JP6547424B2 - Fluorescent image focusing system, focusing method and focusing program - Google Patents

Description

  The present invention relates to a fluorescence image focusing system, a focusing method and a focusing program.

Conventionally, an immunostaining method for detecting an antigen in a tissue sample is known as a cancer diagnostic method. Among the immunostaining methods, in recent years, the fluorescent antibody method has been increasingly used in place of the enzyme antibody method. The “fluorescent antibody method” is a method in which an antibody is labeled with a fluorescent substance, and after staining (antigen-antibody reaction) a tissue sample, the tissue sample is irradiated with an excitation wavelength to cause fluorescence, and this is observed with a fluorescence microscope. is there.
Patent Document 1 discloses an example using a fluorescent antibody method. In particular, Patent Document 1 proposes a method of measuring the level of expression of a biological substance by measuring the number of fluorescent luminescence points and the fluorescence intensity of a fluorescent substance bound to the biological substance using a fluorescence microscope.

  By the way, in order to measure the number of fluorescence points and the fluorescence intensity, an optical system having a high magnification and a numerical aperture (NA) is required. As the magnification and numerical aperture of the optical system become high, the depth of focus becomes narrow, so it is important to adjust the in-focus position. "Focusing" is what is called focusing. If a focus shift occurs, in the microscopic image (fluorescent image) including the fluorescent luminescent spot, the fluorescent luminescent spot may be lost, and even if it is not lost, the luminance of the fluorescent luminescent spot is lowered. The problem is that it becomes low and it is susceptible to background noise.

In this regard, methods of focusing fluorescent images are disclosed in Patent Documents 2 and 3.
In the technique of Patent Document 2, while the user operates the handle (26) to lower the stage (21), the contrast value of the microscope image is calculated, and whether the contrast value is equal to or more than the threshold value is compared to determine the threshold value. When it becomes above, it is comprised so that it may be judged that it focused (paragraph 0018, 0023, 0029-0036, refer FIG. 1, FIG. 3).

  In the technique of Patent Document 3, the stage (11) is moved at a constant speed upward from the bottom in the Z-axis direction and is moved at a constant speed circle in the XY plane to capture a circular fluorescent image by the fluorescent marker (55) ( Paragraphs 0065 to 0082, see FIG. 7). In such a case, the imaging range in the Z-axis direction is divided into a plurality of portions, and a plurality of fluorescent images are captured (see paragraphs 0083 to 0090 and FIGS. 8 to 10). Thereafter, frequency analysis is performed on a plurality of fluorescent images, a fluorescent image having the highest maximum frequency component is selected, an image of a locus of the fluorescent marker (55) is analyzed with respect to the fluorescent image, and an in-focus position is calculated. (Refer to paragraphs 0091 to 0102, FIG. 11, paragraph 0142).

  In addition, there is also a method of calculating an in-focus position by capturing an image each time while switching the focal position at intervals smaller than the focal depth (Z-stack imaging) and analyzing each of the photographed images (paragraph of Patent Document 3) 0010, 0138, 0143 to 0144).

JP, 2013-57631, A JP, 2010-2534, A JP, 2014-98575, A

However, in the technique of Patent Document 2, when the surface of the tissue sample has unevenness and the fluorescent luminescent spots are distributed in the optical axis direction, that is, the fluorescent luminescent spots do not exist on the same plane and the height position of the fluorescent luminescent spots If there is a gap between the two, it is not possible to calculate an accurate in-focus position because the contrast value is used.
In this respect, although the technique of Patent Document 3 can solve the problem of Patent Document 2, it is necessary to perform complicated control in which movement control of the Z-axis direction and XY plane of the stage and imaging and image analysis of fluorescent image are linked. Therefore, an advanced focusing system is required in terms of both hardware and software.
Even in the method using Z-stack imaging, in order to analyze the transition of fluorescent luminescent spots one by one for each captured image, labeling and matching of the same fluorescent luminescent spot between captured images are required, which takes a long time for analysis. There is.
Furthermore, although many biomaterials are expressed at locations where phosphors are densely clustered, which is very important when evaluating the expression level, contrast value and frequency analysis of fluorescence image as described above In the focusing method using the above, there is also a possibility that the weight of the contribution to the expression level evaluation of the portion concerned becomes small at the time of focusing judgment.

  Therefore, the main object of the present invention is to provide a fluorescence image focusing system, a focusing method and a focusing program capable of easily, quickly and quantitatively calculating the focusing position of the fluorescence image.

According to one aspect of the present invention to solve the above-mentioned problems,
A fluorescence microscope for imaging a plurality of fluorescence images while making focal positions of a tissue sample different ;
Generation means for generating a fluorescence image from each of the plurality of fluorescence images;
Extracting means for extracting fluorescent luminescent spots from the fluorescent image;
The luminance value of the luminescent spot area where the fluorescent luminescent spot exists is calculated for each of the luminescent spot areas, and the sum of the luminance values of all the luminescent spot areas on one of the fluorescent image is calculated. Calculating means for calculating the in-focus position;
Equipped with
The calculation means calculates a brightness integral value obtained by summing the brightness values of all the bright spot areas on one fluorescence image, and a focal point in the fluorescence image in which the brightness integral value is maximum among a plurality of the fluorescence images A focusing system for a fluorescence image is provided, characterized in that the position is a focusing position of the fluorescence image.
According to another aspect of the invention,
A fluorescence microscope for imaging a plurality of fluorescence images while making focal positions of a tissue sample different;
Generation means for generating a fluorescence image from each of the plurality of fluorescence images;
Extracting means for extracting fluorescent luminescent spots from the fluorescent image;
The luminance value of the luminescent spot area where the fluorescent luminescent spot exists is calculated for each of the luminescent spot areas, and the sum of the luminance values of all the luminescent spot areas on one of the fluorescent image is calculated. Calculating means for calculating the in-focus position;
Equipped with
The calculation means converts the luminance value of the bright spot area into the number of fluorescent nanoparticles, calculates the total number of fluorescent nanoparticles in all the bright spot areas on one fluorescent image, and a plurality of the fluorescent images The focal position in the fluorescence image at which the total number of the fluorescent nanoparticles is the largest among them is taken as the in-focus position of the fluorescence image.
A focusing system for fluorescence images is provided.

According to another aspect of the invention,
Imaging a plurality of fluorescent images while making focal positions of the tissue sample different ;
Generating a fluorescence image from each of the plurality of fluorescence images;
Extracting fluorescent luminescent spots from the fluorescent image;
The luminance value of the luminescent spot area where the fluorescent luminescent spot exists is calculated for each of the luminescent spot areas, and the sum of the luminance values of all the luminescent spot areas on one of the fluorescent image is calculated. Calculating the in-focus position;
Equipped with
The step of calculating the in-focus position of the fluorescence image calculates a brightness integral value obtained by summing the brightness values of all the bright spot regions on one fluorescence image, and the brightness integral value of the plurality of fluorescence images is calculated. There is provided a method of focusing a fluorescence image, comprising the step of setting the focal position in the fluorescence image at which is the maximum to the in-focus position of the fluorescence image .
According to another aspect of the invention,
Imaging a plurality of fluorescent images while making focal positions of the tissue sample different;
Generating a fluorescence image from each of the plurality of fluorescence images;
Extracting fluorescent luminescent spots from the fluorescent image;
The luminance value of the luminescent spot area where the fluorescent luminescent spot exists is calculated for each of the luminescent spot areas, and the sum of the luminance values of all the luminescent spot areas on one of the fluorescent image is calculated. Calculating the in-focus position;
Equipped with
The step of calculating the in-focus position of the fluorescent image converts the luminance value of the bright spot area into the number of fluorescent nanoparticles, and the total number of fluorescent nanoparticles in all the bright spot areas on one fluorescent image Calculating, and setting a focal position in the fluorescence image that maximizes the total number of the fluorescent nanoparticles among the plurality of fluorescence images as the in-focus position of the fluorescence image
There is provided a method of focusing a fluorescent image characterized by

According to another aspect of the invention,
On the computer
Imaging means for imaging a plurality of fluorescent images while making focal positions of a tissue sample different ;
Generation means for generating a fluorescence image from each of the plurality of fluorescence images;
Extraction means for extracting fluorescent luminescent spots from the fluorescent image
The luminance value of the luminescent spot area where the fluorescent luminescent spot exists is calculated for each of the luminescent spot areas, and the sum of the luminance values of all the luminescent spot areas on one of the fluorescent image is calculated. Calculation means for calculating focus position,
To act as
The calculation means converts the luminance value of the bright spot area into the number of fluorescent nanoparticles, and calculates the total number of fluorescent nanoparticles in all the bright spot areas on one fluorescent image, and a plurality of the fluorescent images There is provided a focusing program of a fluorescence image for setting a focusing position in the fluorescence image in which the total number of the fluorescent nanoparticles is the largest among them, as a focusing position of the fluorescence image.
According to another aspect of the invention,
On the computer
Imaging means for imaging a plurality of fluorescent images while making focal positions of a tissue sample different;
Generation means for generating a fluorescence image from each of the plurality of fluorescence images;
Extraction means for extracting fluorescent luminescent spots from the fluorescent image
The luminance value of the luminescent spot area where the fluorescent luminescent spot exists is calculated for each of the luminescent spot areas, and the sum of the luminance values of all the luminescent spot areas on one of the fluorescent image is calculated. Calculation means for calculating focus position,
To act as
The calculation means converts the luminance value of the bright spot area into the number of fluorescent nanoparticles, and calculates the total number of fluorescent nanoparticles in all the bright spot areas on one fluorescent image, and a plurality of the fluorescent images The focal position in the fluorescence image at which the total number of the fluorescent nanoparticles is the largest among them is set as the in-focus position of the fluorescence image.
A fluorescence image focusing program is provided.

  According to the present invention, the in-focus position of the fluorescence image can be calculated easily and quickly quantitatively.

It is a figure which shows schematic structure of the focusing system of a fluorescence image. It is a flowchart for demonstrating schematically the focusing method (focusing point calculation process) of a fluorescence image. It is a figure which shows an example of a fluorescence image. It is a figure for demonstrating the process which excludes a self-fluorescence area | region from a fluorescence image. It is a figure for demonstrating the process which excludes a self-fluorescence area | region from a fluorescence image. It is a figure for demonstrating the process which carries out hole filling of a part of fluorescent luminescent point. It is a figure for demonstrating the process which deletes a part of fluorescent luminescent point. It is a figure which shows an example of the look-up table which shows the relationship between the area of a luminescent point area | region, and a luminance value. It is a figure for demonstrating the process for calculating an optimal focusing position. It is a figure for demonstrating the modification of FIG. It is a figure for demonstrating the example which designates a fixed area | region from a fluorescence image. FIG. 7 is a diagram showing an example in which a fluorescence image is generated each time the focus position is switched in the first embodiment. FIG. 7 is a view schematically showing calculation results of the in-focus position in the first embodiment. FIG. 10 is a view schematically showing calculation results of a focusing position in Example 2. It is a figure which shows roughly the calculation result of the focusing position in the comparative example 1. FIG. It is a figure for demonstrating the problem in the comparative example 1. FIG.

  Hereinafter, preferred embodiments of the present invention will be described with reference to the drawings.

[Fluorescent image focusing system]
As shown in FIG. 1, the fluorescence image focusing system 1 includes a fluorescence microscope 10, a control device 60 and a display device 70.
The fluorescence microscope 10 includes a stage 12, an objective lens 14, a lens barrel 16, an eyepiece lens 18, and an imaging device 20. At stage 12, a tissue sample 30 after immunostaining is placed. The lens barrel 16 incorporates a lamp 40 and a fluorescent cube 50. The fluorescence cube 50 comprises an excitation filter 52, a dichroic mirror 54 and an absorption filter 56.
The lamp 40 is a lamp that emits excitation light. The excitation filter 52 is a filter that transmits only excitation light. The dichroic mirror 54 is a mirror that reflects or transmits light having a predetermined wavelength as a boundary. Here, the dichroic mirror 54 reflects excitation light and transmits fluorescence. The absorption filter 56 is a filter that blocks excitation light and transmits only fluorescence.
In the fluorescence microscope 10, when the lamp 40 is turned on, excitation light passes through the excitation filter 52, is reflected by the dichroic mirror 54, passes through the objective lens 14, and is irradiated to the tissue specimen 30. As a result, fluorescence is emitted from the tissue sample 30, and the fluorescence is collected by the objective lens 14 and transmitted through the dichroic mirror 54 and the absorption filter 56. Thereafter, the fluorescence is observed through the eyepiece lens 18 as a fluorescence image, and is imaged by the imaging device 20.

A control device 60 for controlling these is connected to the fluorescence microscope 10.
The control device 60 includes a control unit 62 and a storage unit 64.
The control unit 62 is connected to the stage 12 and can control the elevation of the stage 12 to control the focal position (height position). The control unit 42 is connected to the imaging device 20, controls the imaging device 20 to capture a fluorescence image, and can receive the fluorescence image to generate a fluorescence image. The control unit 62 is connected to the lamp 40 and can control lighting and extinguishing of the lamp 40. The storage unit 64 stores a fluorescence image focusing program for executing the focusing position calculation process of FIG.
A display device 70 is connected to the control device 60, and a calculation result by the control device 60 and the like are displayed on the display device 70.

[Tissue sample]
Subsequently, the tissue sample 30 will be described.
The tissue sample 30 is a tissue section containing a target biological material and stained with an immunostaining agent, and the tissue sample 30 after staining is placed on the stage 12 of the fluorescence microscope 10.

(1) Target biological substance Target biological substance is intended for immunostaining using a fluorescent label mainly for detection or quantification from the viewpoint of pathological diagnosis, and is expressed in tissue sections It is a biological substance, in particular a protein (antigen).
Typical target biological substances include biological substances that are expressed on cell membranes of various cancer tissues and can be used as biomarkers.

(2) Immunostaining agent (conjugate of antibody-fluorescent nanoparticle)
As an immunostaining agent, in order to improve the efficiency of the fluorescent labeling and to minimize the time course leading to the deterioration of the fluorescence, the primary antibody and the fluorescent nanoparticle indirectly, that is, an antigen-antibody reaction etc. It is preferred to use a complex linked by a bond of In order to simplify the staining procedure, a complex in which a fluorescent nanoparticle is directly linked to a primary antibody or a secondary antibody can also be used as an immunostaining agent.

Examples of the immunostaining agent include: [Primary antibody to target biological substance] ... [Antibody to primary antibody (secondary antibody)] to [fluorescent nanoparticle].
“...” represents binding by an antigen-antibody reaction, and there is no particular limitation on the mode of binding represented by “-”, for example, covalent bond, ionic bond, hydrogen bond, coordination bond, antigen-antibody bond, Biotin avidin reaction, physical adsorption, chemical adsorption, etc. may be mentioned, and it may be through a linker molecule as required.

(3) Antibody As a primary antibody, an antibody (IgG) that can specifically recognize and bind a protein as a target biological substance as an antigen can be used. For example, when HER2 is a target biological substance, an anti-HER2 antibody can be used, and when HER3 is a target biological substance, an anti-HER3 antibody can be used.
For the secondary antibody, an antibody (IgG) that can specifically recognize and bind the primary antibody as an antigen can be used.
Although both the primary antibody and the secondary antibody may be polyclonal antibodies, monoclonal antibodies are preferred from the viewpoint of quantitative stability. The type of animal producing the antibody (immune animal) is not particularly limited, and may be selected from mice, rats, guinea pigs, rabbits, goats, sheep and the like as in the prior art.

(4) Fluorescent nanoparticle The fluorescent nanoparticle is a nano-sized particle that emits fluorescence upon receiving irradiation of excitation light, and emits fluorescence with an intensity sufficient to represent the target biological substance as a bright spot. It is a possible particle.
As the fluorescent nanoparticles, preferably quantum dots (semiconductor nanoparticles) and fluorescent substance-integrated nanoparticles are used.

(4.1) Quantum dot As a quantum dot, the semiconductor nanoparticle containing a II-VI group compound, a III-V group compound, or a IV group element is used. For example, CdSe, CdS, CdTe, ZnSe, ZnS, ZnTe, InP, InN, InAs, InGaP, GaP, GaAs, Si, Ge and the like can be mentioned.

(4.2) Fluorescent substance accumulation nanoparticle The fluorescent substance accumulation nanoparticle uses a particle made of an organic substance or an inorganic substance as a matrix, and a plurality of fluorescent substances (for example, the above-mentioned quantum dots, fluorescent dyes, etc.) are contained therein. And / or nano-sized particles having a structure adsorbed on the surface thereof.
As the fluorescent substance accumulation nanoparticles, it is preferable that the matrix and the fluorescent substance have substituents or sites having opposite charges to each other, and electrostatic interaction occurs.
As the fluorescent substance integrated nanoparticles, quantum dot integrated nanoparticles, fluorescent dye integrated nanoparticles, etc. are used.

(4.2.1) Base material Among the base materials, as organic substances, resins generally classified as thermosetting resins, such as melamine resin, urea resin, aniline resin, guanamine resin, phenol resin, xylene resin, furan resin, etc. Resins generally classified as thermoplastic resins, such as styrene resin, acrylic resin, acrylonitrile resin, AS resin (acrylonitrile-styrene copolymer), ASA resin (acrylonitrile-styrene-methyl acrylate copolymer), etc .; Other resins such as lactic acid and the like; polysaccharides can be exemplified.
Among the matrix, examples of the inorganic substance include silica, glass and the like.

(4.2.2) Quantum Dot Accumulated Nanoparticle The quantum dot integrated nanoparticle has a structure in which the quantum dot is contained in the matrix and / or adsorbed on the surface thereof.
When the quantum dot is contained in the matrix, the quantum dot may be dispersed in the matrix, and may or may not be chemically bonded to the matrix itself.

(4.2. 3) Fluorescent dye integrated nanoparticle The fluorescent dye integrated nanoparticle has a structure in which a fluorescent dye is contained in the above-mentioned matrix and / or adsorbed on the surface thereof.
Examples of fluorescent dyes include rhodamine dye molecules, squarylium dye molecules, cyanine dye molecules, aromatic ring dye molecules, oxazine dye molecules, carbopyronine dye molecules, and pyromecene dye molecules.
As a fluorescent dye, Alexa Fluor (registered trademark, manufactured by Invitrogen Corporation) dye molecule, BODIPY (registered trademark, manufactured by Invitrogen Corporation) dye molecule, Cy (registered trademark, manufactured by GE Healthcare) dye molecule, HiLyte (registered trademark) Trademark, manufactured by Anapeck Co., Ltd., DyLight (registered trademark, manufactured by Thermo Scientific Co., Ltd.), colored pigment molecule, ATTO (registered trademark, manufactured by ATTO-TEC), dye molecule, MFP (registered trademark, manufactured by Mobitec) ) Dye molecules, CF (registered trademark, manufactured by Biotium) dye molecules, DY (registered trademark, manufactured by DYOMICS company), dye molecules, CAL (registered trademark, manufactured by BioSearch Technologies), etc. can be used. .
When the fluorescent dye is contained in the matrix, the fluorescent dye may be dispersed in the matrix, and may or may not be chemically bonded to the matrix itself.

(5) Staining Method of Tissue Section An example of a staining method will be described.
The method for preparing tissue sections (also referred to simply as "sections" and including sections such as pathological sections) to which this staining method can be applied is not particularly limited, and those prepared according to known procedures can be used.

(5.1) Specimen preparation step (5.1.1) Deparaffinization treatment The sections are 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 more and 30 minutes or less. If necessary, xylene may be replaced during immersion.

  The sections are then 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 more and 30 minutes or less. If necessary, ethanol may be replaced during immersion.

  Immerse the sections in a water container and remove ethanol. The temperature is not particularly limited, but can be performed at room temperature. The immersion time is preferably 3 minutes or more and 30 minutes or less. Also, water may be exchanged during immersion if necessary.

(5.1.2) Activation treatment The target biological material is activated according to a known method. Although the activation conditions are not particularly limited, as an activation solution, 0.01 M citrate buffer (pH 6.0), 1 mM EDTA solution (pH 8.0), 5% urea, 0.1 M Tris-HCl buffer A liquid etc. can be used.
The pH condition is such that the signal is output from the range of pH 2.0 to 13.0 depending on the tissue section to be used, and the condition that roughening of the tissue can be evaluated. Usually, it is carried out at pH 6.0 to 8.0, but in special tissue sections it is also carried out, for example, at pH 3.0.
The heating apparatus can use an autoclave, a microwave, a pressure cooker, a water bath etc. The temperature is not particularly limited, but can be performed at room temperature. The temperature can be 50 to 130 ° C., and the time can be 5 to 30 minutes.

  Then, the section after activation treatment is immersed in a container containing PBS and washed. The temperature is not particularly limited, but can be performed at room temperature. The immersion time is preferably 3 minutes or more and 30 minutes or less. If necessary, the PBS may be replaced during immersion.

(5.2) Immunostaining Step In the immunostaining step, a solution of immunostaining agent containing fluorescent nanoparticles having a site capable of binding directly or indirectly to a target biological substance to stain the target biological substance, Place on a section and react with the target biological material. The solution of the immunostaining agent used in the immunostaining step may be prepared in advance prior to this step.

The conditions for performing the immunostaining step, that is, the temperature and immersion time for immersing the tissue specimen in the immunostaining agent solution, are appropriately adjusted to obtain an appropriate signal according to the conventional immunostaining method. Can.
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 drip a known blocking agent such as BSA-containing PBS or a surfactant such as Tween 20 before performing the treatment as described above.

(5.3) Sample Post-Treatment Step It is preferable that the tissue sample subjected to the immunostaining step is subjected to treatments such as immobilization / dehydration, clearing and encapsulation so as to be suitable for observation.

The immobilization / dehydration treatment may be performed by immersing the tissue specimen in a fixation treatment solution (formalin, paraformaldehyde, glutaraldehyde, acetone, ethanol, methanol, or other crosslinking agent). In the clearing process, a tissue sample which has been fixed and dehydrated may be immersed in a clearing solution (such as xylene). In the sealing process, the tissue sample after the clearing process may be immersed in the sealing solution.
The conditions for performing these treatments, for example, the temperature and immersion time for immersing the tissue specimen in a predetermined treatment solution, may be appropriately adjusted to obtain an appropriate signal according to the conventional immunostaining method. it can.

(5.4) Morphological observation staining step Aside from the immuno staining step, morphological observation staining may be performed to enable observation of the morphology of cells, tissues, organs and the like in the bright field.
The morphological observation staining step can be performed according to a conventional method.
For morphological observation of tissue specimens, staining with eosin in which cytoplasm, stroma, various fibers, red blood cells, and keratinocytes are stained in red to dark red is generally used. Staining with hematoxylin, in which cell nuclei, lime area, cartilage tissue, bacteria and mucus are stained in blue to pale blue, is also used as standard (Hematoxylin and Eosin staining for performing these two staining simultaneously) (Known as HE staining).
When the morphological observation staining step is included, it may be performed after the immunostaining step, or may be performed before the immunostaining step.

[Focusing method of fluorescence image]
Then, the focusing method of a fluorescence image is demonstrated.
Such a method is a method performed when detecting a target biological material from a tissue sample 30 after immunostaining using a focusing system 1 for fluorescence image.

First, the tissue sample 30 after immunostaining is placed on the stage 12 of the fluorescence microscope 10.
Thereafter, using the control device 60, processing for generating a fluorescent image from the tissue sample 30 and processing the image to calculate an optimal in-focus position (in-focus position calculation processing) is executed.

  The focusing position calculation process is executed by cooperation of the control unit 62 and the program stored in the storage unit 64, and the control unit 62 executes the following process according to the program. As such a program, for example, "ImageJ" (open source) can be mentioned. By using such image processing software, a process of extracting fluorescent luminescent spots of a predetermined wavelength (color) from a fluorescent image and calculating the luminance value of the luminescent spot area and the number of fluorescent nanoparticles can be performed semiautomatically and quickly. It can be done.

As shown in FIG. 2, the control unit 62 first adjusts the focal position of the stage 12 to position the tissue sample 30 in the vicinity of the objective lens 14 (step S1).
Thereafter, the control unit 62 turns on the lamp 40 to irradiate the tissue sample 30 with excitation light. As a result, the fluorescent nanoparticle of the tissue sample 30 emits fluorescence and a fluorescent luminescent spot appears. The control unit 62 causes the imaging device 20 to capture the fluorescent image.
Thereafter, the control unit 62 generates a fluorescence image from the fluorescence image based on the imaging result of the imaging device 20 (step S2).
FIG. 3 (a) is an example of a fluorescence image.

Thereafter, the control unit 62 performs preprocessing on the fluorescence image (step S3).
FIG. 3 (b) is an example of a fluorescence image after pre-processing.
The pretreatment includes, for example, treatment for removing the autofluorescent region.
In step S3, for example, when staining with eosin at the time of preparation of tissue specimen 30, when eosin autofluorescence occurs, as shown in FIG. 4, the background region generated by eosin autofluorescence is removed, or As shown in FIG. 5, the autofluorescent region of the tissue sample 30 itself is removed or the like.

Thereafter, the control unit 62 extracts a fluorescent bright spot from the fluorescent image (step S4).
Thereafter, the control unit 62 performs post-processing on the bright spot area (step S5).
The “bright spot area” is an area in which a fluorescent bright spot is present, and in the example of FIG. 3B, is an area corresponding to an open area.
FIG. 3 (c) is an example of a fluorescence image after post-processing.
The post-processing includes, for example, a process of filling in a part of the bright spot area and a process of deleting a part of the bright spot area.
In step S5, for example, as shown in FIG. 6, the inside of the ring-shaped bright spot area is filled, or as shown in FIG. 7, the ring-shaped bright spot area is deleted.

Thereafter, the control unit 62 calculates the brightness value for each bright spot area, and calculates the brightness integral value of all the bright spot areas in the fluorescent image (step S6).
Here, when the light receiving sensitivity of the image sensor 20 does not have a linear relationship with the fluorescence intensity of the bright spot area, the calculated luminance value can not be used as it is. In such a case, the control unit 62 uses the look-up table 66 (see FIG. 8) in which the relationship between the calculated luminance value and the corrected luminance value obtained by correcting the luminance integral value of the bright spot region is previously determined. It may be calculated. That is, the lookup table 66 is stored in the storage unit 64 of the control device 60, and the control unit 62 first calculates the luminance value for each bright spot area and then refers to the lookup table 66 of the storage unit 64. The corrected luminance value for the luminance value is calculated. Finally, the control unit 62 adds the corrected luminance values of all the bright spot areas in the fluorescence image, and sets this as the integrated luminance value.

After that, the control unit 62 determines whether the fluorescence image has reached a predetermined number (step S7), and lowers the focal position of the stage 12 stepwise until the fluorescence image reaches a predetermined number (step S8). The processing of steps S2 to S6 is repeatedly executed each time.
When the fluorescence image reaches a certain number, the control unit 62 compares the luminance integral values at each of the focal positions, and sets the focal position at which the luminance integral value is maximum as the in-focus position of the fluorescence image (step S9). ).
That is, as shown in FIG. 9, in step S6, the luminance integral value of the fluorescence image at the focal position is determined for each focal position, and in step S9, the focal position at which the luminance integral value becomes maximum Focus position.
FIG. 9 shows an example in which the integrated luminance value is calculated at nine different focus positions (●) and the fifth focus position is set as the in-focus position.

  According to the above-described embodiment, the fluorescent image is generated each time the focal position of the stage 12 is switched to calculate the integrated luminance value, and the focal position at which the integrated luminance value becomes maximum is set as the in-focus position of the fluorescent image. Therefore, the in-focus position of the fluorescence image can be calculated easily, quickly and quantitatively.

That is, complicated control in which movement control of the stage 12 and imaging and image analysis of the fluorescence image are interlocked is not necessary, and unlike the technique of Patent Document 3, the focusing position of the fluorescence image can be easily calculated. .
Even if the focal position deviates and the luminance value in the bright spot area decreases, it is reflected on the integral value of the luminance as it is and processing for the fluorescence image is completed. Therefore, unlike the method using Z-stack imaging, the same among the fluorescence images It is not necessary to label and match fluorescent light spots, and the focus position of the fluorescent image can be calculated quickly.
Even in a place where fluorescent nanoparticles are densely clustered, the luminance value of the bright spot area is reflected in the integrated luminance value, and the in-focus position of the fluorescent image can be quantitatively calculated, and the place is It greatly influences the weight of contribution to expression level evaluation, and can also improve evaluation accuracy.

Further, in the present embodiment, the S / N ratio of the fluorescent bright spot can be improved by the pretreatment to the fluorescent light image and the post-treatment to the bright spot area, and the influence of the background noise of the fluorescent light image can be suppressed.
Furthermore, in the present embodiment, since the in-focus position is calculated by the luminance integral value, an event that the fluorescent bright spot is lost in the fluorescent light image can be resolved, and the fluorescent bright spots are distributed in the optical axis direction of the fluorescence microscope 10 Even in this case, the in-focus position can be calculated in consideration of it.

[Modification]
In step S6, the luminance value of the bright spot area is converted to the number of fluorescent nanoparticles, and in step S9, as shown in FIG. 10, the focal position at which the total number of fluorescent nanoparticles is maximum is the focal position of the fluorescent image. It may be
In such a case, in step S6, the luminance value of the bright spot area may be divided by the average luminance value per fluorescent nanoparticle measured in advance using a scanning electron microscope to convert it into the number of fluorescent nanoparticles. For example, when the luminance value of the bright spot area is 50000 and the average luminance value per fluorescent nanoparticle is 10000, the number of fluorescent nanoparticles is five.
FIG. 10 shows an example in which the total number of fluorescent nanoparticles is calculated at nine different focus positions (●) and the fifth focus position is set as the in-focus position.

In the present embodiment (including the modification), for example, when the fluorescence image of FIG. 3A is formed in step S2, the processing of steps S3 to S6 is performed on the entire fluorescence image, and the fluorescence image is generated. The in-focus position of is calculated.
Instead of this, as shown in FIG. 11A, the fluorescence image is divided into a plurality of regions, and the processing of steps S3 to S6 is executed on the designated region 80 designated from among them, and the in-focus position of the fluorescence image is determined. It may be calculated. Or as shown in FIG.11 (b), the process of step S3-S6 may be performed with respect to the designation | designated area | region 82 designated arbitrarily out of the fluorescence image, and the focus position of a fluorescence image may be calculated.
In such a case, as a matter of course, each time the focal position of the stage 12 is switched, the same designated area 80 or 82 is designated among the fluorescence images.

In the present example, Cy5-encapsulated silica nanoparticles were produced as the fluorescent nanoparticles a, and the immunostaining agent A in which an anti-HER2 antibody was bound to the fluorescent nanoparticles a was produced.
Thereafter, immunostaining agent A was used to immunostain tissue sections of human breast tissue, and the in-focus position of the fluorescence image in the tissue sample after immunostaining was calculated.

Example 1
(1) Synthesis of Fluorescent Nanoparticle a The Cy5-encapsulated silica nanoparticle was produced by the method of the following steps (1.1) to (1.5).
Step (1.1): 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.
Step (1.2): 40 mL of ethanol and 10 mL of 14% aqueous ammonia were mixed.
Step (1.3): While stirring the mixture prepared in step (1.2) at room temperature, the mixture prepared in step (1.1) was added. Stirring was performed for 12 hours from the start of the addition.
Step (1.4): The reaction mixture was centrifuged at 10000 G for 60 minutes, and the supernatant was removed.
Step (1.5): Ethanol was added to disperse the precipitate, and centrifugation was performed again. Washing with ethanol and pure water was performed once in the same manner.
When the obtained fluorescent nanoparticles a were observed with a scanning electron microscope (SEM; S-800 manufactured by Hitachi (registered trademark)), the average particle diameter was 110 nm and the variation coefficient was 12%.

(2) Preparation of Immunostaining Agent A An antibody was bound to the fluorescent nanoparticle a by the following steps (2.1) to (2.12).
Step (2.1): 1 mg of fluorescent nanoparticles a was dispersed in 5 mL of pure water. Then, 100 μL of aminopropyltriethoxysilane aqueous dispersion was added and stirred at room temperature for 12 hours.
Step (2.2): The reaction mixture was centrifuged at 10000 G for 60 minutes, and the supernatant was removed.
Step (2.3): Ethanol was added, the precipitate was dispersed, and centrifugation was performed again. Washing with ethanol and pure water was performed once in the same manner.
When the FT-IR measurement of the obtained amino group modified silica nanoparticle was performed, the absorption derived from the amino group could be observed, and it could be confirmed that the amino group was modified.

Step (2.4): The amino group-modified silica nanoparticles obtained in step (2.3) were adjusted to 3 nM with PBS containing 2 mM of EDTA (ethylenediaminetetraacetic acid).
Step (2.5): SM (PEG) 12 (Thermo Scientific Inc., succinimidyl-[(N-maleimidopropionamid) -dodecaethyleneglycolic ester] ester so that the solution prepared in step (2.4) has a final concentration of 10 mM ) Were mixed and allowed to react for 1 hour.
Step (2.6): The reaction mixture was centrifuged at 10000 G for 60 minutes and the supernatant was removed Step (2.7): PBS containing 2 mM EDTA was added, the precipitate was dispersed, and centrifugation was repeated again went. Washing was performed three times according to the same procedure. Finally, redispersion was performed using 500 μL of PBS.

Step (2.8): To 100 μl of anti-HER2 antibody dissolved in 100 μl of PBS, 1 M dithiothreitol (DTT) was added and reacted for 30 minutes.
Step (2.9): Excess DTT was removed from the reaction mixture by gel filtration column to obtain a reduced anti-HER2 antibody solution.

Step (2.10): Using the fluorescent nanoparticle a as a starting material, the particle dispersion obtained in step (2.7) and the reduced anti-HER2 antibody solution obtained in step (2.9) in PBS Mix and react for 1 hour.
Step (2.11): 4 μL of 10 mM mercaptoethanol was added to stop the reaction.
Step (2.12): The reaction mixture was centrifuged at 10000 G for 60 minutes, the supernatant was removed, PBS containing 2 mM EDTA was added, the precipitate was dispersed, and centrifugation was performed again. Washing was performed three times according to the same procedure. Finally, the resultant was re-dispersed with 500 μl of PBS to obtain Cy5-encapsulated silica nanoparticles (immunostain A) to which an anti-HER2 antibody was bound.

(3) Immunostaining of Tissue Specimens Immunostaining was performed on tissue sections of human breast tissue using immunostaining agent A according to the following steps (3.1) to (3. 10). The tissue sections used Cosmo array tissue array slides (CB-A712).

Step (3.1): The tissue section was immersed for 30 minutes in a container containing xylene. On the way, xylene was replaced 3 times.
Step (3.2): The tissue section was immersed in a container containing ethanol for 30 minutes. On the way, ethanol was replaced 3 times.
Step (3.3): The tissue section was immersed in a water container for 30 minutes. We changed the water three times on the way.
Step (3.4): The tissue section was immersed in 10 mM citrate buffer (pH 6.0) for 30 minutes.
Process (3.5): The autoclave process was performed at 121 degree | times for 10 minutes.
Step (3.6): The autoclaved sections were immersed for 30 minutes in a container containing PBS.
Step (3.7): PBS containing 1% BSA was placed on the tissue and left for 1 hour.
Step (3.8): The immunostaining agent A conjugated with anti-HER2 antibody diluted to 0.05 nM in PBS containing 1% BSA was placed on each tissue section and left for 3 hours.
Step (3.9): The stained sections were immersed for 30 minutes in a container containing PBS.
Step (3.10): After dripping Aquatex manufactured by Merck Chemicals, a cover glass was placed and sealed.

(4) Calculation of in-focus position The in-focus position calculation process of FIG. 2 is executed using the same in-focus system of the fluorescence image as in FIG. 1 to generate a fluorescence image from the tissue sample and Calculated.
As a fluorescence microscope, using an upright microscope Axio Imager M2 manufactured by Carl Zeiss, an objective lens is set at 20 ×, excitation light with a wavelength of 630 to 670 nm is irradiated, and a fluorescence image emitted from a tissue specimen is an imaging device (microscope installation The image was taken with a camera, monochrome, and an in-focus position calculation process was executed using image analysis software.
The camera has a pixel size of 6.4 μm × 6.4 μm, 1040 vertical pixels, and 1388 horizontal pixels (imaging area of 8.9 mm × 6.7 mm).
Here, as shown in FIG. 12, while the focal position of the stage was switched stepwise (9 steps), a fluorescent image was generated each time to calculate a luminance integral value.
As a result, as shown in FIG. 13, the integrated luminance value is maximized at the fifth focus position, and the focus position matches the in-focus position visually confirmed from the eyepiece.

Example 2
Under the same conditions as in Example 1, the luminance value of the bright spot area was divided by the average luminance value per fluorescent nanoparticle measured in advance using a scanning electron microscope to convert it into the number of fluorescent nanoparticles, As shown in 14, the total number of fluorescent nanoparticles was the largest at the fifth-step focal position, and the focal position was also in agreement with the in-focus position visually confirmed from the eyepiece.

Comparative Example 1
Under the same conditions as in Example 1, it was determined whether the luminance value of the bright spot area exceeded the threshold value, and the number of fluorescent spots on the bright spot area was counted.
As a result, as shown in FIG. 15, the total number of fluorescent luminescent spots is maximized at the seventh-step focal position, and the focal position is clearly deviated from the in-focus position visually confirmed from the eyepiece.
The cause was considered as follows. Since a plurality of fluorescent nanoparticles adhere to a portion where the amount of expression of the target biological substance is large, the luminance value of the bright spot region becomes high. In such a bright spot area, the luminance value may be higher than that of a single focused fluorescent nanoparticle even in a defocused state. Therefore, as shown in FIG. 16, even if the focus position is switched, the number of fluorescent spots on the ring-shaped bright spot region is counted multiple times, and it becomes impossible to measure the number of accurate fluorescent spots. It was thought that.

DESCRIPTION OF SYMBOLS 1 fluorescence image focusing system 10 fluorescence microscope 12 stage 14 objective lens 16 lens barrel 18 eyepiece lens 20 imaging device 30 tissue specimen 40 lamp 50 fluorescence cube 52 excitation filter 54 dichroic mirror 56 absorption filter 60 control device 62 control unit 64 storage unit 66 Look-up table 70 Display 80, 82 Specified area

Claims (12)

A fluorescence microscope for imaging a plurality of fluorescence images while making focal positions of a tissue sample different ;
Generation means for generating a fluorescence image from each of the plurality of fluorescence images;
Extracting means for extracting fluorescent luminescent spots from the fluorescent image;
The luminance value of the luminescent spot area where the fluorescent luminescent spot exists is calculated for each of the luminescent spot areas, and the sum of the luminance values of all the luminescent spot areas on one of the fluorescent image is calculated. Calculating means for calculating the in-focus position;
Equipped with
The calculation means calculates a brightness integral value obtained by summing the brightness values of all the bright spot areas on one fluorescence image, and a focal point in the fluorescence image in which the brightness integral value is maximum among a plurality of the fluorescence images A focusing system of a fluorescence image , wherein the position is a focusing position of the fluorescence image. In the fluorescence image focusing system according to claim 1 ,
The calculation means may calculate the luminance integral value of the bright spot area using a look-up table in which the relationship between the luminance value of the bright spot area and a corrected luminance value obtained by correcting the luminance value is previously determined. Focusing system for fluorescent images. A fluorescence microscope for imaging a plurality of fluorescence images while making focal positions of a tissue sample different;
Generation means for generating a fluorescence image from each of the plurality of fluorescence images;
Extracting means for extracting fluorescent luminescent spots from the fluorescent image;
The luminance value of the luminescent spot area where the fluorescent luminescent spot exists is calculated for each of the luminescent spot areas, and the sum of the luminance values of all the luminescent spot areas on one of the fluorescent image is calculated. Calculating means for calculating the in-focus position;
Equipped with
The calculation means converts the luminance value of the bright spot area into the number of fluorescent nanoparticles , calculates the total number of fluorescent nanoparticles in all the bright spot areas on one fluorescent image, and a plurality of the fluorescent images The focus position in the fluorescence image in which the total number of the fluorescent nanoparticles is the largest among them is the focus position of the fluorescence image. In the fluorescence image focusing system according to claim 3 ,
The fluorescence is characterized in that the calculation means divides the luminance value of the bright spot area by the average luminance value per fluorescent nanoparticle measured in advance using a scanning electron microscope to convert it into the number of fluorescent nanoparticles. Image focusing system. The fluorescence image focusing system according to any one of claims 1 to 4 .
A focusing system for a fluorescence image, comprising: removing means for removing an autofluorescence area from the fluorescence image. In the fluorescence image focusing system according to any one of claims 1 to 5 ,
A focusing system for a fluorescence image, comprising hole filling means for filling a part of the bright spot area. In the fluorescence image focusing system according to any one of claims 1 to 5 ,
A focusing system for a fluorescence image, comprising: deletion means for deleting a part of the bright spot area. In the fluorescence image focusing system according to any one of claims 1 to 7 ,
A focusing system for a fluorescence image, wherein the calculation means calculates a luminance value of the bright spot area included in the designated area for the designated area of the fluorescence image as a target. Imaging a plurality of fluorescent images while making focal positions of the tissue sample different ;
Generating a fluorescence image from each of the plurality of fluorescence images;
Extracting fluorescent luminescent spots from the fluorescent image;
The luminance value of the luminescent spot area where the fluorescent luminescent spot exists is calculated for each of the luminescent spot areas, and the sum of the luminance values of all the luminescent spot areas on one of the fluorescent image is calculated. Calculating the in-focus position;
Equipped with
The step of calculating the in-focus position of the fluorescence image calculates a brightness integral value obtained by summing the brightness values of all the bright spot regions on one fluorescence image, and the brightness integral value of the plurality of fluorescence images is calculated. A method of focusing a fluorescence image, comprising the step of setting the focal position in the fluorescence image at which x is maximum as the in-focus position of the fluorescence image . Imaging a plurality of fluorescent images while making focal positions of the tissue sample different;
Generating a fluorescence image from each of the plurality of fluorescence images;
Extracting fluorescent luminescent spots from the fluorescent image;
The luminance value of the luminescent spot area where the fluorescent luminescent spot exists is calculated for each of the luminescent spot areas, and the sum of the luminance values of all the luminescent spot areas on one of the fluorescent image is calculated. Calculating the in-focus position;
Equipped with
The step of calculating the in-focus position of the fluorescent image converts the luminance value of the bright spot area into the number of fluorescent nanoparticles, and the total number of fluorescent nanoparticles in all the bright spot areas on one fluorescent image Calculating, and setting a focal position in the fluorescence image that maximizes the total number of the fluorescent nanoparticles among the plurality of fluorescence images as the in-focus position of the fluorescence image
A focusing method of a fluorescence image characterized by On the computer
Imaging means for imaging a plurality of fluorescent images while making focal positions of a tissue sample different ;
Generation means for generating a fluorescence image from each of the plurality of fluorescence images;
Extraction means for extracting fluorescent luminescent spots from the fluorescent image
The luminance value of the luminescent spot area where the fluorescent luminescent spot exists is calculated for each of the luminescent spot areas, and the sum of the luminance values of all the luminescent spot areas on one of the fluorescent image is calculated. Calculation means for calculating focus position,
To act as
The calculation unit is configured to calculate a luminance integral value obtained by summing the luminance values of all the bright spot regions on one of the fluorescence images, and a focal point in the fluorescence image having the largest luminance integral value among the plurality of fluorescence images. A focusing program of a fluorescence image for setting a position as a focusing position of the fluorescence image. On the computer
Imaging means for imaging a plurality of fluorescent images while making focal positions of a tissue sample different;
Generation means for generating a fluorescence image from each of the plurality of fluorescence images;
Extraction means for extracting fluorescent luminescent spots from the fluorescent image
The luminance value of the luminescent spot area where the fluorescent luminescent spot exists is calculated for each of the luminescent spot areas, and the sum of the luminance values of all the luminescent spot areas on one of the fluorescent image is calculated. Calculation means for calculating focus position,
To act as
The calculation means converts the luminance value of the bright spot area into the number of fluorescent nanoparticles, and calculates the total number of fluorescent nanoparticles in all the bright spot areas on one fluorescent image, and a plurality of the fluorescent images The focal position in the fluorescence image at which the total number of the fluorescent nanoparticles is the largest among them is set as the in-focus position of the fluorescence image.
Program for focusing fluorescent images.