JP6578928B2 - Focus position specifying system of fluorescent image, focus position specifying method, and focus position specifying program - Google Patents

Description

  The present invention relates to a focus position specifying system for a fluorescent image, a focus position specifying method, and a focus position specifying program.

Conventionally, an immunostaining method for detecting an antigen in a tissue specimen is known as a cancer diagnosis method. Among immunostaining methods, in recent years, the fluorescent antibody method is increasingly used in place of the enzyme antibody method. The “fluorescent antibody method” is a technique in which a fluorescent substance is labeled on an antibody, and after staining a tissue specimen (antigen-antibody reaction), the tissue specimen is irradiated with excitation light to emit 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 number of fluorescent bright spots and fluorescence intensity of a phosphor bonded to a biological material with a fluorescence microscope to evaluate the expression level of the biological material.

  Incidentally, in order to measure the number of fluorescent bright spots and the fluorescent intensity, an optical system having a high magnification and a numerical aperture (NA) is required. When the magnification and numerical aperture of the optical system are increased, the depth of focus is reduced, so that the adjustment of the focus position is important. “Focus” is so-called focusing. When a focus shift occurs, in a microscope image (fluorescence image) including a fluorescent luminescent spot, there is a possibility that the fluorescent luminescent spot may be missed. There arises a problem that it becomes low and easily affected by background noise.

In this regard, Patent Document 2 discloses a method for specifying a focus position of a fluorescent image.
In Patent Document 2, the imaging range is divided into a plurality of parts in the Z-axis direction, and for each divided imaging range, the stage (11) is moved from the bottom in the Z-axis direction at a constant speed and is moved at a constant speed in the XY plane. Then, a circular fluorescent image is captured by the fluorescent marker (55) (see paragraphs 0048 to 0064 and FIGS. 6 to 9).
Thereafter, frequency analysis is performed on the plurality of fluorescent images, and a fluorescent image having the highest maximum frequency component is selected. Thereafter, the locus image of the fluorescent marker is analyzed for the selected fluorescent image, and distribution information of the plurality of fluorescent markers is calculated based on the analysis result (paragraphs 0065 to 0086, FIGS. 10 to 11).
In Patent Document 2, histogram format data is generated from the distribution information of fluorescent markers, and the in-focus position is specified from the histogram (see paragraphs 0087 to 0096, FIGS. 12 to 14).

JP 2013-57631 A JP 2013-114042 A

In the technique of Patent Document 2, when an in-focus position is specified from a histogram, as an example, the position A with the largest number of fluorescent markers is set as the in-focus position (see paragraph 0091, FIG. 12). However, even though frequency analysis and image analysis of the locus of the fluorescent marker are performed for a plurality of fluorescent images, the fluorescence at position A includes the fluorescence of noise such as dust, and the home of cells in the tissue specimen. It is believed that background fluorescence, including fluorescence, is also included. There is still room for improvement in the method of quantitatively specifying the in-focus position from the fluorescence image in view of the influence of noise and background.
Therefore, a main object of the present invention is to provide a focus position specifying system, a focus position specifying method, and a focus position that can quantitatively specify a focus position from a fluorescent image while suppressing the influence of noise and background. To provide a specific program.

In order to solve the above problems, according to the first aspect of the present invention,
A fluorescence microscope that captures a fluorescent image of a biological sample stained with a staining agent containing a fluorescent marker while stepwise switching the focal position over at least three stages;
Generating means for generating a fluorescent image from the fluorescent image;
Creating means for creating a histogram representing the frequency of occurrence of the fluorescent marker from the fluorescent image;
Specifying means for specifying a focus position of the fluorescent image based on the histogram;
Equipped with a,
The specifying means specifies a focus position of the fluorescent image based on the shape of the histogram, and provides a focus position specifying system for the fluorescent image.
According to a second aspect of the invention,
A fluorescence microscope that captures a fluorescent image of a biological sample stained with a staining agent containing a fluorescent marker while stepwise switching the focal position over at least three stages;
Generating means for generating a fluorescent image from the fluorescent image;
Creating means for creating a histogram representing the frequency of occurrence of the fluorescent marker from the fluorescent image;
Specifying means for specifying a focus position of the fluorescent image based on the histogram;
With
The specifying means is
If the histograms cross each other,
Create an approximate curve based on the histogram, identify a certain approximate curve from the approximate curve as a reference curve, calculate a difference in the occurrence frequency of the fluorescent marker between the reference curve and the other approximate curves,
The focal position of the fluorescent image, which is the identification source of the reference curve, and the focal position of the fluorescent image, which is the creation source of the approximate curve within which a difference in the occurrence frequency of the fluorescent marker is within a certain threshold, A system for specifying a focus position of a fluorescent image characterized by specifying the focus position of a fluorescent image is provided.

According to a third aspect of the invention,
Imaging a fluorescent image of a biological sample stained with a staining agent containing a fluorescent marker while switching the focal position in stages over at least three stages;
Generating a fluorescent image from the fluorescent image;
Creating a histogram representing the frequency of occurrence of the fluorescent marker from the fluorescent image;
Identifying a focus position of the fluorescent image based on the histogram;
Equipped with a,
In the specifying step, a focus position specifying method for a fluorescent image is provided, wherein the focus position of the fluorescent image is specified based on the shape of the histogram .

According to a fourth aspect of the invention,
The computer,
An imaging control means for controlling the fluorescence microscope to capture a fluorescent image of a biological sample stained with a staining agent containing a fluorescent marker while gradually switching the focal position over at least three stages,
Generating means for generating a fluorescent image from the fluorescent image;
Creating means for creating a histogram representing the frequency of occurrence of the fluorescent marker from the fluorescent image;
A specifying means for specifying a focus position of the fluorescent image based on the histogram;
To function as,
The specifying means specifies a focus position of the fluorescent image based on the shape of the histogram, and provides a focus position specifying program for the fluorescent image.

  According to the present invention, it is possible to quantitatively specify a focus position from a fluorescent image while suppressing the influence of noise and background.

It is a figure which shows schematic structure of the focusing position specific system of a fluorescence image. It is a flowchart for demonstrating schematically the focus position specific method (focus position specific process) of a fluorescence image. It is a figure which shows the example of a fluorescence image. It is a figure which shows the example of a histogram. It is a figure which shows the example of the fluorescence image concerning 2nd Embodiment. It is a figure which shows the example of the histogram concerning 2nd Embodiment. FIG. 6 is a histogram created from fluorescent images at the first, third, and fifth stages in FIG. It is a figure for demonstrating the relationship between switching of a focus position and creation of a histogram.

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

[First Embodiment]
[Fluorescence image focusing position identification system]
As shown in FIG. 1, the fluorescence image focus position specifying 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 image sensor 20. The stage 12 is provided with a tissue specimen 30 after immunostaining. The tissue specimen 30 is an example of a biological sample. The lens barrel 16 incorporates a lamp 40 and a fluorescent cube 50. The fluorescent cube 50 includes 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 fluorescent 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 onto the tissue specimen 30. As a result, the tissue specimen 30 emits fluorescence, and the fluorescence is collected by the objective lens 14 and passes through the dichroic mirror 54 and the absorption filter 56. Thereafter, the fluorescence is observed as a fluorescent image through the eyepiece 18 and is imaged on the image sensor 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 raising and lowering of the stage 12 to control the focal position (height position) of the tissue specimen 30 placed on the stage 12. The control unit 62 is connected to the image sensor 20 and can control the image sensor 20 to capture a fluorescent image and receive the fluorescent image to generate a fluorescent 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 fluorescent image focus position specifying program for executing the focus position specifying process of FIG.
A display device 70 is connected to the control device 60, and a calculation result by the control device 60 is displayed on the display device 70.

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

(1) Target biological substance The target biological substance is a target of immunostaining using a fluorescent label for detection or quantification mainly from the viewpoint of pathological diagnosis, and is expressed in tissue sections. Biological substances, especially proteins (antigens).
Typical target biological materials include biological materials that are expressed in cell membranes of various cancer tissues and can be used as biomarkers.

(2) Immunostaining agent (antibody-fluorescent nanoparticle conjugate)
As an immunostaining agent, in order to improve the efficiency of fluorescent labeling and suppress the time lapse leading to the deterioration of fluorescence as much as possible, primary antibodies and fluorescent nanoparticles are used indirectly, that is, other than covalent bonds using antigen-antibody reactions. It is preferable to use a complex linked by these bonds. In order to simplify the staining operation, a complex in which fluorescent nanoparticles are directly bound to a primary antibody or a secondary antibody can also be used as an immunostaining agent.

As an example of the immunostaining agent, [primary antibody against the target biological substance]... [Antibody against primary antibody (secondary antibody)] to [fluorescent nanoparticles] can be mentioned.
“...” represents binding by an antigen-antibody reaction, and the mode of binding indicated by “˜” is not particularly limited. Examples include biotin avidin reaction, physical adsorption, chemical adsorption, and the like, and a linker molecule may be used as necessary.

(3) Antibody As the primary antibody, an antibody (IgG) that specifically recognizes and binds a protein as a target biological substance as an antigen can be used. For example, when HER2 is the target biological material, an anti-HER2 antibody can be used, and when HER3 is the target biological material, an anti-HER3 antibody can be used.
As the secondary antibody, an antibody (IgG) that specifically recognizes and binds to the primary antibody as an antigen can be used.
Both the primary antibody and the secondary antibody may be polyclonal antibodies, but from the viewpoint of quantitative stability, monoclonal antibodies are preferred. The type of animal that produces the antibody (immunized animal) is not particularly limited, and may be selected from mice, rats, guinea pigs, rabbits, goats, sheep, and the like as in the past.

(4) Fluorescent nanoparticles Fluorescent nanoparticles are nano-sized particles that emit fluorescence when irradiated with excitation light, and emit fluorescent light with sufficient intensity to represent the target biological substance as a bright spot one molecule at a time. Possible particles.
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. Examples thereof include CdSe, CdS, CdTe, ZnSe, ZnS, ZnTe, InP, InN, InAs, InGaP, GaP, GaAs, Si, and Ge.

(4.2) Fluorescent substance integrated nanoparticles Fluorescent substance integrated nanoparticles are based on organic or inorganic particles, and a plurality of fluorescent substances (for example, the above quantum dots and fluorescent dyes) are encapsulated therein. Nano-sized particles having a structure that is adsorbed and / or adsorbed on its surface.
As the fluorescent substance-integrated nanoparticles, it is preferable that the matrix and the fluorescent substance have substituents or sites having opposite charges, and an electrostatic interaction works.
As the fluorescent substance integrated nanoparticles, quantum dot integrated nanoparticles, fluorescent dye integrated nanoparticles, and the like 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 resins, acrylic resins, acrylonitrile resins, AS resins (acrylonitrile-styrene copolymers), ASA resins (acrylonitrile-styrene-methyl acrylate copolymers); Examples of other resins such as lactic acid; polysaccharides.
Of the matrix, examples of the inorganic substance include silica and glass.

(4.2.2) Quantum dot integrated nanoparticle The quantum dot integrated nanoparticle has a structure in which the quantum dot is included in the matrix and / or adsorbed on the surface thereof.
When the quantum dots are encapsulated in the matrix, the quantum dots may be dispersed inside 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 included in the 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 pyromesene dye molecules.
As fluorescent dyes, Alexa Fluor (registered trademark, manufactured by Invitrogen) -based dye molecule, BODIPY (registered trademark, manufactured by Invitrogen) -based dye molecule, Cy (registered trademark, manufactured by GE Healthcare) -based dye molecule, HiLite (registered) Trademark, manufactured by Anaspec) dye molecule, DyLight (registered trademark, manufactured by Thermo Scientific) dye 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 DYOMICICS) dye molecules, CAL (registered trademark, manufactured by BioSearch Technologies) dye molecules, and the like can be used. .
Note that when the fluorescent dye is included in the matrix, the fluorescent dye may be dispersed inside 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.
A method for preparing a tissue section to which this staining method can be applied (also simply referred to as “section”, including a section such as a pathological section) is not particularly limited, and a section prepared by a known procedure can be used.

(5.1) Specimen preparation step (5.1.1) Deparaffinization treatment The 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 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. If necessary, ethanol may be exchanged during the immersion.

  Immerse the sections in a container containing water and remove the 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. If necessary, water may be exchanged during the immersion.

(5.1.2) Activation process The activation process of the target biological material 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 A liquid etc. can be used.
The pH condition is such that a signal is output from the range of pH 2.0 to 13.0 depending on the tissue slice to be used, and the roughness of the tissue is such that the signal can be evaluated. Usually, the pH is 6.0 to 8.0, but for special tissue sections, for example, pH 3.0.
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 to 130 ° C. and the time can be 5 to 30 minutes.

  Next, the section after the 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 longer and 30 minutes or shorter. If necessary, PBS may be replaced during the immersion.

(5.2) Immunostaining step In the immunostaining step, in order to stain the target biological material, an immunostaining solution containing fluorescent nanoparticles having sites that can bind directly or indirectly to the target biological material, Place on section and react with target biological material. The immunostaining agent solution used in the immunostaining step may be prepared in advance before this step.

The conditions for performing the immunostaining step, that is, the temperature and immersion time when the tissue specimen is immersed in the immunostaining solution, should be adjusted as appropriate so that an appropriate signal can be obtained according to the conventional immunostaining method. Can do.
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.
Before performing the above-described treatment, it is preferable to add a known blocking agent such as BSA-containing PBS or a surfactant such as Tween 20 dropwise.

(5.3) Specimen post-treatment process The tissue specimen after the immunostaining process is preferably subjected to treatment such as immobilization / dehydration, penetration, 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 (crosslinking agent such as formalin, paraformaldehyde, glutaraldehyde, acetone, ethanol, methanol). In the penetration process, the tissue specimen after the fixation / dehydration process may be immersed in a penetration liquid (xylene or the like). The encapsulating process may be performed by immersing the tissue specimen that has undergone the penetration process in the encapsulating liquid.
The conditions for performing these treatments, for example, the temperature and immersion time when the tissue specimen is immersed in a predetermined treatment solution, can be adjusted as appropriate in accordance with conventional immunostaining methods so as to obtain an appropriate signal. it can.

(5.4) Morphological Observation Staining Step Apart from the immunostaining step, morphological observation staining may be performed so that the morphology of cells, tissues, organs, etc. can be observed in the bright field.
The morphological observation staining step can be performed according to a conventional method.
For morphological observation of tissue specimens, staining using eosin, in which cytoplasm, stroma, various fibers, erythrocytes, and keratinocytes are stained from red to dark red, is typically used. Staining using hematoxylin, in which cell nuclei, lime, cartilage tissue, bacteria, and mucus are stained blue-blue to light blue, is also used as a standard (the method of performing these two stainings simultaneously is hematoxylin and eosin staining). (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.

[Method for specifying focus position of fluorescent image]
Next, a method for specifying the focus position of the fluorescent image will be described.
Such a method is a method performed when a target biological substance is detected from the tissue specimen 30 after immunostaining using the in-focus position specifying system 1 for fluorescent images.

  First, the tissue specimen 30 after immunostaining is placed on the stage 12 of the fluorescence microscope 10. Thereafter, the control device 60 is used to generate a fluorescent image from the tissue specimen 30 and perform image processing to specify an optimum in-focus position (in-focus position specifying process).

  The in-focus position specifying process is executed in cooperation with the control unit 62 and a program stored in the storage unit 64, and the control unit 62 executes the following process according to the program. An example of such a program is “ImageJ” (open source). By using such image processing software, the process of extracting fluorescent luminescent spots of a predetermined wavelength (color) from the fluorescent image and calculating the luminance value of the luminescent spot area and the number of fluorescent nanoparticles is semi-automatically performed quickly. Can be done.

As shown in FIG. 2, the control unit 62 controls the stage 12 of the fluorescence microscope 10 to switch the focal position in stages over at least three stages, and each time the stage is switched, the fluorescence image of the tissue specimen 30 is displayed. Capture at least three fluorescent images (step S1).
When capturing a fluorescent image, the control unit 62 turns on the lamp 40 and irradiates the tissue specimen 30 with excitation light. Then, the fluorescent nanoparticles of the tissue specimen 30 emit fluorescent light, and a fluorescent bright spot appears. The controller 62 causes the image sensor 20 to capture the fluorescent image.

Thereafter, the control unit 62 generates a fluorescent image from the fluorescent image based on the imaging result of the imaging device 20 (step S2).
FIG. 3 shows an example of a fluorescence image generated from the fluorescence image.
Here, a sample in which fluorescent nanoparticles (about 150 nm in diameter) were dispersed on a slide glass as a tissue specimen 30 was prepared, and an 8-bit fluorescent image was generated from the sample using the ImageJ. The focal position was switched in steps of 1.0 μm over 5 stages, and each time the stage was switched, a fluorescent image was captured to generate a total of 5 fluorescent images.

Thereafter, the control unit 62 creates a histogram from the fluorescent image for each generated fluorescent image (step S3).
“Histogram” represents the frequency of occurrence of fluorescent markers in a fluorescent image. For example, as a histogram, a fluorescence image is observed for each pixel and indicates how often a pixel having a certain luminance value is generated.

FIG. 4 shows an example of a histogram created from the five fluorescent images shown in FIG.
In FIG. 4, the horizontal axis represents the luminance value (gradation value) in units of pixels, and the vertical axis represents the number of pixels having the luminance value (frequency). FIG. 4 shows the frequency of occurrence of fluorescent nanoparticles.
As shown in FIG. 4, the histogram created from the fluorescence image at the third stage has a generally high luminance value and a gentle curve shifted to the high luminance side.
On the other hand, the histogram created from the first-stage, second-stage, fourth-stage, and fifth-stage fluorescence images is a curve with a large slope on the low luminance side, particularly the first-stage and fifth-stage fluorescence images. The tendency is remarkable in the histogram created from

Thereafter, the control unit 62 specifies the in-focus position of the fluorescent image based on the created histogram (step S4).
For example, the control unit 62 compares the shapes of the created histograms and specifies the focus position of the fluorescent image from the comparison result.
In the example of FIG. 4, the shapes of histograms created from the first to fifth stages of fluorescence images are compared. As a result, the histogram created from the fluorescence image at the third stage has a generally higher luminance value than the histogram created from the fluorescence images at the first stage, the second stage, the fourth stage, and the fifth stage, and the slope of the curve. Obviously it has become moderate. Therefore, the focal position where the third-level fluorescence image having the highest luminance value is obtained is identified as the in-focus position of the fluorescence image.
Actually, even if the fluorescent images in the first to fifth stages are visually observed (see FIG. 3), the third stage fluorescent image is in focus and the focal positions are ± 1.0 μm and ± 2.0 μm. It can be seen that the image becomes blurred as it increases.
In step S4, in addition to or instead of the control unit 62 automatically comparing the shape of the histogram and specifying the focus position, the histogram created by the control unit 62 is displayed on the display device 70, so that the operator himself The comparison of the shape of the histogram and the identification of the in-focus position may be performed visually.

  Finally, the control unit 62 controls the stage 12 of the fluorescent microscope 10 to move the focal position to the in-focus position of the fluorescent image specified in step S4.

According to the first embodiment described above, the fluorescent image is generated and the histogram is generated while the focal position is switched stepwise, the shapes of the histograms are compared, and the in-focus position of the fluorescent image is specified from the comparison result. is doing.
In this case, even if the fluorescence of noise such as dust is reflected on a pixel, if the number of pixels is about several pixels, the histogram is hardly affected. Even if the fluorescence of noise is strongly reflected in a pixel having a noise, the shape of the whole histogram is hardly changed. Therefore, the comparison result is hardly affected in comparing the shapes of the histograms. In the histogram representing the occurrence frequency of the fluorescent marker as shown in FIG. 4, since the number (frequency) of pixels having the luminance value is calculated for a certain luminance value, the histogram is also applied to a weak signal such as the background. Not easily affected.
From the above, the in-focus position can be quantitatively specified from the fluorescence image while suppressing the influence of noise and background.

[Second Embodiment]
The second embodiment is different from the first embodiment in the following points, and is otherwise the same.

  In steps S <b> 1 and S <b> 2, as in the first embodiment, the control unit 62 captures a fluorescent image of the tissue specimen 30 and generates a fluorescent image each time the stage is switched while switching the focal position in stages. .

FIG. 5 shows an example of a fluorescence image generated from the fluorescence image.
Here, Cy5-encapsulated silica nanoparticles were prepared as the fluorescent nanoparticles a, and an immunostaining agent A in which an anti-HER2 antibody was bound to the fluorescent nanoparticles a was prepared. Thereafter, immunostaining is performed on a tissue section of human breast tissue using immunostaining agent A, and the tissue specimen after immunostaining is prepared as a tissue specimen 30 (sample), and 8-bit fluorescence is prepared from the sample using ImageJ described above. An image was generated. The focal position was switched in steps of 1.0 μm over 5 stages, and each time the stage was switched, a fluorescent image was captured to generate a total of 5 fluorescent images.

  Also in step S3, as in the first embodiment, the control unit 62 creates a histogram from the fluorescence image for each generated fluorescence image.

FIG. 6 shows an example of a histogram created from the five fluorescent images shown in FIG.
In FIG. 6, the horizontal axis represents the luminance value (gradation value) in units of pixels, and the vertical axis represents the number of pixels having the luminance value (frequency). FIG. 6 shows the frequency of occurrence of fluorescent nanoparticles.
As shown in FIG. 6, the histogram created from the fluorescent images in the second to fourth stages has a generally high luminance value and is a gentle curve shifted to the high luminance side, and there is almost no difference.
On the other hand, the histogram created from the first-stage and fifth-stage fluorescence images is a curve with a large slope on the low luminance side.

Also in step S4, basically, as in the first embodiment, the control unit 62 specifies the in-focus position of the fluorescent image based on the created histogram.
In the example of FIG. 6, the shapes of histograms created from the first to fifth stages of fluorescence images are compared. As a result, the histogram created from the fluorescent images at the 2nd to 4th stages has an overall higher brightness value and the slope of the curve is clearly gentler than the histogram created from the 1st and 5th stage fluorescent images. ing. However, the histograms created from the fluorescence images in the second to fourth stages are interlaced with each other.

In such a case, in step S4 according to the second embodiment, first, the control unit 62 creates an approximate curve by approximating the created histogram with an exponential function.
In the example of FIG. 6, approximate curves 101 to 105 are created by approximating a histogram created from the first to fifth stages of fluorescence images with an exponential function. The approximate curve 101 is an approximate curve obtained by approximating a histogram created from the first-stage fluorescence image with an exponential function. The approximate curves 102 to 105 are approximate curves obtained by approximating a histogram created from the fluorescence images in the second to fifth stages with an exponential function.
When the luminance value on the horizontal axis in FIG. 6 is x and the frequency on the vertical axis in FIG. 6 is y, the approximate curves 101 to 105 are expressed by the following exponential functions.
Approximate curve 101; y = 25743e− ^0.032x
Approximate curve 102; y = 14794e ^-0.024x
Approximate curve 103; y = 12343e ^-0.022x
Approximate curve 104; y = 13923e ^-0.023x
Approximate curve 105; y = 19842e ^-0.028x

Thereafter, the control unit 62 compares the approximate curves with each other to identify a certain approximate curve as a reference curve, and calculates a frequency difference between the reference curve and other approximate curves.
In the example of FIG. 6, the approximate curves 101 to 105 are compared, and the approximate curve 103 with the gradual slope on the high luminance side is specified as the reference curve 110. Thereafter, the reference curve 110 is compared with the other approximate curves 101 to 102 and 104 to 105 to calculate a frequency difference. For example, for the approximate curves 101 to 105, the frequency corresponding to the luminance value is calculated for each constant luminance (gradation). Thereafter, the frequency difference between the approximate curves 101 to 102 and 104 to 105 with respect to the frequency of the reference curve 110 is calculated. The frequency difference is calculated by the calculation formula (1).
Difference in frequency [%] = ((frequency y of approximate curve) − (frequency y of reference curve)) / (frequency y of reference curve) × 100 (1)
Table 1 shows the frequency of the approximate curves 101 to 105 calculated for every 50 gradations (in a total of 5 points of luminance values x = 50, 100, 150, 200, and 250).
Table 2 shows the difference between the frequencies of the approximate curves 101 to 012 and 104 to 105 with respect to the frequency of the reference curve 110 (approximate curve 103).

Thereafter, the control unit 62 determines whether or not the calculated frequency difference is within a certain threshold, and the focal position of the fluorescent image that is the identification source of the reference curve and the calculated frequency difference is within the certain threshold. The focal positions of the fluorescent images from which the approximate curve is created are regarded as equivalent, and the focal positions of these fluorescent images are identified as the focal positions of the fluorescent images.
In the example of Table 2, for example, it is determined whether or not the difference between the frequencies of the approximate curves 101 to 012 and 104 to 105 with respect to the frequency of the reference curve 110 is within 30%.
Here, the difference between the frequencies of the approximate curves 101 and 105 with respect to the frequency of the reference curve 110 exceeds 30%, whereas the difference between the frequencies of the approximate curves 102 and 104 with respect to the frequency of the reference curve 110 is within 30%. (Refer to the thick frame).
Therefore, it is considered that the focal position of the fluorescent image from which the reference curve 110 (approximate curve 103) is specified and the focal position of the fluorescent image from which the approximate curves 102 and 104 are created are equivalent, and these fluorescent images. Is determined as the in-focus position of the fluorescent image.
In other words, in the example of FIG. 6, the focal position of the 2nd to 4th stage fluorescence images, which is the origin of the histogram created from the 2nd to 4th stage fluorescence images, is regarded as equivalent, and the focus position of the fluorescence image Identify.
FIG. 7 is a histogram in which the histograms created from the fluorescence images at the 2nd to 4th stages in FIG. 6 are regarded as equivalent. Specifically, the histograms are created from the fluorescence images at the 1st, 3rd and 5th stages. This is a histogram.
Actually, even if the fluorescent images in the first to fifth stages are visually observed (see FIG. 5), the fluorescent images in the second to fourth stages are in focus and the focal position is ± 2.0 μm. Now you can see that the image is out of focus.

In step S4 according to the second embodiment, when calculating the frequency difference (when calculating Table 1 and Table 2), the change in gradation need not be every 50 gradations and can be changed as appropriate. For example, it may be every 10 gradations (total 25 points of luminance value x = 10 to 250).
When determining whether or not the difference in frequency is within a certain threshold value, the threshold value need not be 30% and can be changed as appropriate. For example, the threshold value may be 20%.
It is not necessary to determine whether or not the frequency difference is within a certain threshold value for all gradations. For example, when the frequency difference is calculated with a total of 25 points for every 10 gradations, the frequency is calculated with 20 or more of them. It may be determined whether the difference is within 20%.
That is, the number of gradation changes when calculating the frequency difference, the threshold value when determining the frequency difference, and the number of target points when determining the frequency difference are the accuracy of the in-focus position of the fluorescent image to be specified. It can be appropriately changed depending on the situation.

According to the second embodiment described above, when the histograms cross each other when the shapes of the histograms are compared, the approximate curves are created, the reference curves are specified, and the frequency difference is calculated, and the reference curves are specified. The focus position of the fluorescence image and the focus position of the fluorescence image from which the reference curve whose frequency difference is within a certain threshold are created are identified as the focus position of the fluorescence image.
Therefore, not only when the fluorescent marker exists on the surface (same surface) of the tissue specimen 30, but also when the fluorescent marker exists at different height positions inside the tissue, the in-focus position of the fluorescent image is specified with a certain width. can do.

In addition, this embodiment including the 1st and 2nd embodiment may be improved suitably.
As shown in FIG. 8, the tissue specimen 30 is placed on a slide glass 80 and covered with a cover glass 82. In the present embodiment, the focal position is switched stepwise in at least three stages, a fluorescent image is captured each time, and a plurality of fluorescent images corresponding to the number of focal position switching are generated. In the example of FIGS. 3 and 5, the focal position is switched stepwise in five steps at 1.0 μm intervals along the thickness direction of the tissue specimen 30, and a fluorescent image is captured each time, and a total of five fluorescent images are obtained. Is generated.

  In the present embodiment, the focus position is switched over at least three or more stages along the thickness direction of the tissue specimen 30. (i) As described above, a fluorescence image is captured for each stage to generate a fluorescence image. Alternatively, a histogram may be generated. Alternatively, (ii) a fluorescent image is taken every three stages to generate a fluorescent image, and a histogram is generated. If it is determined that there is no focal position, the same processing may be executed in the next three steps, and such processing may be repeatedly executed every three steps. The focus position is switched in at least three stages, that is, at least three fluorescent images are captured because it is necessary to create and compare at least three histograms in order to specify the focus position of the fluorescent image. It is.

  In the present embodiment, the in-focus position of the fluorescent image is specified using a general-purpose fluorescent microscope 10 (see FIG. 1). A known hole slide scanner may be used in place of the fluorescence microscope 10. According to the hole slide scanner, not only is the focus automatically adjusted in the thickness direction (Z direction) of the tissue specimen 30, but also the stage can be moved in the length and width directions (XY directions) of the tissue specimen 30. Yes, a wide range of fluorescent images can be generated. Even in the hall slide scanner, after the in-focus position of the fluorescent image is specified, the focal position can be automatically moved to the in-focus position of the specified fluorescent image.

  In the present embodiment, a tissue section is targeted as a biological sample, and the tissue specimen 30 is stained with an immunostaining agent containing fluorescent nanoparticles as a fluorescent marker, and the in-focus position of the fluorescent image is specified. The subject of the biological sample may be a cultured cell or a gene (DNA). When the target of the biological sample is a gene, a fluorescent dye can be used as a fluorescent marker. Both fluorescent nanoparticles and fluorescent dyes are examples of fluorescent markers, and other known fluorescent markers may be used.

DESCRIPTION OF SYMBOLS 1 Fluorescence image focusing position specification system 10 Fluorescence microscope 12 Stage 14 Objective lens 16 Lens tube 18 Eyepiece 20 Imaging element 30 Tissue specimen 40 Lamp 50 Fluorescence cube 52 Excitation filter 54 Dichroic mirror 56 Absorption filter 60 Controller 62 Control part 64 Storage unit 70 Display device 101 to 105 Approximate curve 110 Reference curve

Claims (7)

A fluorescence microscope that captures a fluorescent image of a biological sample stained with a staining agent containing a fluorescent marker while stepwise switching the focal position over at least three stages;
Generating means for generating a fluorescent image from the fluorescent image;
Creating means for creating a histogram representing the frequency of occurrence of the fluorescent marker from the fluorescent image;
Specifying means for specifying a focus position of the fluorescent image based on the histogram;
Equipped with a,
The said specifying means specifies the focus position of the said fluorescence image based on the shape of the said histogram, The focus position specification system of the fluorescence image characterized by the above-mentioned . 2. The in-focus position of the fluorescent image according to claim 1, wherein the specifying unit specifies an in-focus position of the fluorescent image by comparing a plurality of shapes of the histograms generated by the generating unit. Specific system. A fluorescence microscope that captures a fluorescent image of a biological sample stained with a staining agent containing a fluorescent marker while stepwise switching the focal position over at least three stages;
Generating means for generating a fluorescent image from the fluorescent image;
Creating means for creating a histogram representing the frequency of occurrence of the fluorescent marker from the fluorescent image;
Specifying means for specifying a focus position of the fluorescent image based on the histogram;
With
The specifying means is
If the histograms cross each other,
Create an approximate curve based on the histogram, identify a certain approximate curve from the approximate curve as a reference curve, calculate a difference in the occurrence frequency of the fluorescent marker between the reference curve and the other approximate curves,
The focal position of the fluorescent image, which is the identification source of the reference curve, and the focal position of the fluorescent image, which is the creation source of the approximate curve within which a difference in the occurrence frequency of the fluorescent marker is within a certain threshold, An in-focus position specifying system for a fluorescent image, characterized by specifying the in-focus position of the fluorescent image. Imaging a fluorescent image of a biological sample stained with a staining agent containing a fluorescent marker while switching the focal position in stages over at least three stages;
Generating a fluorescent image from the fluorescent image;
Creating a histogram representing the frequency of occurrence of the fluorescent marker from the fluorescent image;
Identifying a focus position of the fluorescent image based on the histogram;
Equipped with a,
In the specifying step, the in-focus position of the fluorescent image is specified based on the shape of the histogram . 5. The alignment of the fluorescent image according to claim 4, wherein in the specifying step, the in-focus position of the fluorescent image is specified by comparing the shapes of the plurality of histograms created in the creating step. Focus position identification method. The computer,
An imaging control means for controlling the fluorescence microscope to capture a fluorescent image of a biological sample stained with a staining agent containing a fluorescent marker while gradually switching the focal position over at least three stages,
Generating means for generating a fluorescent image from the fluorescent image;
Creating means for creating a histogram representing the frequency of occurrence of the fluorescent marker from the fluorescent image;
A specifying means for specifying a focus position of the fluorescent image based on the histogram;
To function as,
The fluorescent image in-focus position specifying program characterized in that the specifying means specifies the in-focus position of the fluorescent image based on the shape of the histogram . The in-focus position of the fluorescent image according to claim 6, wherein the specifying unit specifies an in-focus position of the fluorescent image by comparing shapes of the plurality of histograms generated by the generating unit. Specific program.