JP5887823B2 - Organization evaluation method - Google Patents

Description

  The present invention relates to a tissue evaluation method for immunohistochemistry.

  In the pathological diagnosis, dehydration is first performed in order to fix the collected tissue, and processing such as blocking with paraffin is performed. Then, the slice is cut into thin pieces having a thickness of 2 to 8 μm, the paraffin is removed, stained, and observed with a microscope. In this microscopic image, a pathologist makes a diagnosis based on morphological information such as a change in the size and shape of a cell nucleus and a change in a pattern as a tissue, and staining information. With the development of image digitization technology, in the field of pathological diagnosis, information necessary for pathologists to perform pathological diagnosis from pathological images input as digital color images using a microscope, digital camera, etc. Automated pathological diagnosis support devices that extract, measure and display have become widespread.

  For example, Patent Document 1 discloses a nucleus / cytoplasm distribution estimation unit that specifies a cell nucleus region and a cytoplasm region from a pathological image, and a glandular space extraction that specifies a glandular cavity region (a region that hardly includes cellular tissue) from a pathological image. Means, cancer site estimation means for determining whether cancer cells are present, progress determination means for determining the degree of progression of cancer, image display means for displaying a distribution map of cancer cells, progress, etc., A pathological diagnosis support apparatus having the above is disclosed.

  In Patent Document 2, a pathological specimen is stained with two types of dyes that selectively stain a normal site and a cancer site, and the staining density is evaluated from a spectral image using Lambert-Beer's law. A method for detecting cancer cells that determines the presence or absence of cancer cells is disclosed.

  However, regardless of which evaluation method is used, the tissue staining method is a conventional dye staining method (for example, hematoxylin-eosin staining; hereinafter referred to as HE staining) or a dye staining method using an enzyme (for example, DAB staining). The staining concentration greatly depends on environmental conditions such as temperature and time, and does not make full use of the accurate quantitative measurement performance of the pathological diagnosis support apparatus.

  In addition, fluorescent dyes with high quantitative performance are used for tissue staining as a labeling reagent in place of the dye (see Non-Patent Document 1), but the emission luminance is darker than that of the autofluorescence emitted by the tissue. The biomarker cannot be automatically determined by the light emission level.

  Also, antibody therapy using trastuzumab (Herceptin), which is an antibody of HER2 (human epidermal growth factor receptor type 2), has attracted attention as a new drug therapy for breast cancer. In order to determine the indication of this drug, it is necessary to confirm the presence or absence of HER2 overexpression in breast cancer tissue.

  HER2 was discovered as an oncogene having a structure similar to the human epidermal growth factor receptor (EGFR) gene. The HER2 protein encoded by the HER2 gene is a receptor-type glycoprotein that penetrates the cell membrane, and is composed of three domains: extracellular, transmembrane, and intracellular. When a growth factor is bound, it is activated by phosphorylation of tyrosine residues, and is involved in cell growth and malignant transformation through a signal transduction pathway. Overexpression of HER2 is also observed in lung cancer, colon cancer, stomach cancer, bladder cancer and the like. In breast cancer, 90% of the cause of HER2 protein overexpression is amplification of the HER2 gene. In addition, transcription abnormalities and post-transcriptional protein synthesis abnormalities are also considered causes.

  HER2 is considered to be a prognostic factor for breast cancer, and it is known that the prognosis of HER2-positive cases is significantly poor particularly in lymph node metastasis-positive cases. HER2 is attracting attention as an important factor that determines the indication of trastuzumab and as a predictor of the effect of anticancer agents such as anthracyclines and taxanes.

  Trastuzumab is a genetically modified HER2 monoclonal antibody and is used as a therapeutic agent for recurrent breast cancer. In order to reduce the side effects of trastuzumab, Genentic has derived most of the IgG from humans and only the part that binds to the HER2 receptor from mice. The main action of trastuzumab is to bind to the HER2 receptor and suppress the growth of cancer cells, but it is also said to have an action of destroying cancer cells via NK cells and monocytes.

JP 2004-286666 A Special table 2001-525580 gazette

"Pathology and Clinic Vol.25 2007 Special Issue: Immunohistochemistry useful for diagnosis" Bunkodo 2007

  In recent years, with the spread of molecular targeted drug therapy centering on antibody drugs, the need for accurate diagnostic methods is increasing in order to use molecular targeted drugs more efficiently. Also in pathological diagnosis, in order to diagnose a disease more accurately, it is required to quantitatively detect a very small amount of biomarker on a tissue section.

In general, overexpression of HER2 protein is examined by an immunohistochemical method (IHC method), and overexpression of HER2 gene is examined by FISH method. According to HER test guidelines, first, positive, negative, and border areas are selected by a simple IHC method, and if positive, trastuzumab administration is determined. For those in the border region by the IHC method, positive and negative are further selected by the FISH method.
When the IHC method and the FISH method are compared, the IHC method is simple but has a problem of low accuracy. On the other hand, the FISH method has high accuracy, but the work is complicated and the cost is high. In other words, it is desired to develop a technique that can achieve the same accuracy as the FISH method by the IHC method.

  Moreover, the conventional pathological diagnosis method described above requires visual recognition of an irregular cell shape stained in a specific hue. Therefore, in a situation where a subtle hue change is likely to occur depending on the measurement environment (temperature and humidity, etc.), the diagnosis results are likely to vary, and the diagnosis is highly personal, making it difficult to standardize the diagnosis. . Under the circumstances where the number of examinations is expected to increase in the future, it is desired to develop a technique that has low personality and can be automated.

  The present invention has been made in view of the above-described problems in the prior art, and expresses a specific biological material in a tissue slice as a dot-like fluorescence that does not depend on its cell form, thereby enabling the target tissue slice to be expressed. It is an object to make it possible to evaluate the expression level of a specific biological substance easily, efficiently, and with high accuracy.

In order to solve the above problem, the tissue evaluation method of the invention according to claim 1 is:
A staining step of staining a tissue section using a staining reagent in which a biological material recognition site is bound to a spherical particle containing a fluorescent substance;
By irradiating excitation light to the tissue sections together to autofluorescence in the tissue section, an irradiation step of generating a dot-like fluorescent to said spherical particles,
An imaging step of enlarging and imaging the autofluorescence and / or the dot-like fluorescence;
An evaluation step of detecting the dot-like fluorescence from the enlarged image and evaluating the expression level of the biological material corresponding to the biological material recognition site;
It includes,
The spherical particles are fluorescent substance-containing nanoparticles in which a fluorescent substance is dispersed inside the nanoparticles.

The invention according to claim 2 is the invention according to claim 1,
In the staining step, the tissue section is simultaneously stained using a staining reagent in which the biological material recognition site is bound to a spherical particle encapsulating the fluorescent material and an HE (hematoxylin-eosin) staining reagent.

The invention according to claim 3 is the invention according to claim 1 or 2,
The difference in light emission amount between the autofluorescence of the tissue section and the dot-like fluorescence of the spherical particles is the spherical particle fluorescence ≧ 1.1 × autofluorescence.

The invention according to claim 4 is the invention according to any one of claims 1 to 3,
In the imaging step, the autofluorescence and / or the dot-like fluorescence is magnified and imaged on an imaging surface in which a plurality of imaging elements are arranged in a two-dimensional direction, and the image is acquired.
The diameter of the spherical particles and / or the magnification of the dot-like fluorescence is set so that the dot-like fluorescence forms an image across two or more image sensors on the imaging surface.

According to the present invention, the evaluation of the expression level of a specific biological material in a tissue section confirms the presence or absence of dot-like fluorescence or the number (area) of dot-like fluorescence on the tissue section irradiated with excitation light. This is an extremely simple task. Therefore, it is possible to evaluate the expression level of a specific biological material in a tissue section with low personality, easily, efficiently, and with high accuracy. Further, since the same observation form is used regardless of the lesion site, it is preferable for automation and standardization of work necessary for pathological diagnosis.
Further, the present invention can be applied simultaneously with the widely used HE staining. In this case, autofluorescence (background) includes luminescence of the tissue itself when irradiated with excitation light, and luminescence of hematoxylin and eosin dyes by hematoxylin and eosin staining, and does not embed in the background. By securing the amount to the fluorescence indicating the expression level of the biological material, the expression of the biological material can be detected.

It is a figure which shows the preparation procedure of the nanoparticle which includes the fluorescent substance which the biological material recognition site | part couple | bonded. When one single fluorescent substance and one fluorescent substance-containing nanoparticle are observed in focus, (a) the amount of emitted light reaching the microscope imaging plane, and (b) observed from the microscope imaging plane. It is a figure which shows typically the image which acquired the image which is obtained with a microscope installation camera, and (c) 1 pixel of a microscope installation camera. When one fluorescent substance single substance and one fluorescent substance-containing nanoparticle are observed in a state of being out of focus, (a) the amount of emitted light reaching the microscope imaging plane, and (b) observed from the microscope imaging plane. It is a figure which shows typically the image which acquired the image which is acquired with the microscope installation camera. (A) The z-section of the tissue section when a part of the plurality of fluorescent substance-containing nanoparticles overlap each other when viewed from the microscope imaging plane, and (b) the tissue section of FIG. 4 (a) in focus. It is a figure which shows typically the magnitude | size of the light emission amount which reaches | attains a microscope image formation surface when observing a microscope with a microscope, and the image which acquired the image observed from the microscope image formation plane with a microscope installation camera (c). . (A) The z-direction cross section of the tissue section when a part of the plurality of fluorescent substance-containing nanoparticles overlap each other when viewed from the microscope imaging plane, and (b) the tissue section of FIG. It is a figure which shows typically the magnitude | size of the light emission amount which reaches | attains a microscope image formation surface when it observes with a microscope, and the image which acquired the image observed from a microscope image formation surface with a microscope installation camera. (A) The z-direction cross section of the tissue section when a plurality of fluorescent substance-containing nanoparticles are arranged side by side as viewed from the microscope imaging plane, and (b) the tissue of FIG. 6 (a) in focus. A fluorescence amount of the fluorescent substance-containing nanoparticles that reach the microscope imaging plane when the section is observed with a microscope, and (c) an image obtained from the microscope imaging plane by the microscope installation camera. It is a figure shown typically. (A) The z-direction section of the tissue section when a plurality of fluorescent substance-containing nanoparticles are arranged side by side as viewed from the microscope imaging plane, and (b) the tissue of FIG. The figure which shows typically the magnitude | size of the light emission amount which reaches | attains a microscope image formation surface when a section | slice is observed with a microscope, and the image which acquired the image observed from a microscope image formation surface with a microscope installation camera. is there. It is a figure which shows the relationship between the excitation of eosin, light emission wavelength, and fluorescence intensity. It is a figure which shows the microscope image acquired from the microscope installation camera when the labeling material A is used. It is a figure which shows the relationship between the number of bright spots at the time of using the label | marker material A, and a FISH score. It is a figure which shows the relationship between the number of bright spots at the time of using the labeling material B, and a FISH score. It is a figure which shows the relationship between the number of bright spots at the time of using the label | marker material C, and a FISH score. It is a figure which shows the relationship between the number of bright spots at the time of using the label | marker material D, and a FISH score. It is a figure which shows the square value of the correlation coefficient of the number of bright spots measured from the microscope image of each visual field, and a FISH score about each labeling material AD. It is the image acquired from the microscope installation camera when the labeling material d is used. It is the image acquired from the microscope installation camera when the labeling material e is used. It is the image acquired from the microscope installation camera when the labeling material a is used. It is the image acquired from the microscope installation camera when the labeling material b is used. It is the image acquired from the microscope installation camera when the labeling material c is used.

  Hereinafter, although the 1st and 2nd embodiment for implementing this invention is described, this invention is not limited to these.

In the first and second embodiments according to the present invention, nanoparticles enclosing a fluorescent substance bound with a biological substance recognition site (abbreviated as fluorescent substance-containing nanoparticles) are used as a fluorescent labeling material. The fluorescent substance-encapsulating nanoparticles to which the biological substance recognition site is bound are described in detail later, but are generally prepared according to the procedure shown in FIG. First, fluorescent substance-containing nanoparticles are produced by integrating and synthesizing fluorescent substances (S1, S2). Next, bioaffinity is imparted to the fluorescent substance-encapsulating nanoparticles (S3), and the biological substance recognition function is combined with a biological substance recognition site that specifically binds and / or reacts with a target (specific) biological substance. (Molecular recognition function) is given (S4).
In the first embodiment, a tissue section to be observed is stained with fluorescent substance-containing nanoparticles bound with a biological substance recognition site, and the stained tissue section is irradiated with excitation light to form a tissue section. Then, autofluorescence of the tissue and dot-like fluorescence are generated by the fluorescent substance-containing nanoparticles bound to the biological substance recognition site. Then, this fluorescence is magnified and imaged with a microscope, and dot-like fluorescence is detected from the magnified image to evaluate the expression level of the biological material corresponding to the biological material confirmation site.
In the second embodiment, the tissue section to be observed is stained using fluorescent substance-encapsulating nanoparticles combined with a biological substance recognition site and HE staining, and the stained tissue section is irradiated with excitation light. In the tissue section, tissue autofluorescence and eosin autofluorescence, and dot-like fluorescence are generated by the fluorescent substance-containing nanoparticles bound to the biological substance recognition site. Then, this fluorescence is magnified and imaged with a microscope, and dot-like fluorescence is detected from the magnified image to evaluate the expression level of the biological material corresponding to the biological material confirmation site.
The dot-like fluorescence is called a bright spot dot. The staining in the second embodiment is called simultaneous staining.

Here, before describing each embodiment, referring to FIGS. 2 to 7, the emission of fluorescent substance-containing nanoparticles by irradiation with excitation light of a microscope, which is common to the first and second embodiments, and The relationship between the image of the emitted light formed on the microscope observation surface (imaging surface) and the pixel (imaging device) when the image is acquired with a microscope camera will be described.
Here, in each embodiment, the microscope uses an upright microscope Axio Imager M2 manufactured by Carl Zeiss. The camera attached to the microscope (microscope installation camera) has a pixel size of 6.4 μm × 6.4 μm. The fluorescent substance-containing nanoparticles have a diameter of about 100 to 150 nm.

FIG. 2 shows (a) the amount of light emission that reaches the microscope imaging plane when (b) microscope imaging is observed when one fluorescent substance alone and one fluorescent substance-containing nanoparticle are observed in focus. It is a figure which shows typically the image which acquired the image observed from a plane with a microscope installation camera, and (c) 1 pixel of a microscope installation camera.
2 to 7, the microscope imaging plane is shown as the xy plane, and the direction orthogonal to this is shown as the z direction. Further, the fluorescence emission intensity of the fluorescent substance alone and the fluorescent substance-encapsulating nanoparticles is shown by the density of the pattern in the figure (the darker the intensity is higher). Further, TH1 is a light emission amount of “autofluorescence emission amount at the time of simultaneous staining + 10% thereof”.
The fluorescent substance-containing nanoparticles are spherical (including rugby ball shape) particles in which a plurality of fluorescent substances are accumulated as shown in FIG. 1 and have a diameter of about 100 to 150 nm. Since the fluorescence emitted from the fluorescent substance-encapsulating nanoparticles has a strong light emission amount, it diffuses to a larger diameter (diameter 1 to 2.1 μm) as shown in FIG. As shown in FIG. 2B, the diffused diameter is enlarged and imaged by 20 to 40 times according to the magnification of the microscope, and is acquired as a microscope image by a microscope installation camera. The enlarged image has a diameter of about 20 to 40 μm, and this image is a bright dot that is visually recognized by an observer. As described above, since one pixel of the microscope installation camera is 6.4 μm × 6.4 μm, the fluorescent substance-containing nanoparticle image (bright spot dot) formed on the imaging surface of the microscope installation camera is a plurality of pixels ( About 3-7 pixels).

  FIG. 3 shows (a) the amount of emitted light reaching the microscope imaging plane when observing one fluorescent substance single substance and one fluorescent substance-containing nanoparticle in an entirely out-of-focus state, and (b) It is a figure which shows typically the image which acquired the image observed from a microscope image plane with the microscope installation camera. As shown in FIG. 3, when the image is out of focus, the image observed from the microscope image is blurred around the fluorescent substance alone and the fluorescent substance-containing nanoparticles, so that the amount of luminescence reaching the microscope imaging plane is small. Each region becomes large. Therefore, even when the focus is out of focus, the fluorescent substance-containing nanoparticle image (bright spot dot) formed on the imaging surface of the microscope installation camera straddles a plurality of pixels.

  4A is a cross-sectional view in the z direction of a tissue section when a part of a plurality of fluorescent substance-containing nanoparticles overlap each other when viewed from the microscope imaging plane, and FIG. And (c) an image obtained by observing an image observed from the microscope imaging plane with a microscope installation camera. FIG. As shown in FIG. 4, when a part of the plurality of fluorescent substance-encapsulating nanoparticles overlap each other when viewed from the microscope imaging plane, the fluorescence of the fluorescent substance-encapsulating nanoparticles overlaps, and the fluorescence of the fluorescent substance-encapsulating nanoparticles is 1 It becomes fluorescence of an ellipse larger than two fluorescent substance-encapsulating nanoparticles (actually, the size is so small that it looks like a dot). Therefore, the fluorescent substance-encapsulating nanoparticle image (bright dot) formed on the imaging surface of the microscope installation camera extends over a plurality of pixels.

  5A is a cross-sectional view in the z direction of a tissue section when a part of a plurality of fluorescent substance-containing nanoparticles overlap each other when viewed from the microscope imaging plane, and FIG. 5B shows a state where FIG. And (c) an image obtained by observing an image observed from the microscope imaging plane with a microscope installation camera. FIG. As shown in FIG. 5, when the focus is shifted, the periphery of the fluorescent substance-containing nanoparticles is blurred, so that the amount of emitted light reaching the microscope imaging surface is small, but the region is large. Therefore, even when the focus is deviated, the fluorescent substance-encapsulating nanoparticle image (bright spot dot) formed on the imaging surface of the microscope installation camera extends over a plurality of pixels.

  6 (a) shows a z-direction cross section of a tissue section when a plurality of fluorescent substance-containing nanoparticles are arranged side by side when viewed from the microscope imaging plane, and (b) FIG. When the tissue section of a) was observed with a microscope, the fluorescence amount of the fluorescent substance-containing nanoparticles reaching the microscope imaging plane, and (c) the image observed from the microscope imaging plane were acquired with a microscope installation camera. It is a figure which shows an image typically. As shown in FIG. 6, when a plurality of fluorescent substance-containing nanoparticles are arranged side by side when viewed from the microscope imaging plane, a plurality of bright dot dots appear to be aligned. Therefore, the fluorescent substance-encapsulating nanoparticle image (bright dot) formed on the imaging surface of the microscope installation camera extends over a plurality of pixels.

  FIG. 7 shows (a) the z-direction section of the tissue section when a plurality of fluorescent substance-containing nanoparticles are arranged side by side when viewed from the microscope imaging plane, and (b) the state of FIG. A schematic view of the amount of luminescence that reaches the microscope imaging plane when the tissue section of a) is observed with a microscope, and (c) an image acquired by the microscope installation camera from the microscope imaging plane. FIG. As shown in FIG. 7, when the focus is shifted, the periphery of the fluorescent substance-containing nanoparticles is blurred, so that the amount of emitted light reaching the microscope imaging plane is reduced, but the region is increased. Therefore, even when the focus is deviated, the fluorescent substance-encapsulating nanoparticle image (bright spot dot) formed on the imaging surface of the microscope installation camera extends over a plurality of pixels.

From the above, depending on the position where the fluorescent substance-containing nanoparticles are adsorbed to the tissue and the focus adjustment by the observer, both circular and elliptical bright spots exist, but one bright spot dot is at least It is represented by an area of one pixel or more. Therefore, it is possible to automatically detect bright spot dots representing fluorescent substance-containing nanoparticles from the microscope image, for example, by performing binarization processing or the like on the microscope image of the cell slice obtained by the microscope installation camera. Moreover, it cannot be overemphasized that it can recognize visually. However, as will be described later, it is necessary that the difference in the amount of light between the autofluorescence and the fluorescence of the fluorescent substance-containing nanoparticles is not less than a predetermined value, and that the observer can distinguish and recognize both.
In addition, it is known that there are abnormal pixels that always emit high power in individual pixels of a digital camera. In order to prevent the influence of this abnormal pixel (determining that pixel as a bright spot dot), one bright spot dot is connected across a plurality of pixels in at least the x (y) direction or the x and y directions. It is preferable to be configured so as to be imaged. Considering the influence of focus and the like, when the pixel size is 6.4 μm × 6.4 μm, the diameter of the fluorescent substance-containing nanoparticles is preferably set to 100 μm or more when the magnification of the microscope is 20 times.

  In the experiment confirmation by the inventors of the present application, in the case of the method in which HE staining is performed simultaneously (the method of the second embodiment), 10% (1.1%) of the tissue autofluorescence and eosin autofluorescence (background). If the fluorescence of the fluorescent substance-encapsulating nanoparticles (that is, bright dot) has a difference in light emission amount of 2 times or more, either 8 bits (0 to 255 gradations) or 12 bits (0 to 4095 gradations) In this processing system, it was possible to automatically detect bright spot dots from a microscope image. When HE staining is not performed at the same time, if the fluorescent substance-containing nanoparticles have a difference in light emission amount of 10% (1.1 times) or more with respect to the autofluorescence of the tissue, 8 bits (0 to 255 floor) Tone) and 12-bit (0 to 4095 gradation) processing systems, it was possible to automatically detect bright spot dots. However, when the absolute light emission amount is small, visual observation tends to be difficult (requires visual recognition time) even if automatic detection is possible. In consideration of the stability of the automatic detection process and the instantaneous visibility, a light amount difference of 1.5 times or more, more preferably 2 times or more is desired between the autofluorescence and the bright dot.

FIG. 8 shows the relationship between the excitation wavelength, emission wavelength, and fluorescence intensity of eosin.
Eosin is a fluorescent substance, and this fluorescence contributes to improving the visibility of tissue and cell morphology. However, as the excitation light absorption of eosin and the fluorescence (luminescence) intensity of fluorescence increase, the visibility of fluorescence from the fluorescent substance-encapsulating nanoparticles decreases. Therefore, in the case of simultaneous staining in which HE staining is performed simultaneously, as the excitation light wavelength, excessive emission of eosin itself is suppressed, and the visibility of fluorescence from fluorescent substance-containing nanoparticles is increased (autofluorescence including fluorescence of eosin) In order to ensure the difference in fluorescence emission from the fluorescent substance-containing nanoparticles and distinguish them from each other, eosin itself does not absorb excitation light or it is difficult to absorb excitation light (thus, the fluorescence of eosin by excitation light). It is preferable to select a wavelength (which emits less light). Here, as shown in FIG. 8, when the excitation light wavelength is 560 nm or more, the absorption of excitation light of eosin becomes almost zero. On the other hand, the limit (upper limit) at which eosin fluorescence is observed is around 630 nm. Therefore, it is preferable to select the excitation light wavelength in the observation step in the range of 560 to 630 nm. Further, as the fluorescent substance, it is preferable to use a substance that emits fluorescence having a peak in the range of 580 to 690 nm, more preferably in the range of 600 to 630 nm by the excitation light. If a fluorescent substance having a peak in this range is selected, when the excitation light in the above range is selected, the fluorescence difference from the autofluorescence including fluorescence of eosin and the fluorescence from the fluorescent substance-containing nanoparticles is secured to distinguish between the two. This is because it is possible to ensure recognition (difference of 10% (1.1 times or more) between the two light amounts).
In the case where HE staining is not performed at the same time, the autofluorescence of the tissue is weak. Therefore, the wavelength range of the excitation light is not particularly limited to a general range of 200 nm to 700 nm, and the autofluorescence and fluorescent substance-containing nanoparticles are not particularly limited. Thus, it is possible to secure a difference in fluorescence emission from the light source so that both can be distinguished and recognized (a difference in light quantity between the two is 10% (1.1 times or more)).

In addition, when the excitation light is irradiated to the pathological section, the autofluorescence (B) caused by the eosin dye of HE staining is compared to the auto-luminescence (A) inherent to the tissue,
B ≧ 30 × A
It is said that.
Also, since the HE staining concentration itself varies from facility to facility,
B ≧ 50 × A
There will also be cases.
The amount C of autofluorescence that becomes the background of bright dots is C = A + B
Therefore, the luminous intensity is required so that the bright dot is not buried in C. In other words, the luminescence amount of eosin varies greatly depending on the individual, organ, cell site in the tissue section, etc., but in order to satisfy the above conditions (tissue cell specific autofluorescence + eosin autofluorescence 1.1 times or more) In addition, the amount of light emission of approximately 30 times or more of CdSe-Qdot is required for each fluorescent substance-containing nanoparticle.

-First embodiment-
Hereinafter, a first embodiment according to the present invention will be described.
[Fluorescent substance]
Examples of the fluorescent substance used in the first embodiment include fluorescent organic dyes and quantum dots (semiconductor particles). When excited by ultraviolet to near infrared light having a wavelength in the range of 200 to 700 nm, it preferably emits visible to near infrared light having a wavelength in the range of 400 to 1100 nm.

  Examples of fluorescent organic dyes include fluorescein dye molecules, rhodamine dye molecules, Alexa Fluor (Invitrogen) dye molecules, BODIPY (Invitrogen) dye molecules, cascade dye molecules, coumarin dye molecules, and eosin dyes. Examples include molecules, NBD dye molecules, pyrene dye molecules, Texas Red dye molecules, and cyanine dye molecules.

  Specifically, 5-carboxy-fluorescein, 6-carboxy-fluorescein, 5,6-dicarboxy-fluorescein, 6-carboxy-2 ′, 4,4 ′, 5 ′, 7,7′-hexachlorofluorescein, 6 -Carboxy-2 ', 4,7,7'-tetrachlorofluorescein, 6-carboxy-4', 5'-dichloro-2 ', 7'-dimethoxyfluorescein, naphthofluorescein, 5-carboxy-rhodamine, 6-carboxy Rhodamine, 5,6-dicarboxy-rhodamine, rhodamine 6G, tetramethylrhodamine, X-rhodamine, and Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 633, Alexa Fluor 633 , Alexa Fluor 750, BODIPY FL, BODIPY TMR, BODIPY 493/503, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, 30/30 ODIPY 650/665 (all manufactured by Invitrogen), methoxy coumarin, eosin, NBD, pyrene, Cy5, Cy5.5, can be mentioned Cy7 like. You may use individually or what mixed multiple types.

  Quantum dots include II-VI group compounds, III-V group compounds, or quantum dots containing group IV elements as components ("II-VI group quantum dots", "III-V group quantum dots", " Or “Group IV quantum dots”). You may use individually or what mixed multiple types.

  Specific examples include, but are not limited to, CdSe, CdS, CdTe, ZnSe, ZnS, ZnTe, InP, InN, InAs, InGaP, GaP, GaAs, Si, and Ge.

It is also possible to use a quantum dot having the above quantum dot as a core and a shell provided thereon. Hereinafter, as a notation of a quantum dot having a shell in this specification, when the core is CdSe and the shell is ZnS, it is expressed as CdSe / ZnS. For example, CdSe / ZnS, CdS / ZnS, InP / ZnS, InGaP / ZnS, Si / SiO ₂ , Si / ZnS, Ge / GeO ₂ , Ge / ZnS, and the like can be used, but are not limited thereto.
As the quantum dots, those subjected to surface treatment with an organic polymer or the like may be used as necessary. Examples thereof include CdSe / ZnS having a surface carboxy group (manufactured by Invitrogen), CdSe / ZnS having a surface amino group (manufactured by Invitrogen), and the like.

[Fluorescent substance-containing nanoparticles]
In the present embodiment, the fluorescent substance-encapsulating nanoparticles are those in which the fluorescent substance is dispersed inside the nanoparticles, whether the fluorescent substance and the nanoparticles themselves are chemically bonded or not. Good.
The material constituting the nanoparticles is not particularly limited, and examples thereof include polystyrene, polylactic acid, and silica.

  The fluorescent substance-containing nanoparticles used in the present embodiment can be produced by a known method. For example, silica nanoparticles encapsulating a fluorescent organic dye can be synthesized with reference to the synthesis of FITC-encapsulated silica particles described in Langmuir 8, Vol. 2921 (1992). Various fluorescent organic dye-containing silica nanoparticles can be synthesized by using a desired fluorescent organic dye in place of FITC.

  Silica nanoparticles encapsulating quantum dots can be synthesized with reference to the synthesis of CdTe-encapsulated silica nanoparticles described in New Journal of Chemistry, Vol. 33, p. 561 (2009).

  Polystyrene nanoparticles encapsulating a fluorescent organic dye may be copolymerized using an organic dye having a polymerizable functional group described in US Pat. No. 4,326,008 (1982) or polystyrene described in US Pat. No. 5,326,692 (1992). It can be produced using a method of impregnating nanoparticles with a fluorescent organic dye.

  The polymer nanoparticles encapsulating the quantum dots can be prepared by using the method of impregnating the quantum nanoparticles into polystyrene nanoparticles described in Nature Biotechnology, Vol. 19, page 631 (2001).

  Although the average particle diameter of the fluorescent substance-containing nanoparticles used in the present embodiment is not particularly limited, those having a size of about 30 to 800 nm can be used. Further, the coefficient of variation (= (standard deviation / average value) × 100%) indicating the variation in particle diameter is not particularly limited, but it is preferable to use a coefficient of 20% or less. The average particle diameter is obtained by taking an electron micrograph using a scanning electron microscope (SEM), measuring the cross-sectional area of a sufficient number of particles, and taking each measured value as the area of the circle. As sought. In the present application, the arithmetic average of the particle sizes of 1000 particles is defined as the average particle size. The coefficient of variation was also a value calculated from the particle size distribution of 1000 particles.

[Bonding of biological substance recognition sites and fluorescent substance-containing nanoparticles]
The biological material recognition site according to the present embodiment is a site that specifically binds and / or reacts with the target biological material. For example, a nucleotide chain, protein, antibody and the like can be mentioned. Accordingly, substances that bind to the target biological substance include antibodies that recognize the protein as an antigen, other proteins that specifically bind to the protein, and nucleic acids having a base sequence that hybridizes to the nucleic acid. Is mentioned. Specifically, an anti-HER2 antibody that specifically binds to HER2, which is a protein present on the cell surface, an anti-ER antibody that specifically binds to an estrogen receptor (ER) present in the cell nucleus, and actin that forms a cytoskeleton And an anti-actin antibody that specifically binds to. Among them, those in which anti-HER2 antibody and anti-ER antibody are bound to fluorescent substance-encapsulating nanoparticles can be used for selection of breast cancer medication, and are preferable.

  The mode of binding between the biological substance recognition site and the fluorescent substance-encapsulating nanoparticles is not particularly limited, and examples thereof include covalent bonding, ionic bonding, hydrogen bonding, coordination bonding, physical adsorption, and chemical adsorption. A bond having a strong bonding force such as a covalent bond is preferred from the viewpoint of bond stability.

  Moreover, there may be an organic molecule that connects between the biological substance recognition site and the fluorescent substance-encapsulating nanoparticles. For example, in order to suppress nonspecific adsorption with a biological substance, a polyethylene glycol chain can be used, and SM (PEG) 12 manufactured by Thermo Scientific can be used.

  When the biological substance recognition site is bound to the fluorescent substance-encapsulating silica nanoparticles, the same procedure can be applied regardless of whether the fluorescent substance is a fluorescent organic dye or a quantum dot. For example, a silane coupling agent that is a compound widely used for bonding an inorganic substance and an organic substance can be used. This silane coupling agent is a compound having an alkoxysilyl group that gives a silanol group by hydrolysis at one end of the molecule and a functional group such as a carboxyl group, an amino group, an epoxy group, an aldehyde group at the other end, Bonding with an inorganic substance through an oxygen atom of the silanol group. Specifically, mercaptopropyltriethoxysilane, glycidoxypropyltriethoxysilane, aminopropyltriethoxysilane, a silane coupling agent having a polyethylene glycol chain (for example, PEG-silane no. SIM6492.7 manufactured by Gelest), etc. Is mentioned. When using a silane coupling agent, you may use 2 or more types together.

  A known procedure can be used for the reaction procedure of the fluorescent organic dye-encapsulated silica nanoparticles and the silane coupling agent. For example, the obtained fluorescent organic dye-encapsulated silica nanoparticles are dispersed in pure water, aminopropyltriethoxysilane is added, and the mixture is reacted at room temperature for 12 hours. After completion of the reaction, fluorescent organic dye-encapsulated silica nanoparticles whose surface is modified with an aminopropyl group can be obtained by centrifugation or filtration. Subsequently, by reacting an amino group with a carboxyl group in the antibody, the antibody can be bound to the fluorescent organic dye-encapsulated silica nanoparticles via an amide bond. If necessary, a condensing agent such as EDC (1-Ethyl-3- [3-Dimethylaminopropyl] carbohydrate Hydrochloride: manufactured by Pierce (registered trademark)) can also be used.

  If necessary, a linker compound having a site capable of directly binding to a fluorescent organic dye-encapsulated silica nanoparticle modified with an organic molecule and a site capable of binding to a molecular target substance can be used. As a specific example, sulfo-SMCC having both a site that selectively reacts with an amino group and a site that reacts selectively with a mercapto group (Sulfosuccinimidyl 4 [N-maleimidemethyl] -cyclohexane-1-carboxylate: manufactured by Pierce) When used, by binding the amino group of the fluorescent organic dye-encapsulated silica nanoparticles modified with aminopropyltriethoxysilane and the mercapto group in the antibody, antibody-bound fluorescent organic dye-encapsulated silica nanoparticles can be produced.

  When the biological substance recognition site is bound to the fluorescent substance-encapsulated polystyrene nanoparticles, the same procedure can be applied regardless of whether the fluorescent substance is a fluorescent organic dye or a quantum dot. That is, by impregnating polystyrene nanoparticles having functional groups such as amino groups with fluorescent organic dyes and quantum dots, it is possible to obtain fluorescent substance-containing polystyrene nanoparticles having functional groups, and thereafter using EDC or sulfo-SMCC. Thus, antibody-bound fluorescent substance-encapsulated polystyrene nanoparticles can be produced.

  Antibodies that recognize specific antigens include M. actin, MS actin, SM actin, ACTH, Alk-1, α1-antichymotrypsin, α1-antitrypsin, AFP, bcl-2, bcl-6, β-catenin, BCA 225, CA19-9, CA125, calcitonin, calretinin, CD1a, CD3, CD4, CD5, CD8, CD10, CD15, CD20, CD21, CD23, CD30, CD31, CD34, CD43, CD45, CD45R, CD56, CD57, CD61, CD68, CD79a, "CD99, MIC2", CD138, chromogranin, c-KIT, c-MET, collagen type IV, Cox-2, cyclin D1, keratin, cytokeratin (high molecular weight), pankeratin, pankeratin, cytokeratin 5/6, cytokeratin 7, cytokeratin 8, cytokeratin 8/18, cytokeratin 14, cytokeratin 19, cytokeratin 20, CMV, E-cadherin, EGFR, ER, EMA, EBV, factor VIII related antigen, Fassin, FSH, galectin-3, gastrin, GFAP, glucagon, glycophorin A, granzyme B, hCG, hGH, Helicobacter pylori, HBc antigen, HBs antigen, hepatocyte Specific antigen, HER2, HSV-I, HSV-II, HHV-8, IgA, IgG, IgM, IGF-1R, Inhibin, Insulin, Kappa L chain, Ki67, Lambda L chain, LH, Lysozyme, Macrophage, Melan A, MLH-1, MSH-2, Myeloperoxidase, Myogenin, Myoglobin, Myosin, Neurofilament, NSE, p27 (Kip1), p53, p53, P63, PAX 5, PLAP, Pneumocystis carini, Podoplanin (D2-40) , PGR, prolactin, PSA, prostatic acid phosphatase, Renal Cell Carcinoma, S100, somatostatin, spectrin, synaptophysin, TAG-72, TdT, thyroglobulin, TSH, TTF-1, TRAcP, tryptase, villin, vimentin, WT1, Zap- 70 is mentioned.

[Dyeing method]
Hereinafter, the staining method of the first embodiment will be described. The staining method according to the first embodiment is not limited to a pathological section tissue and can also be applied to cell staining.
In addition, a method for preparing a section to which the staining method of the first embodiment can be applied is not particularly limited, and a method prepared by a known method can be used.

1) Deparaffinization process The pathological section is immersed in a container containing xylene to remove paraffin. The temperature is not particularly limited, but can be performed at room temperature. The immersion time is preferably 3 minutes or longer and 30 minutes or shorter. If necessary, xylene may be exchanged during the immersion.
Next, the pathological section is immersed in a container containing ethanol to remove xylene. The temperature is not particularly limited, but can be performed at room temperature. The immersion time is preferably 3 minutes or longer and 30 minutes or shorter. Further, if necessary, ethanol may be exchanged during the immersion.
Next, the pathological section is immersed in a container containing water to remove ethanol. The temperature is not particularly limited, but can be performed at room temperature. The immersion time is preferably 3 minutes or longer and 30 minutes or shorter. Moreover, you may exchange water in the middle of immersion as needed.

2) Activation process The activation process of the target biological substance is performed according to a known method. The activation conditions are not particularly defined, but as the activation liquid, 0.01 M citrate buffer (pH 6.0), 1 mM EDTA solution (pH 8.0), 5% urea, 0.1 M Tris-HCl buffer, etc. are used. be able to. As the heating device, an autoclave, a microwave, a pressure cooker, a water bath, or the like can be used. The temperature is not particularly limited, but can be performed at room temperature. The temperature can be 50-130 ° C. and the time can be 5-30 minutes.
Next, the section after the activation treatment is immersed in a container containing PBS (Phosphate Buffered Saline) and washed. The temperature is not particularly limited, but can be performed at room temperature. The immersion time is preferably 3 minutes or longer and 30 minutes or shorter. If necessary, the PBS may be replaced during the immersion.

3) Staining using fluorescent substance-encapsulated nanoparticles combined with biological substance recognition sites Place PBS dispersion of fluorescent substance-encapsulated nanoparticles combined with biological substance recognition sites on a pathological section and react with the target biological material . By changing the biological material recognition site to be combined with the fluorescent substance-containing nanoparticles, staining corresponding to various biological materials becomes possible. In the case of using fluorescent substance-encapsulating nanoparticles to which several kinds of biological substance recognition sites are bound, each fluorescent substance-encapsulating nanoparticle PBS dispersion may be mixed in advance or separately placed on a pathological section separately. Also good.
The temperature is not particularly limited, but can be performed at room temperature. The reaction time is preferably 30 minutes or more and 24 hours or less.
It is preferable to drop a known blocking agent such as BSA-containing PBS before staining with fluorescent substance-encapsulating nanoparticles.
Next, the stained section is immersed in a container containing PBS, and unreacted fluorescent substance-containing nanoparticles are removed. The temperature is not particularly limited, but can be performed at room temperature. The immersion time is preferably 3 minutes or longer and 30 minutes or shorter. If necessary, the PBS may be replaced during the immersion. A cover glass is placed on the section and sealed. A commercially available encapsulant may be used as necessary.

4) Observation under a fluorescence microscope Using a fluorescence microscope, the number of fluorescent bright spots or emission luminance is measured from a microscope image of a wide field of view with respect to a stained pathological section. An excitation light source and a fluorescence detection optical filter corresponding to the absorption maximum wavelength and fluorescence wavelength of the fluorescent substance used are selected. Measurement of the number of bright spots or emission luminance can be performed using commercially available image analysis software, for example, all bright spot automatic measurement software G-Count manufactured by Zeonstrom Co., Ltd.
Note that image analysis itself using a microscope is well known, and for example, a technique disclosed in Japanese Patent Laid-Open No. 9-197290 can be used.
The field of view of the microscopic image is preferably 3 mm ² or more, more preferably 30 mm ² or more, and further preferably 300 mm ² or more.
Based on the number of bright spots and / or emission luminance measured from the microscope image, the expression level of the target biological substance is evaluated. Specifically, it can be evaluated that the higher the number of bright spots, the higher the expression level of the biological substance. Moreover, it can be evaluated that the higher the emission luminance, the higher the expression level of the biological substance. Visual evaluation is also possible.

  Hereinafter, although an Example is given and 1st Embodiment is described in detail, this invention is not limited to these.

[Synthesis of phosphor-containing nanoparticles]
Synthesis Example 1: Fluorescent Organic Dye-Incorporated Silica: Synthesis of Cy5 Encapsulated Silica Nanoparticles
Cy5-encapsulated silica nanoparticles (hereinafter referred to as nanoparticles 1) were produced by the following steps (1) to (5).
Step (1): 1 mg (0.00126 mmol) of an N-hydroxysuccinimide ester derivative of Cy5 (manufactured by GE Healthcare) and 400 μL (1.796 mmol) of tetraethoxysilane were mixed.
Process (2): Ethanol 40mL and 14% ammonia water 10mL were mixed.
Step (3): The mixture prepared in Step (1) was added to the mixture prepared in Step (2) while stirring at room temperature. Stirring was performed for 12 hours from the start of addition.
Step (4): The reaction mixture was centrifuged at 10,000 G for 60 minutes, and the supernatant was removed.
Step (5): Ethanol was added to disperse the sediment, followed by centrifugation again. Washing with ethanol and pure water was performed once by the same procedure.
The obtained nanoparticles 1 were observed with a scanning electron microscope (SEM; Model S-800, manufactured by Hitachi (registered trademark)). As a result, the average particle size was 110 nm and the variation coefficient was 12%.

(Synthesis Example 2: Quantum dot-encapsulated silica: synthesis of CdSe / ZnS-encapsulated silica nanoparticles having an emission wavelength of 655 nm)
CdSe / ZnS-encapsulated silica nanoparticles (hereinafter referred to as nanoparticles 2) were produced by the following steps (1) to (5).
Step (1): 10 μL of CdSe / ZnS decane dispersion (Invitrogen Qdot655) and 40 μL of tetraethoxysilane were mixed.
Step (2): 4 mL of ethanol and 1 mL of 14% aqueous ammonia were mixed.
Step (3): The mixture prepared in Step (1) was added to the mixture prepared in Step (2) while stirring at room temperature. Stirring was performed for 12 hours from the start of addition.
Step (4): The reaction mixture was centrifuged at 10,000 G for 60 minutes, and the supernatant was removed.
Step (5): Ethanol was added to disperse the sediment, followed by centrifugation again. Washing with ethanol and pure water was performed once by the same procedure.
When the obtained nanoparticles 2 were observed with a scanning electron microscope, the average particle size was 130 nm and the coefficient of variation was 13%.

[Binding of antibody to fluorescent substance-encapsulated silica nanoparticles]
By the method of the following steps (1) to (12), an antibody was bound to the fluorescent substance-containing silica nanoparticles. Here, although the example using the nanoparticle 1 is shown, the same applies to the nanoparticle 2.
Step (1): 1 mg of nanoparticles 1 was dispersed in 5 mL of pure water. Next, 100 μL of an aminopropyltriethoxysilane aqueous dispersion was added and stirred at room temperature for 12 hours.
Step (2): The reaction mixture was centrifuged at 10,000 G for 60 minutes, and the supernatant was removed.
Step (3): Ethanol was added to disperse the sediment, followed by centrifugation again. Washing with ethanol and pure water was performed once by the same procedure.
When the FT-IR measurement of the obtained amino group-modified silica nanoparticles was performed, absorption derived from the amino group was observed, and it was confirmed that the amino group was modified.

Step (4): The amino group-modified silica nanoparticles obtained in step (3) were adjusted to 3 nM using PBS containing 2 mM of EDTA (ethylenediaminetetraacetic acid).
Step (5): SM (PEG) 12 (manufactured by Thermo Scientific, succinimidyl-[(N-maleidopropionamid) -dodecaethyleneglycol] ester) is mixed with the solution prepared in step (4) to a final concentration of 10 mM. The reaction was performed for 1 hour.
Step (6): The reaction mixture was centrifuged at 10,000 G for 60 minutes, and the supernatant was removed. Step (7): PBS containing 2 mM of EDTA was added to disperse the precipitate, and then centrifuged again. The washing | cleaning by the same procedure was performed 3 times. Finally, it was redispersed with 500 μL of PBS.

Step (8): When 100 μg of the anti-HER2 antibody was dissolved in 100 μL of PBS, 1 M dithiothreitol (DTT) was added and reacted for 30 minutes.
Step (9): Excess DTT was removed from the reaction mixture with a gel filtration column to obtain a reduced anti-HER2 antibody solution.

Step (10): Using the nanoparticle 1 as a starting material, the particle dispersion obtained in step (7) and the reduced anti-HER2 antibody solution obtained in step (9) are mixed in PBS and allowed to react for 1 hour. It was.
Step (11): 4 μL of 10 mM mercaptoethanol was added to stop the reaction.
Step (12): The reaction mixture was centrifuged at 10,000 G for 60 minutes, and the supernatant was removed. Then, PBS containing 2 mM of EDTA was added, the precipitate was dispersed, and centrifuged again. The washing | cleaning by the same procedure was performed 3 times. Finally, 500 μL of PBS was redispersed to obtain fluorescent substance-encapsulated silica nanoparticles to which the anti-HER2 antibody was bound.

  Fluorescent substance-encapsulated silica nanoparticles bound with anti-HER2 antibody obtained from nanoparticle 1 as a starting material are labeled material A, and phosphor-encapsulated silica nanoparticles bound with anti-HER2 antibody obtained from nanoparticle 2 as a starting material. This is labeled material B.

[Antibody binding to fluorescent substance]
As a comparative example, an anti-HER2 antibody was bound to Cy5 by the methods of the following steps (1) to (7).
Step (1): When 100 μg of the anti-HER2 antibody was dissolved in 100 μL of PBS, 1M dithiothreitol (DTT) was added and reacted for 30 minutes.
Step (2): Excess DTT was removed from the reaction mixture with a gel filtration column to obtain a reduced anti-HER2 antibody solution.
Step (3): 1 mg (0.00126 mmol) of an N-hydroxysuccinimide ester derivative of Cy5 (manufactured by GE Healthcare) was adjusted to 3 nM using PBS containing 2 mM of EDTA (ethylenediaminetetraacetic acid).
Step (4): SM (PEG) 12 (manufactured by Thermo Scientific, succinimidyl-[(N-maleidopropionamidyl) -dodecaethyleneglycol] ester) is mixed with the solution prepared in step (3) to a final concentration of 10 mM. The reaction was performed for 1 hour.

Step (5): The reaction mixture obtained in step (4) was mixed with the reduced anti-HER2 antibody solution obtained in step (2) in PBS and allowed to react for 1 hour.
Step (6): 10 mM mercaptoethanol μL4 was added to stop the reaction.
Step (7): Excess mercaptoethanol was removed by a gel filtration column to obtain a reduced anti-HER2 antibody solution (labeling material D) bound to Cy5.

  In the same manner as in Cy5, a labeling material C is obtained by binding an anti-HER2 antibody to CdSe.

[Tissue staining using fluorescent substance-containing nanoparticles]
Using the produced antibody-binding labeling materials A to D by the methods of the following steps (1) to (10), immunostaining was performed using adjacent sections of human breast tissue whose FISH score was measured in advance. As a stained section, a tissue array slide (CB-A712) manufactured by Cosmo Bio was used. Twenty-four sections with a FISH score of 1-9 were used.

Step (1): The pathological section was immersed in a container containing xylene for 30 minutes. The xylene was changed three times during the process.
Step (2): The pathological section was immersed in a container containing ethanol for 30 minutes. The ethanol was changed three times during the process.
Step (3): The pathological section was immersed in a container containing water for 30 minutes. The water was changed three times along the way.
Step (4): The pathological section was immersed in 10 mM citrate buffer (pH 6.0) for 30 minutes.
Step (5): Autoclaving was performed at 121 degrees for 10 minutes.
Step (6): The section after autoclaving was immersed in a container containing PBS for 30 minutes.
Step (7): PBS containing 1% BSA was placed on the tissue and left for 1 hour.
Step (8): Labeling materials A to D bound with anti-HER2 antibody diluted to 0.05 nM with PBS containing 1% BSA were placed on each tissue section and left for 3 hours.
Step (9): The stained sections were each immersed for 30 minutes in a container containing PBS.
Step (10): Aquex manufactured by Merck Chemicals was dropped, and then a cover glass was placed and sealed.

〔Experimental result〕
About the tissue section dye | stained using each labeling material AD, a visual field (observation area) was changed and several microscope images were acquired, and the number of the fluorescence luminescent spots was measured from each microscope image with image analysis software. As an example, FIG. 9 shows a microscope image obtained from a microscope installation camera when the labeling material A is used.
The microscope is an upright microscope Axio Imager M2 manufactured by Carl Zeiss, the objective lens is set to 20 times, and excitation light having a wavelength of 630 to 670 nm is irradiated to form an image of fluorescence emitted from the tissue section. Then, a microscope image (image data) was acquired with a microscope installation camera (monochrome), and the number of bright spots was measured with image analysis software. The camera has a pixel size of 6.4 μm × 6.4 μm, a vertical pixel count of 1040, and a horizontal pixel count of 1388 (imaging area 8.9 mm × 6.7 mm).
For each of the labeling materials A to D, the correlation coefficient R between the measured number of bright spots and the FISH score was calculated in each visual field. The FISH score corresponds to the overexpression level of the HER2 gene, and the larger the value of the FISH score, the higher the overexpression level of the HER2 gene.

FIG. 10 shows the number of bright spots measured from microscopic images of a plurality of different visual fields (0.3 mm ² , 3 mm ² , 32 mm ² , 324 mm ² ) and FISH when the labeling material A (Cy5 inclusion labeling material) is used. It is a figure which shows the relationship with a score. The value of R ² shown in the figure is the square value of the correlation coefficient between the number of bright spots and the FISH score.

FIG. 11 shows the number of bright spots measured from microscopic images of a plurality of different fields of view (0.3 mm ² , 3 mm ² , 32 mm ² , 324 mm ² ) when using the labeling material B (CdSe-containing labeling material), and FISH It is a figure which shows the relationship with a score.

FIG. 12 shows the number of bright spots measured from microscopic images of a plurality of different visual fields (0.3 mm ² , 3 mm ² , 32 mm ² , 324 mm ² ) and the FISH score when the labeling material C (CdSe) is used. It is a figure which shows a relationship.

FIG. 13 shows the number of bright spots measured from microscopic images of a plurality of different visual fields (0.3 mm ² , 3 mm ² , 32 mm ² , 324 mm ² ) and the FISH score when the labeling material D (Cy5) is used. It is a figure which shows a relationship.

Table 1 and FIG. 14 show the square value (R ² ) of the correlation coefficient between the number of bright spots and the FISH score measured from the microscopic images of each visual field (observation area) for each of the labeling materials A to D.

When a tissue section is stained with the labeling material A and the number of bright spots is measured from a microscopic image of a field of 0.3 mm ² , the square value (R ² ) of the correlation coefficient between the number of bright spots and the FISH score is The correlation was not found between the number of bright spots and the FISH score. This is probably because the field of view of 0.3 mm ² is too narrow and the variation is large.

When a tissue section is stained with the labeling material A and the number of bright spots is measured from a microscopic image of a 3 mm ² visual field, the square value (R ² ) of the correlation coefficient between the number of bright spots and the FISH score is 0. 5387. This value is approximately 0.734 when converted to the correlation coefficient R, and it can be said that there is a strong correlation between the number of bright spots and the FISH score.

When a tissue section is stained with the labeling material A and the number of bright spots is measured from a microscope image of a field of view of 32 mm ² , the square value (R ² ) of the correlation coefficient between the number of bright spots and the FISH score is 0. 9011. When the visual field is 32 mm ² , it can be said that the correlation is stronger than when the visual field is 3 mm ² .

When the tissue section was stained with the labeling material A and the number of bright spots was measured from a microscopic image of a field of view of 324 mm ² , the square value (R ² ) of the correlation coefficient between the number of bright spots and the FISH score was 0. It was 9887. When the field of view is 324 mm ² , it can be said that the correlation is stronger than when the field of view is 32 mm ² .

Similarly, when the labeling material B was used, it was found that there was a correlation between the number of bright spots and the FISH score in the field of view of 3 mm ² or more, and the correlation coefficient increased as the field of view increased.

Further, from the results when the labeling materials A and B were used, it was found that the square value (R ² ) of the correlation coefficient between the number of bright spots and the FISH score was sufficiently close to 1 in the field of view of 324 mm ² .

  On the other hand, when the tissue section was stained using the labeling material C or the labeling material D, no correlation was found between the number of bright spots and the FISH score.

  Moreover, even when the focus of the microscope was slightly shifted above or below the thickness (usually several μm) of the tissue section to be observed, no significant difference was observed in the above situation.

From the above results, when the tissue sections are observed with a wide field of view using the labeling materials A and B, the correlation between the number of bright spots and the FISH score is good, and the expression level of HER2 is evaluated based on the number of bright spots. Was found to be possible. That is, the expression level of a specific biological substance can be evaluated by measuring the number of bright spots from an image with a field of view of 3 mm ² or more without using a time-consuming method such as the FISH method. It is an effective alternative. Therefore, an immunohistochemical image can be evaluated efficiently.

Since the labeling materials A and B use particles containing a fluorescent substance and are brighter than the labeling materials C and D using a single fluorescent substance, each point of the bright spot is captured from the image. Easy to measure the number of bright spots with high accuracy.
In this way, by using a fluorescent labeling material with high brightness as a labeling material for immunohistochemistry, even in a general-purpose fluorescence microscope system, a labeling material that binds to a target molecule even in a low-magnification image of about 20 times the objective lens Can be recognized.
In addition, by combining a plurality of low-magnification fluorescent images, a large area on the tissue section can be observed. As a result, it is considered that the detection and quantitative evaluation of a trace amount of biological material that has been conventionally overlooked are possible.

  In addition, although the said Example demonstrated the case where the expression level of HER2 was evaluated based on the number of fluorescent luminescent spots, the light emission brightness | luminance of a fluorescent luminescent spot was measured from a microscope image, and the expression level of HER2 based on light emission brightness | luminance. Can also be evaluated.

-Second Embodiment-
Next, a second embodiment according to the present invention will be described.
In the second embodiment, in order to observe the morphology of tissue cells, a case is shown in which HE staining is used together (simultaneous staining) with staining using fluorescent substance-encapsulating nanoparticles bound with a biological substance recognition site.
The fluorescent substance, the fluorescent substance-encapsulating nanoparticles, the biological substance recognition site, and the binding between the biological substance recognition site and the fluorescent substance-encapsulating nanoparticles according to the second embodiment are the same as those described in the first embodiment. Therefore, explanation is used. That is, also in the second embodiment, a fluorescent labeling material (fluorescent substance-encapsulating nanoparticles to which a biological substance recognition site is bound) as described in the first embodiment is used.

Here, the autofluorescence of tissue and eosin, for example, emits fluorescence having a wavelength ranging from about 400 to 600 nm (having a peak at about 440 nm) when irradiated with an excitation wavelength of 350 nm, and is approximately when irradiated with an excitation wavelength of 490 nm. Fluoresce at wavelengths ranging from 500 to 650 nm (with a peak at about 540 nm). Therefore, a common cut filter can be used by selecting, as a fluorescent substance for a fluorescent labeling material, a substance that emits fluorescence whose wavelength overlaps with the autofluorescence of tissue and eosin when irradiated with excitation light of the same wavelength. It is possible to observe their fluorescence in the same visual field. In this case, since the fluorescence emitted from the fluorescent substance-containing nanoparticles is high in brightness, it can be identified without being buried in autofluorescence (background) of tissues or the like. The intensity of the autofluorescence emitted by the tissue and the intensity of the fluorescence emitted by the fluorescent substance-encapsulating nanoparticles can be appropriately determined by selecting the wavelength of the excitation light and the fluorescent substance so that the overlapping state of the fluorescent wavelengths is preferable. It is preferable to adjust so that it may have a balance.
The fluorescent substance in the present embodiment is a fluorescent substance having a fluorescence wavelength of 580 to 800 nm when irradiated with an excitation light wavelength of 550 to 700 nm.
A preferred fluorescent material is a fluorescent material having a fluorescent wavelength of 580 to 650 nm when irradiated with an excitation light wavelength of 575 to 620 nm.
A more preferable fluorescent substance is a fluorescent substance having a fluorescent wavelength of 600 to 650 nm when irradiated with an excitation light wavelength of 575 to 600 nm.
The most preferable fluorescent substance is a fluorescent substance having a fluorescent wavelength of 600 to 620 nm when irradiated with an excitation light wavelength of 580 to 595 nm.

  The immunohistochemical method according to the second embodiment includes a step of staining a tissue section using a fluorescent labeling material as described above, and a step of performing HE staining as necessary (referred to as a “staining step”). And a step of obtaining predetermined information by irradiating excitation light (referred to as “observation step”).

[Dyeing process]
In the staining step in the immunohistochemical method of the second embodiment, a tissue section is stained using a specific fluorescent labeling material as described above. The object to be stained is not limited to a pathological section tissue, and can also be applied to cell staining. The method for preparing a section to which the staining method in the second embodiment can be applied is not particularly limited, and a section prepared by a known method can be used. For example, when using a paraffin-embedded section that is widely used as a pathological section, it may be stained by the following procedure.

1) Deparaffinization process The deparaffinization process is the same as that described in the first embodiment, and thus the description is incorporated herein.

2) Activation process Since the activation process is the same as that described in the first embodiment, description thereof is incorporated.

3) Staining treatment with fluorescent labeling material A PBS dispersion liquid of fluorescent labeling material provided with a biological substance recognition site and fluorescent substance-containing nanoparticles is prepared, placed on a pathological section, and reacted with the target biological substance. When staining multiple biological materials, prepare PBS dispersions of fluorescent labeling materials with biological material recognition sites corresponding to each biological material and different fluorescent material-containing nanoparticles, and use them as pathological sections. It is only necessary to place it and react with the target biological substance. When placing on a pathological section, a PBS dispersion of each fluorescent labeling material may be mixed in advance, or may be placed separately and sequentially.

  The temperature is not particularly limited, but can be performed at room temperature. The reaction time is preferably from 30 minutes to 24 hours. It should be noted that a known blocking agent such as BSA-containing PBS is preferably dropped before staining with a fluorescent labeling material.

  Next, the stained section is immersed in a container containing PBS to remove unreacted fluorescent substance-containing nanoparticles. The temperature is not particularly limited, but can be performed at room temperature. The immersion time is preferably 3 minutes or longer and 30 minutes or shorter. If necessary, PBS may be exchanged during immersion.

Further, in the second embodiment, since it is possible to observe the morphology of the tissue using the autofluorescence emitted from the tissue itself, it is not necessary to use other staining solution or the like. HE (hematoxylin-eosin) staining is performed together. Hematoxylin is a blue-violet pigment that stains cell nuclei, bone tissue, part of cartilage tissue, serous components, etc. (basophilic tissue, etc.). Eosin is a red to pink pigment that stains cytoplasm, soft tissue connective tissue, red blood cells, fibrin, endocrine granules, and the like (eosinophilic tissue, etc.). Of these, eosin emits autofluorescence. When HE staining is performed together with staining with the predetermined fluorescent labeling material in the present invention, eosin that stains cytoplasm and the like together with autofluorescence emitted by tissues and the like when irradiated with excitation light. Due to autofluorescence emitted from, it becomes easier to obtain information on the morphology of cells or tissues.
After the staining process, a cover glass is placed on the section and sealed. A commercially available encapsulant may be used as necessary.

[Observation process]
The observation step irradiates the tissue section stained in the above step with excitation light, thereby acquiring cell or tissue morphology information (cell morphology information) based on the tissue autofluorescence and eosin autofluorescence, and a fluorescent substance. This is a step of obtaining information (referred to as antigen molecule information in the second embodiment) indicating the expression level of a specific biological substance in a cell or tissue based on fluorescence by the encapsulated nanoparticles.

  The excitation light should have an appropriate wavelength such that the tissue and eosin used as necessary emit autofluorescence of a desired wavelength, and the fluorescent substance in the phosphor-containing nanoparticle emits fluorescence of the desired wavelength. The irradiation means for the excitation light is not particularly limited. For example, a stained tissue section may be irradiated with excitation light having an appropriate wavelength and output from a laser light source included in a fluorescence microscope using a filter that selectively transmits a predetermined wavelength as necessary.

  Cell morphology information and antigen molecule information are acquired in the same field, i.e., both the autofluorescence of the tissue and the fluorescence emitted by the fluorescent labeling material obtained from a single stained section are included in the same field of view. It is preferable to distinguish and recognize them and acquire cell morphology information and antigen molecule information based on each of them. Of course, if necessary, for example, by using an appropriate filter that can sufficiently reduce one of tissue autofluorescence or fluorescence emitted by a fluorescent labeling material, only cell morphology information is acquired in a certain visual field, You may make it acquire antigen molecule information in a visual field.

  In addition, cell morphology information and antigen molecule information may be obtained from a (fluorescence) microscope tube for quick observation, or an image taken by a camera installed in the (fluorescence) microscope is displayed separately. You may make it acquire by displaying on a means (a monitor etc.) and observing it. Depending on the fluorescent substance to be used, there may be a case where antigen molecule information can be obtained from an image taken by a camera even if antigen molecule information cannot be sufficiently obtained by visual observation from a microscope barrel.

  Obtaining the antigen molecule information includes, for example, measuring the number of target antigen molecules or the target antigen luminance per cell based on the number of fluorescent bright spots or emission luminance from a microscope camera. What is necessary is just to select the excitation light source and the optical filter for fluorescence detection corresponding to the absorption maximum wavelength and fluorescence wavelength of the used fluorescent substance. For measurement of the number of bright spots or emission luminance, it is preferable to use commercially available image analysis software (for example, G-Count, a total bright spot automatic measurement software manufactured by Zeonstrom Inc.), but the measuring means is particularly limited. It is not a thing. From this measurement result, the expression level of the target biological substance (here, antigen) can be evaluated. Specifically, it can be evaluated that the higher the number of bright spots, the higher the expression level of the biological substance. Moreover, it can be evaluated that the higher the emission luminance, the higher the expression level of the biological substance. Visual evaluation is also possible.

An example (Example 2) in the second embodiment will be described below.
Synthesis Example 1 Synthesis of Fluorescent Organic Dye (Cy5) Encapsulated Silica Nanoparticles Silica nanoparticles encapsulating Cy5 as a fluorescent organic dye by the methods of the following steps (1) to (4) (hereinafter referred to as “nanoparticle A”). Was made.
Step (1): 1 mg (0.00126 mmol) of an N-hydroxysuccinimide ester derivative of Cy5 (manufactured by GE Healthcare) and 400 μL (1.796 mmol) of tetraethoxysilane were mixed.
Process (2): Ethanol 40mL and 14% ammonia water 10mL were mixed.
Step (3): The mixture prepared in Step (1) was added to the mixture prepared in Step (2) while stirring at room temperature. Stirring was performed for 12 hours from the start of addition.
Step (4): The reaction mixture was centrifuged at 10,000 g for 60 minutes, and the supernatant was removed. Ethanol was added to disperse the sediment and centrifuged again. In the same procedure, washing with ethanol and pure water was performed once.
When 1000 pieces of the obtained nanoparticles A were observed with a scanning electron microscope (SEM; model S-800 manufactured by Hitachi, Ltd.), the average particle size was 110 nm and the variation coefficient was 12%.

Synthesis Example 2 Synthesis of Quantum Dot (CdSe / ZnS) Encapsulated Silica Nanoparticles Silica nanoparticles encapsulating CdSe / ZnS as quantum dots (hereinafter referred to as “nanoparticle B”) by the methods of the following steps (1) to (4). .) Was produced. Note that the peak of the emission wavelength of CdSe / ZnS is 655 nm.
Step (1): 10 μL of CdSe / ZnS decane dispersion (Invitrogen Qdot655) and 40 μL of tetraethoxysilane were mixed.
Step (2): 4 mL of ethanol and 1 mL of 14% aqueous ammonia were mixed.
Step (3): The mixture prepared in Step (1) was added to the mixture prepared in Step (2) while stirring at room temperature. Stirring was performed for 12 hours from the start of addition.
Step (4): The reaction mixture was centrifuged at 10,000 g for 60 minutes, and the supernatant was removed. Ethanol was added to disperse the sediment and centrifuged again. Washing with ethanol and pure water was performed once by the same procedure.
When SEM observation was performed on 1000 of the obtained nanoparticles B, the average particle size was 130 nm and the coefficient of variation was 13%.

[Synthesis Example 3] Synthesis of fluorescent organic dye (Texas Red) -encapsulated silica nanoparticles Using Texas Red sulfonyl chloride instead of Cy5 N-hydroxysuccinimide ester derivative (manufactured by GE Healthcare) as a raw material in Step (1) Except for the above, in the same manner as in Synthesis Example 1, silica nanoparticles (hereinafter referred to as “nanoparticles C”) containing Texas Red as a fluorescent organic dye were produced.
When 1000 pieces of the obtained nanoparticles C were observed with a scanning electron microscope (SEM; model S-800 manufactured by Hitachi, Ltd.), the average particle size was 108 nm and the coefficient of variation was 11%.

[Preparation Example 1] Preparation of labeling material in which anti-HER2 antibody is bound to nanoparticle A Anti-HER2 antibody is bound to nanoparticle A prepared in Synthesis Example 1 according to the following procedure, and labeling material ("labeling material a ").
Step (1): 1 mg of nanoparticles A was dispersed in 5 mL of pure water. 100 μL of aminopropyltriethoxysilane aqueous dispersion was added and stirred at room temperature for 12 hours.
Step (2): The reaction mixture was centrifuged at 10,000 g for 60 minutes, and the supernatant was removed.
Step (3): Ethanol was added to disperse the sediment, followed by centrifugation again. Washing with ethanol and pure water was performed once by the same procedure. When the product was subjected to FT-IR measurement, absorption derived from an amino group was observed, and it was confirmed that the nanoparticle A could be modified with an amino group.
Step (4): The amino group-modified nanoparticles A obtained in step (3) were adjusted to 3 nM using PBS (phosphate buffered saline) containing 2 mM of EDTA (ethylenediaminetetraacetic acid). .
Step (5): SM (PEG) 12 (manufactured by Thermo Scientific, succinimidyl-[(N-maleomidopropionamid) -dodecaethyleneglycol] ester) is mixed with the solution prepared in step (4) to a final concentration of 10 mM. The reaction was performed for 1 hour.
Step (6): The reaction mixture was centrifuged at 10,000 g for 60 minutes and the supernatant was removed. Step (7): PBS containing 2 mM of EDTA was added, the precipitate was dispersed, and centrifuged again. The washing | cleaning by the same procedure was performed 3 times. Finally, it was redispersed using 500 μL PBS.
Step (8): When 100 μg of anti-HER2 antibody was dissolved in 100 μL of PBS, 1M dithiothreitol (DTT) was added and reacted for 30 minutes.
Step (9): Excess DTT was removed from the reaction mixture with a gel filtration column to obtain a reduced anti-HER2 antibody solution.
Step (10): Using the nanoparticle A as a starting material, the particle dispersion obtained in step (7) and the reduced anti-HER2 antibody solution obtained in step (9) are mixed in PBS and reacted for 1 hour. I let you.
Step (11): 4 μL of 10 mM mercaptoethanol was added to stop the reaction.
Step (12): The reaction mixture was centrifuged at 10,000 g for 60 minutes, the supernatant was removed, PBS containing 2 mM of EDTA was added, the precipitate was dispersed, and centrifuged again. The washing | cleaning by the same procedure was performed 3 times. Finally, nanoparticles A (labeling material a) to which anti-HER2 antibody was redispersed using 500 μL PBS were obtained.

[Production Example 2] Production of labeling material in which anti-HER2 antibody is bound to nanoparticle B Nanoparticle B was used in place of nanoparticle A, except that nanoparticle B was used, and anti-HER2 was applied to nanoparticle B. A labeling material to which an antibody was bound (referred to as “labeling material b”) was prepared.

[Preparation Example 3] Preparation of labeling material in which anti-HER2 antibody is bound to nanoparticle C Nanoparticle C was used in place of nanoparticle A, except that nanoparticle C was used. A labeling material to which the antibody was bound (referred to as “labeling material c”) was prepared.

[Production Example 4] Production of labeling material in which anti-HER2 antibody is bound to fluorescent organic dye (Cy5) A labeling material in which anti-HER2 antibody is bound to Cy5 (referred to as "labeling material d") is produced by the following procedure. did.
Step (1): When 100 μg of the anti-HER2 antibody was dissolved in 100 μL of PBS, 1M dithiothreitol (DTT) was added and reacted for 30 minutes.
Step (2): Excess DTT was removed from the reaction mixture with a gel filtration column to obtain a reduced anti-HER2 antibody solution.
Step (3): PBS (phosphate buffered saline) containing 1 mg (0.00126 mmol) of N-hydroxysuccinimide ester derivative of Cy5 (GE Healthcare) and 2 mM of EDTA (ethylenediaminetetraacetic acid) And adjusted to 3 nM.
Step (4): SM (PEG) 12 (manufactured by Thermo Scientific, succinimidyl-[(N-maleomidopropionamid) -dodecaethyleneglycol] ester) is mixed with the solution prepared in step (3) to a final concentration of 10 mM. The reaction was performed for 1 hour.
Step (5): The reduced anti-HER2 antibody solution obtained in step (2) was mixed with the reaction mixture obtained in step (4) in PBS and allowed to react for 1 hour.
Step (6): 4 μL of 10 mM mercaptoethanol was added to stop the reaction.
Step (7): Excess mercaptoethanol was removed by a gel filtration column to obtain a reduced anti-HER2 antibody (labeling material d) solution labeled with Cy5.

[Production Example 5] Production of labeling material in which anti-HER2 antibody is bound to quantum dot (CdSe / ZnS) According to the protocol of Qdot Antibody Conjugation Kit manufactured by Life Technology, anti-HER2 antibody is bound to quantum dot (CdSe / ZnS). A labeling material (referred to as “labeling material e”) was prepared. The specific procedure is as follows. The anti-HER2 antibody was reduced with 20 mM dithiothreitol (DTT), and excess DTT was removed by a gel filtration column to obtain a reduced antibody solution. On the other hand, the quantum dot obtained the maleimide-ized quantum dot which can react with a reduced antibody by removing excess SMCC with a gel filtration column after reacting with SMCC. The obtained reduced antibody and maleimidated quantum dots were mixed and reacted for 1 hour, and then the reaction was stopped by adding mercaptoethanol to 100 μM. The solution after the reaction and the reaction was stopped by gel filtration to obtain an anti-HER2 antibody bound with quantum dots.

<Tissue immunostaining and observation using labeling materials a to e>
Using the labeling materials a to e prepared in the above preparation examples, immunostaining of adjacent sections of human breast tissue was performed according to the following procedure. The adjacent sections are 24 sections selected from a tissue array slide (CB-A712) manufactured by Cosmo Bio and having a FISH score of 1 to 9 measured in advance.
1) A pathological section was immersed in a container containing xylene for 30 minutes. The xylene was changed three times during the process.
2) The pathological section was immersed in a container containing ethanol for 30 minutes. The ethanol was changed three times during the process.
3) The pathological section was immersed in a container containing water for 30 minutes. The water was changed three times along the way.
4) The pathological section was immersed in 10 mM citrate buffer (pH 6.0) for 30 minutes.
5) Autoclaving was performed at 121 degrees for 10 minutes.
6) The section after autoclaving was immersed in a container containing PBS for 30 minutes.
7) 1% BSA-containing PBS was placed on the tissue and left for 1 hour.
8) Labeling materials a to e bound with anti-HER2 antibody diluted to 0.05 nM with PBS containing 1% BSA were placed on each tissue section and left for 3 hours.
9) Each section after staining was immersed in a container containing PBS for 30 minutes.
10) Fixation treatment was performed with a 4% neutral paraformaldehyde solution for 10 minutes, and then HE staining was performed.
11) Aquex made by Merck Chemicals was dropped, and then a cover glass was placed and sealed.

  About the section | stain dye | stained by the said procedure, while irradiating the excitation light which has a predetermined wavelength using the upright microscope Axio Imager M2 by Carl Zeiss, about the fluorescence which has a predetermined wavelength, the microscope image from a microscope installation camera is shown. Acquisition and observation from a microscope column were performed. The excitation wavelength and the emission wavelength of the observed fluorescence are 630 nm and 680 nm, respectively, when using Cy5 as a fluorescent substance (whether it is a simple substance or encapsulated in fluorescent substance-containing nanoparticles), and CdSe / ZnS is used. In the case of using Texas Red, the wavelength is 590 nm and 620 nm, respectively. Table 2 shows the results of the identification of fluorescence from the fluorescent label.

15 to 19 show images acquired from the microscope installation camera.
FIG. 15 is an image acquired from a microscope installation camera when the labeling material d is used.
The Although cell morphology information by weak emission (autofluorescence) derived from eosin can be observed, antigen molecule information is buried in the cell morphology information and cannot be identified. A similar observation image is obtained when observed from a microscope barrel.
FIG. 16 is an image acquired from a microscope installation camera when the labeling material e is used. Although cell morphology information by weak emission (autofluorescence) derived from eosin can be observed, antigen molecule information is buried in the cell morphology information and cannot be identified. A similar observation image is obtained when observed from a microscope barrel.
FIG. 17 is an image acquired from a microscope installation camera when the labeling material a is used. It is in a state where it is possible to distinguish antigen molecule information that looks like a bright spot and cell shape information that is weakly emitted (autofluorescence) derived from eosin that is slowly connected. Note that antigen molecule information and cell morphology information could not be identified from the microscope column.
FIG. 18 is an image acquired from a microscope installation camera when the labeling material b is used. It is in a state where it is possible to distinguish antigen molecule information that looks like a bright spot and cell shape information that is weakly emitted (autofluorescence) derived from eosin that is slowly connected. A similar observation image is obtained when observed from a microscope barrel.
FIG. 19 is an image obtained from a microscope installation camera when the labeling material c is used. It is in a state where it is possible to distinguish antigen molecule information that looks like a bright spot and cell shape information that is weakly emitted (autofluorescence) derived from eosin that is slowly connected. A similar observation image is obtained when observed from a microscope barrel.

  When a microscope image of a tissue section on a single slide loaded in the microscope is acquired by a microscope installation camera (monochrome), as shown in Table 2, when labeling materials a to c are used, the background (tissue) Autofluorescence + eosin fluorescence) and fluorescence from fluorescent substance-containing nanoparticles are clearly distinguishable. Therefore, by analyzing a microscopic image of a tissue section obtained using these fluorescent labeling materials and measuring the number of target antigen molecules or target antigen luminance per cell based on the number of fluorescent bright spots or emission luminance, this From the measurement result, the expression level of the target biological substance (here, antigen) can be evaluated. Specifically, it can be evaluated that the higher the number of bright spots, the higher the expression level of the biological substance. Moreover, it can be evaluated that the higher the emission luminance, the higher the expression level of the biological substance. Visual evaluation is also possible.

  In Example 2, by using high-intensity fluorescent substance-containing nanoparticles as a fluorescent labeling material for immunohistochemistry, morphological information (cell autofluorescence + eosin autofluorescence) in the same section and in the same fluorescent field of view. ) And information reflecting the expression level of the antigen molecule (fluorescence from the fluorescent substance-encapsulating nanoparticles) can be observed, so that the diagnostic accuracy and convenience of the pathologist can be improved. Even when a monochrome camera is used, high-intensity fluorescence emitted by fluorescent substance-containing nanoparticles bound to antigen molecules can be distinguished from autofluorescence emitted by cells or the like. Moreover, since noise cutting by excitation light using an optical filter is not hindered, highly sensitive measurement can be maintained.

  As described above, the first embodiment and the second embodiment have been described. In the present invention, although the biological material recognition site needs to be different depending on the target lesion (cancer) type, the pathology Observation of information on the expression level of the target biological material using the slice is a common matter of confirming the bright dot. Therefore, it is possible to standardize pathological slice observations within each facility or across facilities regardless of the lesion type.

  In addition, in the conventional breast cancer diagnosis method using three slides including the FISH method which requires skill and cost, the method of the present invention can be adopted as a method to replace the FISH method, and the cost can be reduced.

  Also, in the case of performing pathological diagnosis based on two slides, ie, cell morphology information of a HE-stained slide and antigen molecule information by an immunostained (DAB) slide, the antigen molecule information by the latter slide is used as the antigen molecule according to the present invention. It can be replaced with information.

  In addition, in addition to the diagnosis using the information of the three slides described above, the developing pathologist adds information on the slide stained according to the present invention, and uses the information on the number of bright spots as teaching data. You can learn to improve your diagnostic ability.

In addition, as in Japanese Patent Application No. 2011-059172 filed separately by the applicant of the present application, HE staining and staining of the present invention were simultaneously performed using one slide, and after setting the slide on the microscope, the light source of the microscope (excitation) The wavelength of the light is first set to display a familiar HE image (color image), and the line of sight is concentrated on the region of interest in the HE image. If the dot dot is caused to emit light, it is possible to visually recognize the presence of the bright dot in the region of interest where the line of sight is concentrated at a glance in a short time.
If the field of view that can be viewed at a time of the microscope is relatively narrow, the region to be diagnosed on the pathological section will be divided into several observations. Even if the slide fixing stage of the microscope is adjusted so that the field of view becomes the observation range of the microscope and the light source wavelength is switched, the same effect as described above can be obtained.

  First, the microscope slide fixing stage is sequentially moved in the x-direction or y-direction, and each field of view is repeatedly photographed by the microscope installation camera. From the image data of each field of view obtained by the photographing, bright dot (number, Alternatively, based on a predetermined rule, for example, from one with more or less bright dots, or from a predetermined number of ranges, the microscope is calculated. It is also possible to adjust the slide fixing stage so that the corresponding visual field position is in step, and observe each visual field from the microscope barrel. That is, it is possible to variably control the order in which the cell morphology information should be observed in accordance with the feature quantity of bright spot dots (number or integrated value of number × luminance).

  Note that the description in the above embodiment is a preferred example of the present invention, and is not limited thereto, and can be changed as appropriate without departing from the spirit of the invention.

Claims (4)

A staining step of staining a tissue section using a staining reagent in which a biological material recognition site is bound to a spherical particle containing a fluorescent substance;
By irradiating excitation light to the tissue sections together to autofluorescence in the tissue section, an irradiation step of generating a dot-like fluorescent to said spherical particles,
An imaging step of enlarging and imaging the autofluorescence and / or the dot-like fluorescence;
An evaluation step of detecting the dot-like fluorescence from the enlarged image and evaluating the expression level of the biological material corresponding to the biological material recognition site;
It includes,
The tissue evaluation method, wherein the spherical particles are fluorescent substance-containing nanoparticles in which a fluorescent substance is dispersed inside the nanoparticles. 2. The staining step according to claim 1, wherein in the staining step, the tissue section is co-stained using a staining reagent in which the biological material recognition site is bound to a spherical particle encapsulating the fluorescent substance and an HE (hematoxylin-eosin) staining reagent. Organization evaluation method. 3. The tissue evaluation method according to claim 1, wherein a difference in light emission amount between the autofluorescence of the tissue section and the dot-like fluorescence of the spherical particles is the spherical particle fluorescence ≧ 1.1 × autofluorescence. In the imaging step, the autofluorescence and / or the dot-like fluorescence is magnified and imaged on an imaging surface in which a plurality of imaging elements are arranged in a two-dimensional direction, and the image is acquired.
The diameter of the spherical particles and / or the magnification of the dot-like fluorescence is set so that the dot-like fluorescence is imaged across two or more image sensors on the imaging surface. The tissue evaluation method according to any one of the above.