stain, the stained-area extracting unit  366  extracts a stained area due to the target stain from a corrected single stain image that is obtained after the single-stain image correcting unit  367  performs a correction process on the single stain image of the target stain. 
     The single-stain image correcting unit  367  performs, on all the second stains in accordance with the processing rank, a correction process to correct the single stain image by excluding, from the single stain image of the target stain, stained areas of all the stains that rank higher than the target stain in the processing rank set by the processing-rank setting unit  365 . 
     The analyzing unit  368  analyzes an image based on a stained area extracted by the stained-area extracting unit  366 . 
     Operation of the Imaging System 
     Next, operation of the above-described imaging system  1  is explained. 
       FIG. 6  is a flowchart that illustrates operation of the imaging system  1 . 
     First, the image-acquisition controller  321  controls operation of the imaging device  2  so as to acquire a multiband image of the stained specimen S (Step S). The multiband image acquired by the imaging device  2  is stored in the storage unit  33  (the image-data storage unit  332 ) via the image acquiring unit  31 . 
     It is assumed below that two types of stains, cell nucleus immunostain and cell nucleus counterstain, have been applied to the stained specimen S. That is, for example, as illustrated in  FIG. 18 , on a multiband image  100  acquired by the imaging device  2 , the positive cell PC and the negative cell NC are visualized by the DAB dye and the H dye, respectively. For the convenience of the following explanation, the cell nucleus immunostain is described as DAB stain, and the cell nucleus counterstain as H stain. 
     After Step S 1 , the calculating unit  36  reads the multiband image  100  stored in the image-data storage unit  332  and recognizes the type of stains (here, the DAB stain and the H stain) applied to the stained specimen S based on the metadata attached to the multiband image  100 . Then, the calculating unit  36  executes image processing on the multiband image  100  in accordance with each recognized stain (Step S 2 ). 
       FIG. 7  is a flowchart that illustrates the procedure at Step S 2 . Here, the flowchart illustrated in  FIG. 7  is equivalent to the image processing method according to the disclosure. 
     The single-stain image calculating unit  361  calculates single stain images each representing the stained state of each of the stains, the DAB stain and the H stain, from the multiband image  100  (Step S 2 A: a single-stain image calculation step). 
     Here, according to the first embodiment, the single-stain image calculating unit  361  calculates, as a single stain image, a dye-quantity image in which a pixel value is a quantity of dye. 
       FIG. 8  is a flowchart that illustrates the procedure of the single-stain image calculation step S 2 A. 
     The spectral-transmittance estimating unit  362  estimates the spectral transmittance of each pixel from the multiband image  100  by using for example Wiener estimation (the above-described Equations (1) to (6)) (Step S 2 A 1 ). 
     After Step S 2 A 1 , the dye-quantity estimating unit  363  estimates the quantity of dye in each pixel with regard to each of the stains, the DAB stain and the H stain, by using the spectral transmittance estimated by the spectral-transmittance estimating unit  362  according to for example the Lambert-Beer law (the above-described Equations (7) to (16)) (Step S 2 A 2 ). 
     After Step S 2 A 2 , the calculating unit  364  calculates the DAB dye-quantity image  200  ( FIG. 19 ) in which each pixel value is the quantity of dye in each pixel with regard to the DAB stain, estimated by the dye-quantity estimating unit  363 , and the H dye-quantity image  300  ( FIG. 20 ) in which each pixel value is the quantity of dye in each pixel with regard to the H stain, estimated by the dye-quantity estimating unit  363  (Step S 2 A 3 ). 
     After Step S 2 A, the processing-rank setting unit  365  sets the processing ranks of the DAB stain and the H stain based on specificity information stored in the specificity-information storage unit  333  (Step S 2 B). Specifically, at Step S 2 B, the processing-rank setting unit  365  sets the first processing rank to the DAB stain (the first stain) and the second processing rank to the H stain (the second stain). 
     After Step S 2 B, the stained-area extracting unit  366  performs an extraction process to extract a DAB stained area due to the DAB stain (the first stain), which is set as the first processing rank by the processing-rank setting unit  365 , from the DAB dye-quantity image  200  of the DAB stain (Step S 2 C: a stained-area extraction step). 
       FIG. 9  is a diagram that illustrates an example of the histogram of pixel values of the DAB dye-quantity image  200  calculated at the stained-area extraction step S 2 C. Here, (a) to (c) of  FIG. 9  are histograms corresponding to (a) to (c) of  FIG. 21 .  FIG. 10  is a diagram that illustrates an example of an image  500  of DAB stained areas ArD (portions illustrated in white) extracted at the stained-area extraction step S 2 C. 
     Specifically, at Step S 2 C, the stained-area extracting unit  366  calculates the histogram ( FIG. 9 ) of pixel values of the DAB dye-quantity image  200 . Then, the stained-area extracting unit  366  applies Otsu&#39;s method, K-means algorithm, or the like, to the calculated histogram to automatically calculate a threshold for binarization and then extracts the DAB stained area ArD ( FIG. 10 ) due to the DAB stain. 
       FIG. 11  is a diagram that illustrates an example of an H corrected dye-quantity image  301  calculated at a single-stain image correction step S 2 D. In the illustration of  FIG. 11 , a part closer to white has a larger quantity of H dye. 
     After Step S 2 C, the single-stain image correcting unit  367  calculates the H corrected dye-quantity image  301  ( FIG. 11 ) that is obtained after the H dye-quantity image  300  of the H stain (the second stain) that is set as the second processing rank by the processing-rank setting unit  365  is corrected by excluding, from the H dye-quantity image  300 , the DAB stained area ArD of the DAB stain (the first stain) that is set as the first processing rank by the processing-rank setting unit  365  (Step S 2 D: a single-stain image correction step). 
     Moreover, the DAB stained area ArD excluded from the H corrected dye-quantity image  301  is a portion that is not the processing target for the subsequent process (Step S 2 E). 
     After Step S 2 D, the stained-area extracting unit  366  performs an extraction process to extract an H stained area due to the H stain (the second stain), which is set as the second processing rank by the processing-rank setting unit  365 , from the H corrected dye-quantity image  301  of the H stain (Step S 2 E: a stained-area extraction step). 
       FIG. 12  is a diagram that illustrates an example of the histogram of pixel values of the H corrected dye-quantity image  301  calculated at a stained-area extraction step S 2 E. Here, (a) to (c) of  FIG. 12  are histograms corresponding to (a) to (c) of  FIG. 21 .  FIG. 13  is a diagram that illustrates an example of an image  600  of H stained areas ArH (portions illustrated in white) extracted at the stained-area extraction step S 2 E. 
     Specifically, at Step S 2 E, the stained-area extracting unit  366  calculates the histogram ( FIG. 12 ) of pixel values of the H corrected dye-quantity image  301 . Then, the stained-area extracting unit  366  applies Otsu&#39;s method, K-means algorithm, or the like, to the calculated histogram to automatically calculate a threshold for binarization and then extracts the H stained area ArH ( FIG. 13 ) of the H stain. 
     After Step S 2 E, the analyzing unit  368  analyzes the image based on the DAB stained area ArD and the H stained area ArH (Step S 2 F: an analysis step). 
     For example, at Step S 2 F, the analyzing unit  368  performs morphology processing such as expansion, contraction, or compensation, on the DAB stained area ArD to correct the shape of positive cells and counts the positive cells. Furthermore, the analyzing unit  368  performs the above-described morphology processing on the H stained area ArH to correct the shape of negative cells and counts the negative cells. Then, the analyzing unit  368  makes a positive/negative determination. 
     After Step S 2 , the control unit  32  causes the display unit  35  to display processing results (images representing the DAB stained area ArD and the H stained area ArH, the count values of positive cells and negative cells, a result of positive/negative determination, and the like) obtained by the calculating unit  36  (Step S 3 ). 
     The first embodiment described above produces the following advantages. 
     The image processing device  3  according to the first embodiment sequentially performs an extraction process to extract a stained area due to the target stain from the target dye-quantity image with regard to every stain applied to the stained specimen S, starting from the stain having high specificity. Furthermore, the image processing device  3  sequentially performs a correction process, on all of the second stains in at least one type except for the first stain having the highest specificity among the stains applied to the stained specimen S, to correct the dye-quantity image of the target stain by excluding stained areas of all the stains having higher specificity than the target stain from the dye-quantity image, starting from the stain having the highest specificity. Furthermore, to extract a stained area due to the second stain, the image processing device  3  extracts a stained area due to the target stain from the corrected single stain image that is obtained after the correction process is performed on the dye-quantity image of the target stain. Then, the image processing device  3  analyzes the image based on the extracted stained area. 
     That is, with the image processing device  3 , as it is understood from the comparison between the histogram illustrated in  FIG. 12  and the histogram illustrated in  FIG. 21 , it is possible to remove artifacts that are present in the range of a large quantity of dye from the H corrected dye-quantity image  301  used to extract the H stained area ArH. Thus, application of Otsu&#39;s method, or the like, to the histogram of pixel values of the H corrected dye-quantity image  301  enables automatic calculation of a threshold in a desirable manner. 
     Therefore, as it is understood from the comparison between the H stained area ArH illustrated in  FIG. 13  and the H stained area ArH′ illustrated in  FIG. 22 , the image processing device  3  according to the first embodiment enables extraction of the H stained area ArH in a desirable manner and prevents the positive cell PC from being extracted as the negative cell NC. That is, there is an advantage such that cells are extractable in a desirable manner. 
     Furthermore, the image processing device  3  according to the first embodiment sets the processing rank of each stain applied to the stained specimen S for an extraction process and a correction process based on specificity information stored in the specificity-information storage unit  333 . This allows processing at an appropriate processing rank during an extraction process and a correction process, proper extraction of a stained area, and proper calculation of a corrected dye-quantity image. 
     Furthermore, to calculate a dye-quantity image, the image processing device  3  according to the first embodiment estimates the spectral transmittance of each pixel from a multiband image by using Wiener estimation, or the like, and estimates the quantity of dye in each pixel with regard to each stain by using the spectral transmittance according to the Lambert-Beer law. This allows high-accuracy estimation of the quantity of dye in each pixel with regard to each stain. 
     Second Embodiment 
     Next, a second embodiment is explained. 
     In the following explanation, the same configuration and step as those in the above-described first embodiment are attached with the same reference numeral, and their detailed explanations are omitted or simplified. 
     In the above-described first embodiment, an explanation is given of a process of the calculating unit  36  when two types of stains are applied to the stained specimen S. 
     In the second embodiment, an explanation is given of a process of the calculating unit  36  when three types of stains are applied to the stained specimen S. 
       FIG. 14  is a flowchart that illustrates an image processing method according to the second embodiment. Here,  FIG. 14  is a flowchart that illustrates the procedure of Step S 2 , corresponding to  FIG. 7 .  FIG. 15  is a diagram that illustrates an example of a multiband image (stained-specimen image)  700  of the stained specimen S to which three types of stains, cell nucleus immunostain, cytoplasmic immunostain, and cell nucleus counterstain, have been applied. 
     It is assumed below that three types of stains, cell nucleus immunostain, cytoplasmic immunostain, and cell nucleus counterstain, have been applied to the stained specimen S. Specifically, on the multiband image  700  acquired by the imaging device  2 , as illustrated in  FIG. 15 , for example, there are portions visualized by a cell nucleus immunostain St 1 , portions visualized by a cytoplasmic immunostain St 2 , and portions visualized by a cell nucleus counterstain St 3 . 
     At Step S 2 , the single-stain image calculating unit  361  first calculates single stain images each representing the stained state of each of the stains, the cell nucleus immunostain St 1 , the cytoplasmic immunostain St 2 , and the cell nucleus counterstain St 3 , from the multiband image  700  during the same process as that at Step S 2 A explained in the above-described first embodiment (Step S 2 G: a single-stain image calculation step). Specifically, at Step S 2 G, the single-stain image calculating unit  361  calculates a first dye-quantity image (not illustrated) in which each pixel value is the quantity of dye in each pixel with regard to the cell nucleus immunostain St 1 , a second dye-quantity image (not illustrated) in which each pixel value is the quantity of dye in each pixel with regard to the cytoplasmic immunostain St 2 , and a third dye-quantity image (not illustrated) in which each pixel value is the quantity of dye in each pixel with regard to the cell nucleus counterstain St 3 . 
     After Step S 2 G, the processing-rank setting unit  365  sets the processing ranks of the cell nucleus immunostain St 1 , the cytoplasmic immunostain St 2 , and the cell nucleus counterstain St 3  based on specificity information stored in the specificity-information storage unit  333  during the same process as that at Step S 2 B explained in the above-described first embodiment (Step S 2 H). Specifically, at Step S 2 H, the processing-rank setting unit  365  ranks the cell nucleus immunostain (the first stain) St 1  as the first processing rank, the cytoplasmic immunostain (the second stain) St 2  as the second processing rank, and the cell nucleus counterstain (the second stain) St 3  as the third processing rank. 
     After Step S 2 H, the stained-area extracting unit  366  performs an extraction process to extract a first stained area Ar 1  (see  FIG. 16 ) of the cell nucleus immunostain St 1  (the first stain), which is set as the first processing rank by the processing-rank setting unit  365 , from the first dye-quantity image of the cell nucleus immunostain St 1  during the same process as that at Step S 2 C explained in the above-described first embodiment (Step S 2 I: a stained-area extraction step). 
       FIG. 16  is a diagram that illustrates an example of a second corrected dye-quantity image  800  that is calculated at a single-stain image correction step S 2 J. 
     After Step S 2 I, the single-stain image correcting unit  367  calculates the second corrected dye-quantity image  800  ( FIG. 16 ) obtained after the second dye-quantity image is corrected by excluding the first stained area Ar 1  of the cell nucleus immunostain St 1 , which is set as the first processing rank by the processing-rank setting unit  365 , from the second dye-quantity image of the cytoplasmic immunostain (the second stain) St 2 , which is set as the second processing rank by the processing-rank setting unit  365 , during the same process as that at Step S 2 D explained in the above-described first embodiment (Step S 2 J: a single-stain image correction step). 
     Here, the first stained area Ar 1  excluded from the second corrected dye-quantity image  800  is a portion that is not the processing target for the subsequent process (Step S 2 K). 
     After Step S 2 J, the stained-area extracting unit  366  performs an extraction process to extract a second stained area Ar 2  (see  FIG. 17 ) of the cytoplasmic immunostain St 2  (the second stain), which is set as the second processing rank by the processing-rank setting unit  365 , from the second corrected dye-quantity image  800  of the cytoplasmic immunostain St 2  during the same process as that at Step S 2 E explained in the above-described first embodiment (Step S 2 K: a stained-area extraction step). 
       FIG. 17  is a diagram that illustrates an example of a third corrected dye-quantity image  900  that is calculated at a single-stain image correction step S 2 L. 
     After Step S 2 K, the single-stain image correcting unit  367  calculates the third corrected dye-quantity image  900  (see  FIG. 17 ) obtained after the third dye-quantity image of the cell nucleus counterstain (the second stain) St 3 , set as the third processing rank by the processing-rank setting unit  365 , is corrected by excluding the first and the second stained areas Ar 1 , Ar 2  of the cell nucleus immunostain St 1  and the cytoplasmic immunostain St 2 , which are set as the first and the second processing ranks by the processing-rank setting unit  365 , respectively, from the third dye-quantity image during the same process as that at the above-described Step S 2 J (Step S 2 L: a single-stain image correction step). 
     Here, the first and the second stained areas Ar 1 , Ar 2  excluded from the third corrected dye-quantity image  900  are portions that are not the processing targets for the subsequent process (Step S 2 M). 
     After Step S 2 L, the stained-area extracting unit  366  performs an extraction process to extract a third stained area Ar 3  ( FIG. 17 ) of the cell nucleus counterstain (the second stain) St 3 , which is set as the third processing rank by the processing-rank setting unit  365 , from the third corrected dye-quantity image  900  of the cell nucleus counterstain St 3  during the same process as that at Step S 2 I, S 2 K (Step S 2 M: a stained-area extraction step). 
     After Step S 2 M, the analyzing unit  368  analyzes the image based on the first to the third stained areas Ar 1  to Ar 3  in the same manner as Step S 2 F explained in the above-described first embodiment (Step S 2 N: an analysis step). 
     The above-described second embodiment produces the advantage similar to that of the above-described first embodiment even when three types of stains are applied to the stained specimen S. 
     Other Embodiments 
     Although the embodiments for implementing the disclosure are explained above, the disclosure does not need to be limited to only the above-described first and second embodiments. 
     According to the above-described first and second embodiments, dye-quantity images, in which a pixel value is a quantity of dye, is used as a single stain image according to the disclosure; however, this is not a limitation. 
     For example, at Step S 2 A 3 , after the quantity of dye in each pixel is estimated with regard to each stain, a single-stain color reproduction image, in which a pixel value (G*(x)) is an RGB value, is calculated based on the quantity of dye by using the above-described Equations (17) to (26), and the single-stain color reproduction image may be used as a single stain image according to the disclosure. Furthermore, to calculate a single-stain color reproduction image of the target stain, in the above-described Equation (20) and Equation (21), the coefficient α is 1 for the quantity of dye with regard to the target stain, and the coefficient α is 0 for the quantity of dye with regard to other stains. 
     Furthermore, the average value of bands with regard to the above-described single-stain color reproduction image is calculated, and the obtained gray-scaled image may be used as a single stain image according to the disclosure. 
     Moreover, a single-stain color reproduction image of each stain, in which a pixel value is an RGB value, is calculated directly based on the pixel value of each pixel of a multiband image by using a look-up table or a transformation matrix, and the single-stain color reproduction image may be used as a single stain image according to the disclosure. 
     According to the above-described first and second embodiments, at Step S 2 A 3 , the spectral transmittance of each pixel is estimated from a multiband image, and the quantity of dye in each pixel is estimated with regard to each stain by using the spectral transmittance; however, this is not a limitation. 
     For example, the quantity of dye in each pixel may be estimated with regard to each stain directly based on the pixel value of each pixel of a multiband image by using a look-up table or a dye-quantity estimation matrix obtained by regression analysis. 
     According to the above-described first and second embodiments, the image processing device  3  recognizes the type of stain applied to the stained specimen S based on the metadata attached to a multiband image and, based on specificity information stored in the specificity-information storage unit  333 , sets the processing rank of each stain for an extraction process and a correction process; however, this is not a limitation. 
     For example, the input unit  34  receives input of specificity information as well as the type of stain applied to the stained specimen S in accordance with user&#39;s operation. Then, the control unit  32  adds, as metadata, the information indicating the input type of stain and the specificity information to a multiband image stored in the image-data storage unit  332 . That is, the processing-rank setting unit  365  sets the processing rank of each stain for an extraction process and a correction process based on the metadata (the information indicating the type of stain and the specificity information). Therefore, with this configuration, the specificity-information storage unit  333  may be omitted. 
     Furthermore, in accordance with user&#39;s operation, the input unit  34  receives input of the processing rank of each stain as well as the type of stain applied to the stained specimen S. Then, the control unit  32  adds, as metadata, the information indicating the input type of stain and the information indicating the processing rank of each stain to a multiband image stored in the image-data storage unit  332 . That is, the stained-area extracting unit  366  and the single-stain image correcting unit  367  perform an extraction process and a correction process in accordance with the processing rank described in the metadata. Therefore, with this configuration, the processing-rank setting unit  365  and the specificity-information storage unit  333  may be omitted. 
     According to the above-described first and second embodiments, the image processing device  3  recognizes the type of stain applied to the stained specimen S based on the metadata attached to a multiband image; however, this is not a limitation. 
     For example, the type of stain applied to the stained specimen S may be determined based on the color distribution, or the like, of a multiband image. 
     In the above-described first and second embodiments, the combination of stains applied to the stained specimen S may be not only the combinations described in the first and the second embodiments but also other combinations. Furthermore, there may be four or more types of stains. 
     According to the above-described first and second embodiments, at Steps S 2 C, S 2 E, S 2 I, S 2 K, and S 2 M, Otsu&#39;s method, or the like, is applied to the calculated histogram to automatically calculate a threshold; however, this is not a limitation. For example, a fixed percentage, approximately the top 5 to 20%, for the quantity of dye with regard to the whole number of pixels may be used as the threshold. 
     The image processing device, the image processing method, and the image processing program according to the disclosure produce an advantage in that cells are extractable in a desirable manner. 
     Additional advantages and modifications will readily occur to those skilled in the art. Therefore, the disclosure in its broader aspects is not limited to the specific details and representative embodiments shown and described herein. Accordingly, various modifications may be made without departing from the spirit or scope of the general inventive concept as defined by the appended claims and their equivalents.