KR20180062411A - Method of acquiring an image of a biological sample - Google Patents

Abstract

The present invention related to a method of providing digital data from a three-dimensional image of a sample taken for a biopsy or the like, and the method includes a sample light-transparency step, a dyeing step, a two-dimensional fluorescence image acquiring step, a three-dimensional fluorescence image generating step, and an image converting step. In the sample light-transparency step, the light transmittance of a sample taken from an organism or tissue is increased. In the dyeing step, the sample is dyed with a fluorescent material. In the two-dimensional fluorescence image acquiring step, excitation light is irradiated to a cross section of the sample, and the fluorescence material present in the cross section of the sample is excited, so that a two-dimensional fluorescence image of the sample is acquired from an irradiated fluorescence signal. In the three-dimensional fluorescence image generating step, a two-dimensional fluorescence image for each cross section of the sample is acquired, and the acquired two-dimensional fluorescence images are sequentially laminated, so that a three-dimensional fluorescence image for the sample is generated. In the image converting step, the three-dimensional fluorescent image is converted into a virtual white light image by using the intensity of the fluorescent signal of each point of the three-dimensional fluorescent image.

Description

[0001] The present invention relates to a method for acquiring a three-dimensional image of a biological sample,

The present invention relates to a method for acquiring a three-dimensional image of a biological sample. More particularly, the present invention relates to a method for acquiring a three-dimensional image of a biological sample that provides a three-dimensional image of a sample taken for biopsy or the like as digital data.

In the diagnosis of diseases such as cancer, it is essential to take a tissue of suspected lesion, make a slide, make a stain, and then microscopically examine the lesion.

Depending on what kind of test tool is used, the histopathologic examination can be performed by a long and thin needle biopsy histopathologic examination, a surgical pathology examination for removing organs by general anesthesia, a skin punch test , An endoscopic pathology examination that involves inserting an instrument with a camera through the body entrance, such as the mouth through which the air passes, the anus, the nostril, and the like, and clipping the site.

In general, biopsies are used to fix tissue samples taken from the human body to check for lesions, and are used for washing, dehydration, clearing, paraffin infiltration for tissue dissolution, After microscopic embedding, microtome cutting and staining, very thin samples are made into tens or hundreds of slides and examined with an optical microscope to determine whether they are lesions.

Such a conventional tissue inspection method has a problem in that the time from the sampling to the confirmation of the lesion usually takes about 2 to 5 days to delay the operation and treatment of the patient.

In addition, when we wanted to share the results of the biopsy performed at the lower hospitals with the higher hospitals, the pathologists of the higher hospitals judged based on the photographs and handwriting records written by the pathologists of the lower hospitals. It was very inefficient because a higher-level hospital had to re-organize or re-read samples from lower hospitals.

In addition, when a fluorescence microscope is used to observe a sample, a high contrast ratio can be obtained as compared with a general optical microscope, but there is a disadvantage that a standardized lesion reading method can not be applied to an optical microscope.

Korean Registered Patent No. 10-1239066 (entitled " Method for Detecting Self-Nano Particles and Observing Cells or Tissues Using a Confocal Microscope, "

SUMMARY OF THE INVENTION Accordingly, it is an object of the present invention to solve the above-mentioned problems, and it is an object of the present invention to quickly determine whether a lesion of a sample is lesioned and to generate a 3D image of a sample as digital data, And a method of acquiring a three-dimensional image of a biological sample, in which white light is transmitted through an end surface of a sample to convert the image into an image to be displayed and to which an existing reading method can be applied.

According to an aspect of the present invention, there is provided a method for acquiring a three-dimensional image of a biological sample, the method comprising: a sample light transparency step of increasing a light transmittance of a sample taken from an organism or tissue; Dyeing the sample with a fluorescent material; A two-dimensional fluorescence image acquiring step of irradiating excitation light to the cross-section of the sample and acquiring a two-dimensional fluorescence image of the sample from a fluorescence signal excited and emitted by the fluorescence substance present in the cross section of the sample; A three-dimensional fluorescence image generation step of acquiring a two-dimensional fluorescence image for each cross-section of the sample and successively laminating the obtained two-dimensional fluorescence images to generate a three-dimensional fluorescence image for the sample; And an image transforming step of transforming the three-dimensional fluorescence image into a virtual white light image by using intensity of a fluorescent signal of each point of the three-dimensional fluorescence image.

In the method for acquiring three-dimensional images of a biological sample according to the present invention, the image conversion step may include: a concentration calculating step of calculating a concentration of the fluorescent substance from intensity of a fluorescent signal of each point of the three-dimensional fluorescent image; An estimation step of estimating an absorption spectrum absorbed in the fluorescent material among the continuous spectrum of the white light using the calculated concentration of the fluorescent material; And a reducing step of reducing a hue corresponding to the estimated absorption spectrum to each point of the three-dimensional fluorescence image.

In the method for acquiring three-dimensional images of a biological sample according to the present invention, the sample light transparency may include a first washing step of immersing the sample in a washing solution, a step of replacing the washing solution with a rinsing solution, A second culture step of replacing the rinsing solution with a combination solution in which the rinsing solution and the rinsing solution are mixed at a constant ratio to cultivate the sample for a predetermined period of time; A second washing step of immersing the sample in the washing solution and immersing the sample in a stabilizing solution and incubating the same for a predetermined time to deactivate the rinsing solution remaining in the sample; And a car washing step.

A method for obtaining a three-dimensional image of a biological sample according to the present invention, wherein the sample comprises cells consisting of a nucleus and a cytoplasm, wherein the nucleus contains 4'6-diamidino-2-phenylindole (4 ' 6-diamidino-2-phenylindole, DAPI), and the cytoplasm can be stained with eosin.

In the three-dimensional image acquisition method of the biological sample of the present invention, the excitation light has a fluorescence absorption wavelength range corresponding to the 4'-6-diamidino-2-phenylindole or a fluorescence absorption wavelength range corresponding to the eosin , The nucleus and the existing region existing in the cytoplasm are excited by the excitation light to emit a fluorescence signal and the remaining region other than the existing region may not be excited by the excitation light and emit a fluorescence signal.

In the method for acquiring three-dimensional images of a biological sample according to the present invention, an image storing step of storing the virtual white light image in the form of digital data may be further included.

A method for acquiring a 3D image of a biological sample of the present invention, comprising the steps of: extracting a cross-sectional image of each cross-section of the virtual white light image, And classifying the cross-sectional image into a read object image group if the match rate is greater than or equal to a predetermined reference match rate, and classifying the cross-sectional image into a read-out image group if the match rate is less than a reference match rate; As shown in FIG.

According to the three-dimensional image acquisition method of the biological sample of the present invention, it is possible to promptly determine whether the sample is lesion.

Further, according to the method for acquiring three-dimensional images of a biological sample of the present invention, it is possible to acquire a fluorescent image of better quality by adjusting the wavelength range of the excitation light to the fluorescence absorption wavelength range corresponding to the fluorescent material. The reliability of the lesion judgment can be increased.

In addition, according to the method for acquiring three-dimensional images of a biological sample of the present invention, noise can be largely reduced and light bleaching can be prevented since the light can be irradiated only to a section that the user wants to actually observe.

According to the method for acquiring 3D images of a biological sample according to the present invention, a 3D fluorescence image is converted into a virtual white light image and provided to doctors, thereby shortening the time required for the lesion reading and increasing the read reliability.

According to the three-dimensional image acquisition method of the biological sample of the present invention, it is possible to remotely check an accurate diagnosis result of a lesion of a patient without moving to a remote hospital, and to pay a burden And it is possible to increase the reliability of the lesion diagnosis by crossing the lesion of the sample through the stored three-dimensional image of the pathologists.

According to the method for acquiring a three-dimensional image of a biological sample of the present invention, a cross-sectional image having a lesion is firstly filtered through a read program, and only a cross-sectional image suspected of lesion is read secondarily by a doctor, Time and manpower can be saved, and the reliability of the lesion diagnosis can be increased.

FIG. 1 is a view schematically showing a process of performing a method of acquiring a three-dimensional image of a biological sample according to an embodiment of the present invention,
FIG. 2 is a block diagram illustrating a method of acquiring a three-dimensional image of the biological sample of FIG. 1,
FIG. 3 is a diagram illustrating a method of remotely diagnosing a lesion of a sample using the 3D image acquisition method of the biological sample of FIG. 1,
FIG. 4 is a view for explaining a process of converting a virtual white light image in the three-dimensional image acquisition method of the biological sample of FIG. 1,
FIG. 5 is a block diagram showing a detailed procedure of a sample light transparency step included in the three-dimensional image acquisition method of the biological sample of FIG. 1,
FIG. 6 is a view showing selective emission of fluorescence signal regions by excitation light having a specific fluorescence absorption wavelength range in the 3D image acquisition method of the biological sample of FIG. 1,
7 is a block diagram illustrating a method for acquiring a three-dimensional image of a biological sample according to another embodiment of the present invention,
8 is a flowchart illustrating a discrimination algorithm applied to an image classification step included in the 3D image acquisition method of the biological sample of FIG.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, embodiments of a method for acquiring a three-dimensional image of a biological sample according to the present invention will be described in detail with reference to the accompanying drawings.

2 is a block diagram illustrating a method of acquiring a three-dimensional image of a biological sample of FIG. 1, and FIG. 2 is a block diagram of a method of acquiring a three-dimensional image of a biological sample according to an embodiment of the present invention. FIG. 4 is a diagram for explaining a method for acquiring a virtual white light image in a three-dimensional image acquisition method of the biological sample of FIG. 1; FIG. FIG. 5 is a block diagram showing a detailed procedure of a sample light transparency step included in the method of acquiring a three-dimensional image of the biological sample of FIG. 1, and FIG. 6 is a flowchart of a three- And selectively emitted by the excitation light having a specific fluorescence absorption wavelength range according to the fluorescence signal region in the image acquisition method.

1 to 6, a method for acquiring a three-dimensional image of a biological sample according to an exemplary embodiment of the present invention is a method for providing a three-dimensional image of a sample, A three-dimensional fluorescence image generating step S4, an image converting step S5, an image storing step S6, a three-dimensional fluorescence image generating step S4, a two-dimensional fluorescence image acquiring step S3, .

The sample light transparency step S1 is a step of increasing the light transmittance of the sample S taken from the organism or tissue. As the excitation light L penetrates deeply into the sample S, it is possible to clearly determine whether the excitation light is lesioned. The excitation light L can not pass through the sample S, So that it becomes difficult to observe the sample S.

That is, since the excitation light L can not transmit or transmit the sample S, the virtual white light image Ir of the desired sample S decreases as the emission intensity of the fluorescence signal N decreases. Can not be obtained.

The sample light transparency step S1 includes a first washing step S1a for immersing the sample S in the washing solution W1 as shown in FIG. 1 or 5, a washing step S1a for washing the washing solution W1 with a rinsing solution A first culture step (S1b) of culturing the sample (S) for a predetermined period of time by replacing the rinsing solution (W1) with the rinsing solution (W2) A second washing step (S1d) of immersing the sample (S) in the washing solution (W1) and washing again, and a second washing step (S1d) of immersing the sample (S) in the washing solution (W1) The stabilization step S1e and the sample S for immersing the rinsing solution W2 remaining in the sample S in the cleaning solution W1 by soaking the stabilizing solution S in the stabilizing solution W4 and culturing the solution for a predetermined time to deactivate the rinsing solution W2 remaining in the sample S And a tertiary cleaning step S1f for cleaning the substrate.

The cleaning solution W1 was prepared by mixing 50 wt% of phosphate buffer saline, 25 wt% of hydrochloric acid having an acidity (PH) of 3 to 5 and 25 wt% of potassium hydrogen phthalate solution And the rinse solution W2 may be a mixed solution of 4 to 10% by weight of glutamic acid and 90 to 96% by weight of water, and the combination solution W3 may be 1 to 4% by weight of the rinse solution, (W4) may be a solution in which 4% by weight of glycine, 4% by weight of acetamide and 92% by weight of water is mixed.

As the sample light transparency method, various methods other than the above-described method can be used. For example, a benzyl alcohol / benzyl benzoate (BABB) method, a Scale method, a 3D imaging of solvent-cleared organs (3DISCO) method, a ClearT method, a deep brain method, a CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging (CIBIC), PACT (passive CLARITY technique), perfusion-assisted agent release in situ (PARS), and Immunolabeling-enabled (iDISCO) 3D imaging of solvent-cleared organs, ACT-PRESTO (active clearing technique-pressure related efficient and stable transfer of macromolecules into organs) method, SWITCH (system-wide control of interaction time and kinetics of chemicals) . These methods are obvious to those of ordinary skill in the art and will not be described in detail.

The dyeing step (S2) is a step of dyeing the sample (S) with the fluorescent material (M). Assuming that the sample S contains a cell Ce consisting of a nucleus Ce1 and a cell Ce2, the nucleus Ce1 is 4'6-diamidino-2-phenylindole (4'6 -diamidino-2-phenylindole, DAPI), and cytoplasmic (Ce2) can be stained with eosin. Because hematoxylin, which is mainly used in conventional histological examinations, can stain the nucleus (Ce1) but does not have a fluorescent property, the contrast between the nucleus (Ce1) and the cytoplasm (Ce2) is clear This is because it can appear.

Here, the excitation light (L) has a fluorescence absorption wavelength range corresponding to 4'6-diamidino-2-phenylindole or a fluorescence absorption wavelength range corresponding to eosin, and as shown in FIG. 6, The excitation region A in which the excitation light Ce2 is present is excited by the excitation light L1 to emit the fluorescence signal N and the remaining region A ' So that the fluorescence signal N is not emitted.

A good fluorescence image means that the remaining area A where the fluorescent material M does not exist is dark and black and the fluorescence substance M emits the fluorescence signal N and the existing area A is bright, (signal to noise ratio). The present embodiment can acquire fluorescence images of better quality by adjusting the wavelength range of the excitation light L to the fluorescence absorption wavelength range corresponding to the fluorescent material M, thereby increasing the reliability of the lesion reading .

The two-dimensional fluorescence image acquiring step S3 irradiates the excitation light L to the cross section of the sample S and irradiates the fluorescence material M Dimensional fluorescence image I2 of the sample S from the fluorescence signal N which is excited and emitted.

The excitation light L to be irradiated on the cross section of the sample S may be irradiated parallel to the cross section of the sample S as shown in Fig.

In the two-dimensional fluorescence image acquisition step S3, various types of image acquisition devices may be used. Typically, the image acquisition device is a total internal reflection fluorescence microscopy (TIRF), a high-inclinometry and a laminated optical sheet microscopy (HILO) , Real-time confocal fluorescence microscopy, and selective plane illumination microscopy (SPIM).

The total reflection microscope is based on the phenomenon that the light is reflected from the interface of the medium when the incident angle is larger than the critical angle when the light travels from a medium having a high refractive index to a medium having a low refractive index and thus the information inside the cell can not be obtained at all, have.

The HILO microscope is made by making the incident angle smaller than the critical angle in the total reflection method. Although it can be seen in the cell, the noise is relatively severe and it is not applicable to the thick sample.

A real-time confocal fluorescence microscope is a method of reducing noise by using a lens to make a position that shares the focus and focus of an objective lens and placing a small hole at the position so as to prevent a signal out of the focus plane from passing through the hole. It takes a lot of time to scan two-dimensionally to obtain one cross-section image.

All of the above-mentioned microscopes come into the sample S in a vertical direction since the light enters through the rear portion of the objective lens, so that light is also incident on a portion not observed by us. Noise is relatively inevitable.

On the other hand, the selective planar irradiation microscope is configured such that the excitation light L is irradiated parallel to the end face of the sample S by disposing an additional lens in the optical system in a direction perpendicular to the objective lens 20 to which the fluorescence signal N is input do. Since the excitation light L can be irradiated only to the section to be actually observed by the user, the noise can be greatly reduced, and the optical bleaching phenomenon occurring in the confocal microscope (when the excitation light is applied to the fluorescent material, The fluorescence signal can not be emitted any more and the fluorescence signal can no longer be emitted.

The three-dimensional fluorescence image generation step S4 acquires a two-dimensional fluorescence image I2 for each cross-section of the sample S, sequentially laminates the obtained two-dimensional fluorescence images I2, Dimensional fluorescence image (I3).

In the process of acquiring the two-dimensional fluorescence image I2 for each cross section of the sample S, two-dimensional (two-dimensional) fluorescence image I2 is obtained for each cross section of the sample S while moving the sample S in the vertical direction, It is possible to acquire the fluorescence image I2 and acquire the two-dimensional fluorescence image I2 for each cross section of the sample S while rotating the sample S about the rotation axis inside the sample S have.

The three-dimensional fluorescence image (I3) can be obtained by collecting the two-dimensional fluorescence images (I2) successively obtained by a computer program. The correction can obtain a clearer three-dimensional fluorescence image (I3) And may represent the depth of the three-dimensional fluorescent image I3.

The image conversion step S5 is a step of converting the three-dimensional fluorescent image I3 into a virtual white light image Ir using the intensity of the fluorescent signal N of each point of the three-dimensional fluorescent image I3.

The actual white light image means an image which is transmitted through the white light WL on the cross section of the sample S and is directly seen on the user's eye E as shown in FIG. Means an image in which the three-dimensional fluorescent image I3 is transformed so that the white light WL appears to be transmitted through the cross section of the sample S.

The image conversion step S5 includes a concentration calculation step, an estimation step, and a reduction step.

The concentration calculating step calculates the concentration of the fluorescent material M from the intensity of the fluorescent signal N at each point of the three-dimensional fluorescent image I3. The estimation step estimates an absorption spectrum (P1 or P2) to be absorbed by the fluorescent material (M) in the continuous spectrum (T) of the white light (WL) using the calculated concentration of the fluorescent material. The reduction step reduces the hue (C1 or C2) corresponding to the estimated absorption spectrum to each point of the three-dimensional fluorescent image (I3).

For example, as shown in Fig. 1 or Fig. 4, one of the cross sections of the three-dimensional fluorescent image I3 is divided longitudinally and laterally into a plurality of regions, and the first region A1 of the plurality of regions The first concentration of the fluorescent material M in the region is calculated from the first fluorescent signal N1 and the ratio of the absorption of the white light WL to the fluorescent material M is inversely proportional to the thickness and concentration of the sample S The spectrum of the specific wavelength band of the continuous spectrum T included in the white light WL while the white light WL is transmitted through the first region A1 based on the thickness, the first concentration and the proportional constant of the sample given in advance The position and width of the dark line) is absorbed by the fluorescent material to exhibit the first absorption spectrum P1. The color C1 corresponding to the estimated first absorption spectrum P1 is returned as a data value and these are all combined to obtain a virtual white light image Ir for either one of the three-dimensional fluorescent image I3 will be.

That is, a three-dimensional fluorescence image (I3) obtained so as to apply a standardized lesion reading method to a general optical microscope is converted into a virtual white light image (Ir). The types of diseases, including cancer, vary widely, and most of the manuals for determining the types of lesions are based on optical microscopes, and doctors are trained on pathologic testing. Therefore, if the three-dimensional fluorescent image I3 is provided to doctors without being converted into a virtual white light image Ir, the lesion readout is also difficult and the time taken to read out becomes long.

The image storage step S6 stores the virtual white light image Ir in the form of digital data.

Since the 3D image of the sample S can be provided as digital data through the image storing step S6, the medical information can be shared among the hospitals. As shown in FIG. 3, a virtual white light image (Ir) obtained by performing a biopsy for a lesion suspected to a patient in a lower hospital is remotely transmitted to a higher-level hospital in the form of digital data, and the higher- Provide the diagnosis results to the patient.

Therefore, it is possible to remotely check the accurate diagnosis result of the patient's own lesion without moving the patient to the remote hospital, reduce the burden of collecting the sample again from the patient, The reliability of the lesion diagnosis can be increased.

FIG. 7 is a block diagram illustrating a method for acquiring a three-dimensional image of a biological sample according to another embodiment of the present invention. FIG. 8 is a flowchart illustrating a method of acquiring a three- Fig. In Figs. 7 and 8, the members denoted by the same reference numerals as those shown in Figs. 1 to 6 have the same configuration and function, and a detailed description thereof will be omitted.

Referring to FIGS. 7 and 8, a method for acquiring a three-dimensional image of a biological sample according to another embodiment of the present invention may further include an image classification step S7.

The image classifying step S7 is performed subsequent to the image transforming step S5 to extract a cross-sectional image of each cross-section of the virtual white light image Ir and to classify a pattern of the cross-sectional image and a pattern of the pre- If the matching rate is equal to or greater than a preset reference matching rate, the sectional image is classified into the reading target image group, and if the matching rate is less than the reference matching ratio, the sectional image is classified into the reading excluding image group.

Conventionally, since tens or hundreds of slides (not shown) having the sample S discontinued are all read to check whether the sample S is lesioned, the loss of time and attraction is reduced accordingly. Since it is usually very inefficient to read all the slides, the reliability of the lesion diagnosis has been compromised because only a few specimens are selected in actual hospitals. On the other hand, in the present embodiment, a cross-sectional image having a lesion is primarily filtered through an image classification step S7, and only a cross-sectional image suspected of lesion is read secondarily by a doctor, thereby saving time and manpower And the reliability of the lesion diagnosis can be enhanced.

The three-dimensional image acquisition method of the biological sample of the present invention configured as described above can quickly determine whether a sample is lesioned by quickly making the light transparent without damaging the sample tissue.

Further, in the method of acquiring the three-dimensional image of the biological sample constructed as described above, it is possible to acquire a fluorescent image of better quality by adjusting the wavelength range of the excitation light to the fluorescence absorption wavelength range corresponding to the fluorescent substance, It is possible to increase the reliability of the judgment of the lesion.

In addition, since the three-dimensional image acquisition method of a biological sample configured as described above can irradiate light only to a section that the user actually wants to observe, noise can be greatly reduced and an effect of preventing optical bleaching can be obtained .

In addition, the 3D image acquisition method of the biological sample constructed as described above converts the three-dimensional fluorescence image into the virtual white light image and provides it to the physicians, thereby shortening the time required for the lesion reading and increasing the read reliability Can be obtained.

In addition, the three-dimensional image acquisition method of the biological sample constructed as described above can remotely confirm the accurate diagnosis result of the lesion of the patient without moving to the remote hospital, The burden can be alleviated, and the reliability of the lesion diagnosis can be improved by cross-determining whether or not the lesion is present in the sample through the 3D image stored by the pathologists.

In addition, in the 3D image acquisition method of the biological sample constructed as described above, the cross-sectional image having a possibility of lesion is primarily filtered through the readout program, and only the cross-sectional image suspected of lesion is read secondarily by the doctor , Time and manpower can be saved, and the reliability of the lesion diagnosis can be enhanced.

The scope of the present invention is not limited to the above-described embodiments and modifications, but can be implemented in various forms of embodiments within the scope of the appended claims. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present invention as defined by the appended claims.

S1: sample optical transparency step
S2: Dyeing step
S3: 2D fluorescence image acquisition step
S4: Three-dimensional fluorescent image acquisition step
S5: Image conversion step
L: excitation light
M: Fluorescent material
N: Fluorescent signal
S: Sample

Claims (7)

A sample light transparency step of increasing the light transmittance of the sample taken from the organism or tissue;
Dyeing the sample with a fluorescent material;
A two-dimensional fluorescence image acquiring step of irradiating excitation light to the cross-section of the sample and acquiring a two-dimensional fluorescence image of the sample from a fluorescence signal excited and emitted by the fluorescence substance present in the cross section of the sample;
A three-dimensional fluorescence image generation step of acquiring a two-dimensional fluorescence image for each cross-section of the sample and successively laminating the obtained two-dimensional fluorescence images to generate a three-dimensional fluorescence image for the sample; And
And converting the three-dimensional fluorescence image into a virtual white light image using the intensity of the fluorescence signal of each point of the three-dimensional fluorescence image. The method according to claim 1,
Wherein the image conversion step comprises:
A concentration calculating step of calculating the concentration of the fluorescent material from the intensity of the fluorescent signal of each point of the three-dimensional fluorescent image;
An estimation step of estimating an absorption spectrum absorbed in the fluorescent material among the continuous spectrum of the white light using the calculated concentration of the fluorescent material; And
And reducing the hue corresponding to the estimated absorption spectrum to each point of the three-dimensional fluorescence image. The method according to claim 1,
Wherein the sample light transparency step comprises:
A first washing step of immersing the sample in the washing solution and washing the sample,
A primary culture step of replacing the washing solution with a rinsing solution and culturing the sample for a predetermined time,
A second culture step in which the rinsing solution is replaced with a combination solution in which the rinsing solution and the rinsing solution are mixed at a predetermined ratio to cultivate the sample for a predetermined period of time,
A second washing step of immersing the sample in the washing solution and washing the sample again,
A stabilization step of immersing the sample in a stabilizing solution and incubating the sample for a certain period of time to inactivate the rinsing solution remaining in the sample and
A third washing step of immersing the sample in the washing solution and washing the sample again. The method according to claim 1,
The sample includes cells consisting of nucleus and cytoplasm,
In the staining step, the nucleus is stained with 4'6-diamidino-2-phenylindole (DAPI), and the cytoplasm is stained with eosin A method for acquiring a three-dimensional image of a biological sample. 5. The method of claim 4,
Wherein the excitation light has a fluorescence absorption wavelength range corresponding to the 4'6-diamidino-2-phenylindole or a fluorescence absorption wavelength range corresponding to the eosin,
Wherein the nucleus and the existing region existing in the cytoplasm are excited by the excitation light to emit a fluorescence signal and the remaining region other than the existing region is excited by the excitation light and does not emit a fluorescence signal A three dimensional image acquisition method of a biological sample. The method according to claim 1,
And storing the virtual white light image in the form of digital data. The method according to claim 1,
After the image conversion step,
Extracting a cross-sectional image of each cross-section of the virtual white light image, determining a match rate between a pattern of the cross-sectional image and a pattern of a previously entered lesion image, and if the match rate is equal to or greater than a preset reference match rate, And classifying the cross-sectional image into a read-out image group if the coincidence rate is less than a reference coincidence rate.

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