CN115656120A - Dark field reflection ultraviolet optical microscopic imaging method and system - Google Patents
Dark field reflection ultraviolet optical microscopic imaging method and system Download PDFInfo
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Abstract
The invention relates to the technical field of rapid pathological detection, and discloses a dark field reflection ultraviolet optical microscopic imaging method, which comprises the following steps: s10: obtaining a tissue sample; s20: placing the tissue sample on an object stage, and obliquely irradiating ultraviolet light generated by an ultraviolet light source onto the tissue sample; and collecting ultraviolet reflected light from the tissue sample by using an ultraviolet optical system, and obtaining an ultraviolet dark field reflection image through a bright signal of the superficial diffuse reflected light of the tissue sample and a dark signal of ultraviolet incident light absorbed by cell nucleus. Dark field illumination based on ultraviolet light provides image contrast by utilizing dark signals generated by ultraviolet light absorbed by cell nuclei and bright signals generated by diffuse reflection of superficial layers of biological tissues under the condition of eliminating interference of specular reflection light and scattered light. The method does not need fluorescent staining, the sample does not need special section processing, the pathological tissue imaging result can be provided quickly and accurately with high signal-to-back ratio, and a quick and accurate reference basis is provided for intraoperative margin assessment.
Description
Technical Field
The invention relates to the technical field of rapid pathological detection, in particular to a dark field reflection ultraviolet optical microscopic imaging method and system.
Background
Histopathological examination is an important tool for biomedical diagnosis, and the conventional histopathological examination process includes: firstly, a piece of tissue cut off in the operation is taken, then the cut tissue is embedded in a paraffin block, sliced, stained by Hematoxylin-eosin (HE), and finally observed by a microscope to obtain a corresponding pathological diagnosis result. However, conventional paraffin sections are long in pathological detection time, usually 3-4 days, and cannot help intraoperative margin assessment requiring rapid diagnosis results.
At present, the paraffin-embedded section is often replaced by a frozen section in clinical operation, which comprises sampling, embedding, freezing, sectioning, staining and observing diagnosis, and the required time is generally 30 minutes or more. However, the freezing process often causes small broken holes in the tissue sample, which affects the judgment and diagnosis of the pathologist, and greatly limits the use of the frozen section technique in the incision edge assessment during the operation.
Intraoperative margin assessment: evaluation of cryosections of biopsies obtained during surgery can be a routine part of clinical care, but is fraught with difficulties. These difficulties include the time involved in embedding, freezing, cutting, staining, and viewing the resulting stained sections. This process may take 10 minutes or more for each sample. Furthermore, the quality of most frozen samples may not be optimal and is generally lower than that of formalin-fixed paraffin-embedded ("FFPE") samples. The resulting delays and analytical challenges limit the use of intraoperative biopsy or surgical margin assessment. And if the margin assessment is not done intraoperatively, additional surgery may be required. For example, 20% to 40% of breast cancer surgeries must be performed anew to remove residual cancer present at or near the surgical margin; it would be desirable to be able to perform a rapid, accurate assessment of pathology during the initial surgical procedure to ensure complete removal of the cancer deposits, and to reduce the pain and risk associated with re-surgery.
In patent document CN201580061933.4, a system and method for controlling imaging depth in tissue using fluorescence microscopy under uv excitation after staining with fluorescent agents is disclosed, which labels biological tissue with multiple exogenous fluorophores, then illuminates the biological tissue with uv light, and then probes multiple fluorescence signals that can characterize tissue information. Although this method can also image thick tissue without the need for thin-slicing processing, the fluorescence labeling process is not only time consuming, but can also contaminate biological tissue, affecting subsequent molecular analysis of tissue samples.
Disclosure of Invention
The invention aims to provide a dark field reflection ultraviolet optical microscopic imaging method and system, and aims to solve the problem that a method and a system for quickly and accurately acquiring pathological images are lacked in intraoperative margin assessment in the prior art.
The invention is realized in this way, a dark field reflection ultraviolet optical microscopic imaging method, comprising the following steps:
s10: obtaining a tissue sample;
s20: placing the tissue sample on an object stage, and obliquely irradiating ultraviolet light generated by an ultraviolet light source onto the tissue sample;
and collecting ultraviolet reflected light from the tissue sample by using an ultraviolet optical system, and obtaining an ultraviolet dark field reflection image through a bright signal of the superficial diffuse reflected light of the tissue sample and a dark signal of ultraviolet incident light absorbed by cell nucleus.
Optionally, in step S20, an ultraviolet light source having a wavelength band selected from a wavelength band of light having a center located at or near the following wavelength is used: 290nm;280nm;270nm;260nm;250nm;245nm;240nm;235nm;230nm;225nm;220nm;210nm;200nm;
wherein the ultraviolet light source comprises at least one of the following: an LED; a laser; a tunable laser; or a continuous source including, but not limited to, at least one of a continuous laser light source, an arc lamp, a laser ignition arc lamp, a krypton-bromine excimer lamp.
Optionally, the ultraviolet optical system includes an objective lens and an image sensor, and the ultraviolet reflected light is transmitted through the objective lens and received by the image sensor.
Optionally, in step S20, the normalization process is performed on the image formed by the ultraviolet reflection light, and the normalization process includes the following steps:
s21: taking a blank area image without structural features on a tissue sample at each collection wavelength, and taking the blank area image as a normalized reference image;
s22: and dividing the intensity value of each pixel on the reference image by the maximum intensity value on the reference image to obtain each pixel coefficient, and correspondingly distributing the obtained pixel coefficients to each actually shot image.
Optionally, in step S10, after the tissue sample is washed, a certain amount of refractive index matching solution is slowly dropped on the tissue sample, and then a cover glass is covered on the surface of the tissue sample, so that the refractive index matching solution is filled between the surface of the tissue sample and the cover glass; the cover glass has high transmittance to ultraviolet light, and the refractive index matching fluid is close to the refractive index of the tissue sample and has high transmittance to ultraviolet light.
Optionally, in step S20, the oblique incident angle of the ultraviolet light is: the included angle between the oblique incident ultraviolet light and the plane of the tissue sample is between 30 and 70 degrees.
Optionally, in S20, the ultraviolet light excites an autofluorescence reaction of the tissue sample, and the autofluorescence signal from the tissue sample is collected by the ultraviolet optical system to obtain an autofluorescence image of the tissue sample.
Optionally, the ultraviolet optical system includes a filter wheel, the filter wheel includes a first filter and a second filter, the first filter selects ultraviolet light to pass through, and the second filter selects fluorescence to pass through; and respectively obtaining an ultraviolet dark field reflection image and an autofluorescence image of the tissue sample through the switching of the filter wheel.
Optionally, performing virtual HE staining on the ultraviolet dark-field reflectance image of the tissue sample, including the following steps:
1) Carrying out signal inversion on the collected ultraviolet dark field reflection image to obtain an inversion image, and using the inversion image as an H channel in virtual HE dyeing;
2) Using the collected autofluorescence image as an E channel in virtual HE staining;
3) And distributing the signal values of the H channel and the E channel to R, G, B channels in a virtual dyeing map according to a certain proportion, and then combining signals of R, G, B channels to obtain a virtual HE image corresponding to the ultraviolet dark field reflection image.
Optionally, in step 3), the signal values of the H channel and the E channel are assigned to the R, G, B channel in the virtual stain map according to the following formula,
a dark-field reflective ultraviolet optical microscopy imaging system comprising:
the ultraviolet light source is used for generating ultraviolet light in an ultraviolet waveband, and the ultraviolet light is obliquely incident on the tissue sample;
a microscope for providing optical information about the tissue sample;
an image acquisition system for generating one or more images from optical information provided by the microscope.
Optionally, the system further comprises an image processing system and a display system, wherein the image processing system is used for processing the image obtained by the image acquisition system, and the display system is used for displaying the processed image for analysis.
Compared with the prior art, the dark-field reflection ultraviolet optical microscopic imaging method provided by the invention has the advantages that ultraviolet light is obliquely incident to the tissue sample, dark-field illumination (oblique incidence) based on the ultraviolet light (mainly 260nm, and the wavelength is the absorption peak of cell nucleus) is realized, and under the condition of eliminating interference of specular reflection light and scattered light, dark signals generated by ultraviolet light absorbed by the cell nucleus and bright signals generated by diffuse reflection of the superficial layer of the biological tissue are utilized to provide image contrast. The method does not need fluorescent staining, the sample does not need special section processing (such as frozen section), the pathological tissue imaging result can be provided rapidly, accurately and with high signal-to-back ratio, and a rapid and accurate reference basis is provided for intraoperative margin assessment.
Drawings
FIG. 1 is a schematic flow chart of an embodiment of a dark-field reflection ultraviolet optical microscopy imaging method provided by the present invention;
FIG. 2 is a schematic diagram of a dark field reflection UV optical microscopy imaging method according to the present invention;
FIG. 3 is a schematic diagram of an optical path of a dark field reflection ultraviolet optical microscopic imaging system provided by the present invention;
FIG. 4 is a process flow diagram of virtual staining of a dark field reflective UV optical microscopy imaging method provided by the present invention;
FIG. 5 is a comparison of an ultraviolet dark field reflection image, an autofluorescence image and a fluorescence image after dapi dyeing of a dark field reflection ultraviolet optical microscopic imaging method provided by the invention;
FIG. 6 is a comparison chart of signal-to-back ratio imaging of an ultraviolet dark field reflection image, an autofluorescence image and a fluorescence image after dapi dyeing of a dark field reflection ultraviolet optical microscopic imaging method provided by the invention.
Description of reference numerals:
100-a tissue sample; 200-ultraviolet light, 210-ultraviolet light source, 220-optical filter, 300-ultraviolet optical system, 310-objective table, 311-glass slide, 312-protective plate, 313-cover glass, 314-refractive index matching liquid, 320-objective lens, 330-image sensor, 340-filter wheel and 350-cylindrical mirror.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The following describes the implementation of the present invention in detail with reference to specific embodiments.
The same or similar reference numerals in the drawings of the present embodiment correspond to the same or similar components; in the description of the present invention, it should be understood that if there is an orientation or positional relationship indicated by the terms "upper", "lower", "left", "right", etc. based on the orientation or positional relationship shown in the drawings, it is only for convenience of describing the present invention and simplifying the description, but it is not intended to indicate or imply that the referred device or element must have a specific orientation, be constructed in a specific orientation, and be operated, and therefore, the terms describing the positional relationship in the drawings are only used for illustrative purposes and are not to be construed as limiting the present patent, and the specific meaning of the terms may be understood by those skilled in the art according to specific circumstances.
Referring to fig. 1-6, preferred embodiments of the present invention are shown.
A dark field reflection ultraviolet optical microscopic imaging method comprises the following steps:
s10: obtaining a tissue sample 100;
s20: placing the tissue sample 100 on the stage 310 with the ultraviolet light 200 generated by the ultraviolet light source 210 obliquely incident on the tissue sample 100;
the ultraviolet reflected light from the tissue sample 100 is collected using the ultraviolet optical system 300, and an ultraviolet dark-field reflection image is obtained by a bright signal of the diffuse reflected light of the superficial layer of the tissue sample 100 and a dark signal of the absorption of the ultraviolet incident light by the cell nucleus.
In the dark-field reflection ultraviolet optical microscopic imaging method provided by the embodiment, through oblique incidence of ultraviolet light 200 to the tissue sample 100, dark-field illumination (oblique incidence) based on ultraviolet light (mainly 260nm, the wavelength is the absorption peak of the cell nucleus) provides image contrast by using a dark signal generated by ultraviolet light absorbed by the cell nucleus and a bright signal generated by diffuse reflection of a superficial layer of biological tissue under the condition of eliminating interference of specular reflection light and scattered light. The method does not need fluorescent staining, the sample does not need special section processing (such as frozen section), the pathological tissue imaging result can be provided rapidly, accurately and with high signal-to-back ratio, and a rapid and accurate reference basis is provided for intraoperative margin assessment.
In the prior art, it is difficult to quickly and accurately evaluate the tissue sample 100, and if the ultraviolet light 200 is directly irradiated to the tissue sample 100, dark signals of the ultraviolet incident light absorbed by the cell nucleus of the tissue sample 100 cannot be obtained because the interference of the specular reflected light and the scattered light is very strong.
The ultraviolet optical system 300 of the present embodiment reduces loss and absorption of ultraviolet light during transmission as much as possible, and optical elements involved in the ultraviolet optical system 300 include a transmission element (mainly plays a role of transmission in an optical path), and the transmission element has a high transmittance for ultraviolet light, so as to avoid excessive loss of ultraviolet light during transmission; alternatively, the ultraviolet optical system 300 may further include a reflective element (mainly having a reflection function in the optical path) having a high reflectivity with respect to the ultraviolet light, so as to avoid excessive loss of the ultraviolet light upon reflection.
The ultraviolet optical system 300 includes an objective lens 320 and an image sensor 330, and ultraviolet reflected light is transmitted through the objective lens 320 and received by the image sensor 330.
The ultraviolet optical system 300 in this embodiment is a dark field reflective wide field microscope, 1) all optical components in the system have high transmittance to ultraviolet light, typically over 95% transmittance; 2) A light source section: LED light or laser of 260nm can be used, and LED light or laser of 200-400nm can also be used.
Optionally, the oblique incidence angle of the ultraviolet light 200 is: the oblique incidence of the ultraviolet light 200 is between 30 ° and 70 ° from the tissue sample plane, for example, the ultraviolet light 200 is obliquely incident on the tissue sample surface at an angle of 45 ° or 60 °. To better eliminate the adverse effects of specularly reflected light.
As shown in fig. 2, fig. 2 (a) shows the uv reflected light component from a tissue sample 100 under bright field illumination conditions of the prior art; fig. 2 (b) shows the principle of dark field reflection uv optical microscopy imaging of the present invention.
In fig. 2 (a), under the conventional bright field illumination condition, i.e., ultraviolet incident light is perpendicularly incident on the cover glass 313 above the tissue sample 100, and is reflected by the cover glass 313 and the tissue sample 100, mainly including specular reflected light (1), diffuse reflected light (2) on the superficial surface of the tissue sample 100, and scattered light (3) caused by non-uniform refractive index of the interface of the tissue sample 100. Which is received by the ultraviolet optical system 300
I detect ＝①+②+③-④，
Wherein (4) indicates the dark signal absorbed by the cell nucleus under the condition of ultraviolet incident light, which characterizes the distribution of cell nucleus in the tissue sample 100 and is also the key information in the pathological examination.
Under the traditional bright field illumination, the specular reflection light (1) and the scattered light (3) are too strong to submerge a dark signal (4) absorbed by ultraviolet incident light by a cell nucleus, so that the cell structure of biological tissues cannot be observed in a common bright field and dark field reflection light microscope.
As shown in fig. 2 (b), under dark field illumination conditions, i.e., ultraviolet incident light is obliquely incident on the cover glass 313 above the tissue sample 100. In the present embodiment, on the basis of the dark field illumination, the influence of the specular reflection can be eliminated as much as possible because the ultraviolet light 200 is incident obliquely and the specular reflection light (1) thereof is also emitted obliquely, so that the specular reflection light (1) thereof is not received by the ultraviolet optical system 300.
Further, in the present embodiment, the tissue sample 100 is flattened as much as possible by the cover glass 313, and a layer of refractive index matching fluid 314 is filled between the cover glass 313 and the tissue sample 100 to eliminate the influence of stray light (3), and image contrast is obtained mainly based on the dark signal (4) of ultraviolet absorption of cell nuclei and the diffuse reflected light (2) of the superficial layer of the tissue sample 100. The optical signal received by the ultraviolet optical system 300 at this time: I.C. A detect = 2) - (4), the effect of specular reflected light and stray light overwhelming the dark signal too strongly is eliminated.
Wherein, the cover glass 313 is covered on the tissue sample 100; the cover glass 313 is used for flattening the sample as much as possible, so that a clear image can be obtained in a large visual field range, and defocusing influence caused by the height of the outline of the tissue sample is reduced.
Specifically, in step S10, the obtained tissue sample 100 is subjected to a washing process and then placed on the slide 311. For example, the surface of the biological tissue sample 100 to be observed and the opposite surface thereof may be first slightly flattened with a scalpel, and then washed with phosphate-buffered saline (PBS) for 5 seconds, and then the tissue may be placed on the slide 311. After cleaning treatment, the subsequent ultraviolet microscopic imaging effect can be prevented from being influenced by stains.
Specifically, in step S10, the washed tissue sample 100 is placed on the slide 311, a certain amount of the refractive index matching fluid 314 is slowly dropped on the tissue sample 100, and then the cover glass 313 is covered on the surface of the tissue sample 100, so that the refractive index matching fluid 314 is filled between the surface of the tissue sample 100 and the cover glass 313; wherein the cover glass 313 has a high transmittance to ultraviolet light. The refractive index matching fluid 314 can eliminate the influence on the optical detection result caused by the material change of the interface at the joint, so that the cover glass 313 and the tissue sample 100 have better fitting property and light transmission property, the reflection loss related to the air contact surface of the tissue sample 100 is greatly reduced, the ultraviolet microscopic imaging effect of the tissue sample 100 is greatly improved, and the ultraviolet dark field reflection image of the tissue sample 100 is clearer.
For example, a certain amount of PBS solution or glycerol is slowly dropped on the biological tissue to avoid bubbles, and then a cover glass 313 is covered on the surface of the biological tissue to ensure that the space between the biological tissue and the cover glass 313 is filled with the PBS solution or glycerol and no bubbles exist. It is to be noted that the slide glass 311 and the cover glass 313 used herein are both quartz glass slides, and have high transmittance for ultraviolet band light; if the cover glass 313 has low transmittance for ultraviolet band light, the acquisition of the ultraviolet dark field reflection image is greatly affected.
Specifically, at least two guard plates 312 are vertically disposed on the slide 311, the tissue sample is located between the two guard plates 312, and the cover glass 313 abuts on the top of the guard plates 312.
Preferably, two protection plates 312 with equal height are arranged in parallel on the carrier sheet, the tissue sample 100 is placed between the two protection plates 312, two sides of the cover glass 313 abut against the tops of the two protection plates 312, for example, two small glass slides may be additionally cut as protection plates, fixed (by bonding) on the original glass slide 311 and located at two sides of the tissue sample 100 (forming a groove structure in which the tissue sample 100 is placed), and then two sides of the cover glass 313 are attached to the two small glass slides to obtain a relatively flat observation surface.
A plurality of equal-height guard plates 312 are connected end to form a receptacle with the slide 311 for receiving the tissue sample 100, and a cover glass 313 covers the top of the receptacle.
For example, four shields 312 may be disposed on the slide 311, the four shields 312 being secured to the slide, the four shields 312 being arranged in a square, the four shields 312 and the slide 311 together forming an open square receptacle. Placing the tissue sample 100 in the square cavity and then covering the cover glass 313 at the opening of the square cavity can obtain a relatively flat observation surface and facilitate filling the refractive index matching fluid 314 without being lost.
Optionally, in step S20, an ultraviolet light source 210 having a wavelength band selected from a wavelength band of light having a center located at or near the following wavelength is used: 290nm;280nm;270nm;260nm;250nm;245nm;240nm;235nm;230nm;225nm;220nm;210nm;200nm;
wherein the ultraviolet light source 210 includes at least one of: an LED; a laser; a tunable laser; or a continuous source comprising at least one of a continuous laser light source, an arc lamp, a laser ignition arc lamp, a krypton-bromine excimer lamp, or other source having sufficient brightness in the desired spectral range.
Specifically, in step S20, the normalization process is performed on the image formed by the ultraviolet reflection light, and the normalization process includes the following steps:
s21: taking a blank area image without structural features on a tissue sample 100 at each collection wavelength as a normalized reference image;
s22: and dividing the intensity value of each pixel on the reference image by the maximum intensity value on the reference image to obtain each pixel coefficient, and correspondingly distributing the obtained pixel coefficients to each actually shot image.
Through the normalization processing, the influence of non-uniform illumination is eliminated, and the contrast of the image is optimized.
Further, the normalization process may include the steps of:
i) Taking a blank area image without structural features on a sample at each collection wavelength as a normalized reference image (I) ref (x, y), I being the measured intensity value, (x, y) being the corresponding pixel coordinate);
ii) the intensity value of each pixel on the reference map is then divided by the maximum intensity value of the reference map to obtain a calibration matrix C (where C (x, y) = I:) ref (x,y)÷max(I ref (x,y)))；
iii) Multiplying the obtained calibration matrix C with each actually shot picture to eliminate the influence of non-uniform illumination;
iv) the set image at the central collection wavelength is the resulting normalized ultraviolet dark field reflectance image (e.g., a central collection wavelength of 260 nm) in which dark negative signals represent nuclei and bright positive signals are from diffuse reflectance of the superficial layer of the tissue sample 100, which can clearly identify nuclear structures.
Optionally, in S20, the ultraviolet light excites an autofluorescence reaction of the tissue sample 100, and the autofluorescence signal from the tissue sample 100 is collected by the ultraviolet optical system 300, so as to obtain an autofluorescence image of the tissue sample 100. Autofluorescence is the light that biological structures (e.g., mitochondria and lysosomes) naturally emit when they absorb light, and is used to distinguish light originating from artificially added fluorescent labels (fluorophores). Autofluorescence images also do not require fluorescent staining, but autofluorescence is generally weak and does not provide sufficient contrast of bright and dark signals, and can be used as an auxiliary detection means.
The ultraviolet optical system 300 includes a filter wheel 340, the filter wheel 340 is disposed between the cylindrical mirror 350 and the objective lens 320, or between the cylindrical mirror 350 and the image sensor 330, the filter wheel 340 includes a first filter and a second filter, the first filter selects the ultraviolet light to pass through, so that the ultraviolet light can pass through the first filter as much as possible; the second filter is used for selecting the passing of fluorescence, so that the autofluorescence generated by the tissue sample can pass through the second filter as much as possible; by switching the filter wheel 340, the ultraviolet dark field reflection image and the autofluorescence image of the tissue sample 100 are obtained respectively.
For example, the reflected light and autofluorescence signals from the superficial layer of tissue sample 100 are collected by objective lens 320 of UV optical system 300, and then passed through a motorized wheel (filter wheel 340) equipped with filters having center wavelengths of 260nm (first filter) and 357nm (second filter), respectively. The filter wheel 340 will distinguish the signals in the actual image acquisition where the 260nm channel corresponds to the uv dark field reflectance map and the 357nm channel corresponds to the auto fluorescence map.
Optionally, performing virtual HE staining on the ultraviolet dark-field reflectance image of the tissue sample 100 includes the following steps:
1) Carrying out signal inversion on the collected ultraviolet dark field reflection image to obtain an inversion image, and using the inversion image as an H channel in virtual HE dyeing;
2) Using the collected autofluorescence image as an E channel in virtual HE staining;
3) And distributing the signal values of the H channel and the E channel to a R, G, B channel in the virtual dyeing map according to a certain proportion, and then combining signals of a R, G, B channel to obtain a virtual HE image corresponding to the ultraviolet dark field reflection image.
The cell nucleus detected by the ultraviolet dark field reflection image is a black negative signal, when virtual staining is carried out, the reflection image needs to be subjected to signal inversion (namely, the dark cell nucleus is lightened, and the diffuse reflection bright signal is darkened; the method can be directly realized through imagej. In terms of algorithm, the method takes an 8-bit image as an example, the original reflection image is I 1 After signal inversion is I 2 ＝255-I 1 )。
Further, in step 3), the signal values of the H channel and the E channel are assigned to the R, G, B channel in the virtual staining map according to the following formula,
the virtual HE staining is virtual Hematoxylin-eosin (HE) staining, and has the effect of converting a black and white image (an ultraviolet dark field reflection image) detected by reflection into a virtual HE staining effect, so that a pathologist can conveniently make judgment. High signal-to-back ratio nuclear imaging (similar to dapi staining) is achieved by virtual HE staining, which is much stronger than the contrast brought by autofluorescence. This virtual result can be compared to the actual HE staining profile.
A dark field reflection ultraviolet optical microscopic imaging system based on a dark field reflection ultraviolet optical microscopic imaging method comprises:
an ultraviolet light source 210, the ultraviolet light source 210 being configured to generate ultraviolet light 200 in an ultraviolet band, the ultraviolet light 200 being incident obliquely on the tissue sample 100;
a microscope for providing optical information about the tissue sample 100;
an image acquisition system for generating one or more images from optical information provided by the microscope.
The ultraviolet light source 210 generates ultraviolet light 200, the ultraviolet light 200 obliquely enters the cover glass 313 through the optical filter 220 and the short-focus lens, the ultraviolet light 200 transmits through the cover glass 313, passes through the refractive index matching fluid 314 and then irradiates on the surface of the tissue sample 100, part of the light is reflected by the cover glass 313, and the reflected light obliquely exits and is not received by the ultraviolet optical system 300; part of the light is diffusely reflected by the tissue sample 100 and received by the uv optical system 300; also dark signals of the cell nuclei upon absorption of the uv incident light can be detected by the uv optical system 300.
Optionally, the system further comprises an image processing system and a display system, wherein the image processing system is used for processing the image obtained by the image acquisition system, and the display system is used for displaying the processed image for analysis.
In an embodiment of implementing the above dark-field reflection-based ultraviolet optical microscopic imaging method, a dark-field reflection ultraviolet optical microscopic imaging system for rapid pathological detection is adopted, and includes an ultraviolet light source 210 and an ultraviolet optical system 300, where the ultraviolet optical system 300 includes a stage 310, a cover glass 313, an objective lens 320, a tube lens 350 and an image sensor 330, the stage 310 is used for bearing a tissue sample 100 to be detected, and the cover glass 313 is covered on the tissue sample 100; the cover glass 313 is used for flattening the sample as much as possible, so that a clear image can be obtained in a large visual field range, and defocusing influence caused by the height of the outline of the tissue sample is reduced.
The ultraviolet light source 210 is arranged deviating from the receiving optical path of the ultraviolet optical system 300, the ultraviolet light 200 emitted by the ultraviolet light source 210 is obliquely incident to the cover glass 313, and the ultraviolet reflected light reflected by the tissue sample 100 passes through the objective lens 320 and the tube lens 350 in sequence and is received by the image sensor 330, so as to obtain the ultraviolet dark field reflection image of the tissue sample 100. In which the specular reflection light reflected by the cover glass 313 is not received by the ultraviolet optical system.
According to the dark-field reflection ultraviolet optical microscopic imaging system for rapid pathology detection provided by the embodiment, the ultraviolet light 200 emitted by the ultraviolet light source 210 is obliquely incident on the cover glass 313 to form dark-field illumination (oblique incidence) of the ultraviolet light 200, and under the condition of eliminating interference of specular reflection light and scattered light, dark signals generated by the ultraviolet light 200 absorbed by cell nuclei and bright signals generated by diffuse reflection of a superficial layer of biological tissues are utilized to provide image contrast. Therefore, in the detection process of the tissue sample 100, fluorescent staining is not needed, special section processing (such as frozen section) is not needed for the sample, the pathological tissue imaging result can be provided rapidly and accurately with high signal-to-back ratio, and a rapid and accurate reference basis is provided for intraoperative margin assessment.
Specifically, the tissue sample 100 is placed on a slide 311, the slide 311 is placed on a stage 310, a cover glass 313 is arranged above the tissue sample 100, and the cover glass 313 has high transmittance to ultraviolet light; an index matching fluid 314 is filled between the tissue sample 100 and the cover glass 313, and the index matching fluid 314 is a fluid having a refractive index close to that of the tissue sample 100 and a high transmittance to ultraviolet light. For example, the refractive index matching fluid 314 includes, but is not limited to, currently used PBS (phosphate-buffered saline) solution or glycerol, and these solutions may be replaced by a uv-transparent fluid having a refractive index close to that of the biological tissue to be measured.
In the following embodiments, a dark-field reflective ultraviolet optical microscopy imaging method and system is provided that can perform fast, label-free imaging of thick pathological tissue samples 100 without sectioning processing to facilitate fast and accurate pathological diagnosis and provide fast and accurate basis for intraoperative margin assessment. As shown in fig. 2, the specific method flow is as follows:
1. the biological tissue sample 100 after fresh excision or formalin fixation is taken, the surface to be observed and the opposite surface thereof are first slightly flattened with a scalpel (for easy detection), and then washed with phosphate-buffered saline (PBS) for 5 seconds, and then the tissue is placed on a slide 311.
2. A certain amount of PBS solution or glycerol is slowly dripped on the biological tissue to avoid bubbles, and then a cover glass 313 is covered on the surface of the biological tissue to ensure that the space between the biological tissue and the cover glass 313 is filled with the PBS solution or the glycerol and no bubbles exist. It is to be noted that the slide glass 311 and the cover glass 313 used herein are both quartz glass slides, and have high transmittance for ultraviolet band light.
3. The prepared biological sample is fixed on an object stage 310, such as a three-dimensional displacement stage, and then dark-field illumination is performed by ultraviolet light of 260nm, and then reflected light signals with a central wavelength of 260nm and autofluorescence signals with a central wavelength of 357nm are collected by an ultraviolet optical system 300, respectively.
Wherein, the ultraviolet LED light source with the wavelength of 260nm obliquely enters the sample surface after passing through the optical filter 220 with the central wavelength of 260nm and one to two short-focus optical lenses. The oblique incidence angle is 30-70 degrees with the sample plane, and the illumination area on the sample plane is 5-10mm 2 The intensity of the illumination of the single light source is about 20mW.
4. The image taken by the camera is normalized to eliminate the influence of non-uniform illumination: firstly, shooting a blank area image without structural features on a sample under each collection wavelength, and taking the blank area image as a normalized reference image; then dividing the intensity value of each pixel on the reference image by the maximum intensity value of the reference image, and correspondingly distributing the obtained pixel coefficients to each actually shot image. The image with the central collection wavelength of 260nm is the obtained ultraviolet dark field reflection image, and the nuclear structure can be clearly identified.
5. As shown in fig. 4, the collected black and white ultraviolet dark field reflection image may be further subjected to virtual HE staining, which specifically includes: i) Carrying out signal inversion on an ultraviolet dark field reflection map corresponding to the central collection wavelength of 260nm, and taking the inversion map as a cell nucleus channel (H channel) in HE staining; ii) collecting the auto-fluorescence map corresponding to 357nm wavelength at the center as cytoplasmic channel (E-channel) in HE staining; iii) And distributing the signal values of the H channel and the E channel to a R, G, B channel (formula (1)) in the virtual staining map according to a certain proportion, and combining the three RGB channels to obtain a virtual HE image corresponding to the black-white ultraviolet dark field reflection map.
R＝10 -(0.644E+0.093H)
G＝10 -(0.717E+0.954H)
B＝10 -(0.267E+0.283H) (1)
Wherein, the reflected light and autofluorescence signals from the sample surface are collected by objective lens 320, and pass through an electric wheel (filter wheel 340) equipped with filters with center wavelengths of 260nm and 357nm, respectively. The filter wheel 340 will distinguish the signals in the actual image acquisition where the 260nm channel corresponds to the uv dark field reflectance map and the 357nm channel corresponds to the auto fluorescence map.
The optical signal then passes through a tube lens 350, which is matched to the objective lens 320, and is then collected by a camera that is sensitive to ultraviolet light.
The currently used 260nm ultraviolet LED light source can be expanded to an LED light source with a wave band of 200-400nm or laser, and the wave band of the collected reflected light is not limited to 260nm, and can be correspondingly expanded to a wave band of 200-400 nm. Meanwhile, the refractive index matching fluid 314 is not limited to the currently used PBS solution or glycerol, and the solution can be replaced by a UV-transparent fluid with a refractive index close to that of the biological tissue to be detected.
Compared with the prior art, the method has the greatest advantages of high imaging speed and high signal-to-back ratio of the image.
1) The imaging speed is high: the time taken from sample preparation to actual image acquisition is about 2-3 minutes based on the method of the invention, and the algorithms for image normalization and virtual staining are also extremely simple (calculation time less than 1 second). The existing MUSE (ultraviolet surface excited fluorescence microscopic imaging technology) technology needs dyeing, so that a sample can be polluted, the dyeing process is time-consuming, and an image can be obtained in 10 minutes or more. While the CHAMP (ultraviolet illumination autofluorescence imaging) technique does not need to be dyed, but the technique needs to acquire 36 frames of images and perform calculation and reconstruction to obtain a final image, and the whole process is time-consuming, has poor image quality, and is difficult to be used as a reliable basis for intraoperative margin evaluation.
2) High imaging signal-to-back ratio: as shown in FIG. 5, the method of the present invention can perform high signal-to-back ratio imaging on cell nuclei, wherein FIG. 5-a corresponds to the ultraviolet dark field reflection image obtained by the method of the present invention, FIG. 5-b corresponds to the autofluorescence image of the tissue sample, and FIG. 5-c corresponds to the fluorescence image after dapi staining. In comparison, the signal contrast of the ultraviolet dark field reflection image shown in fig. 5-a is much higher than the autofluorescence image shown in fig. 5-b, because the autofluorescence signal of the biological tissue sample is generally weaker, and the bright signal of the ultraviolet reflection light and the dark signal absorbed by the cell nucleus of the tissue sample have higher signal contrast.
In an actual contrast experiment, imaging verification is carried out by using a mouse brain tissue sample 100, and an ultraviolet dark field reflection image (figure 5-a) and an autofluorescence image (figure 5-b) are respectively measured; the nuclei of the samples were then dapi stained (4', 6-diamidino-2-phenylindole), a common fluorescent staining procedure, with a filter with a center wavelength of 447nm added to the filter wheel 340 of the uv optical system, and the sample images were again taken under this filter (fig. 5-c). Referring to fig. 6, it can be found through comparison of experimental results that, in the case of no staining, the contrast of the nuclear signal of the ultraviolet dark field reflection image obtained by the method provided by the invention is close to the fluorescence staining level with extremely high specificity and sensitivity, and is far higher than the signal-to-back ratio in the autofluorescence image, and the nuclear signal can be used as a reference for intraoperative margin assessment. And the method provided by the invention is very quick in implementation process (the whole process only needs 2-3 minutes), does not need to be dyed or frozen, does not cause damage to a tissue sample, and is very suitable for intraoperative incisal edge assessment.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (12)
1. A dark field reflection ultraviolet optical microscopic imaging method is characterized by comprising the following steps:
s10: obtaining a tissue sample;
s20: placing the tissue sample on an objective table, and obliquely irradiating ultraviolet light generated by an ultraviolet light source onto the tissue sample;
ultraviolet reflected light from the tissue sample is collected by an ultraviolet optical system, and an ultraviolet dark field reflection image is obtained through a bright signal of shallow surface diffuse reflected light of the tissue sample and a dark signal of ultraviolet incident light absorbed by cell nucleus.
2. The dark-field reflection ultraviolet optical microscopy imaging method according to claim 1, characterized in that in step S20, an ultraviolet light source having a wavelength band selected from the group consisting of wavelengths of light having a center located at or near the following wavelengths is used: 290nm;280nm;270nm;260nm;250nm;245nm;240nm;235nm;230nm;225nm;220nm;210nm;200nm;
wherein the ultraviolet light source comprises at least one of the following: an LED; a laser; a tunable laser; or a continuous source including, but not limited to, at least one of a continuous laser light source, an arc lamp, a laser ignition arc lamp, a krypton-bromine excimer lamp.
3. The dark-field reflective ultraviolet optical microscopy imaging method as set forth in claim 2, wherein the ultraviolet optical system includes an objective lens and an image sensor, the ultraviolet reflected light being transmitted through the objective lens and received by the image sensor.
4. The dark-field reflection ultraviolet optical microscopy imaging method according to claim 3, characterized in that in step S20, normalization processing is performed on the image formed by the ultraviolet reflection light, and the normalization processing comprises the following steps:
s21: taking a blank area image without structural features on a tissue sample at each collection wavelength, and taking the blank area image as a normalized reference image;
s22: and dividing the intensity value of each pixel on the reference image by the maximum intensity value on the reference image to obtain each pixel coefficient, and correspondingly distributing the obtained pixel coefficients to each actually shot image.
5. The dark-field reflection ultraviolet optical microscopy imaging method as claimed in claim 3, wherein in step S10, after the tissue sample is cleaned, a certain amount of refractive index matching fluid is slowly dripped on the tissue sample, and then a cover glass is covered on the surface of the tissue sample, so that the refractive index matching fluid is filled between the surface of the tissue sample and the cover glass; the cover glass has high transmittance to ultraviolet light, and the refractive index matching fluid is close to the refractive index of the tissue sample and has high transmittance to ultraviolet light.
6. The dark-field reflection ultraviolet optical microscopy imaging method as claimed in claim 3, wherein in step S20, the oblique incidence angle of the ultraviolet light is as follows: the included angle between the oblique incident ultraviolet light and the plane of the tissue sample is between 30 and 70 degrees.
7. The dark-field reflection ultraviolet optical microscopy imaging method according to any one of claims 1-6, wherein in S20, the ultraviolet light excites an autofluorescence reaction of the tissue sample, and autofluorescence signals from the tissue sample are collected using the ultraviolet optical system to obtain an autofluorescence image of the tissue sample.
8. The dark field reflection uv light microscopy imaging method according to claim 7, wherein the uv optical system comprises a filter wheel comprising a first filter and a second filter, the first filter selected to pass uv light and the second filter selected to pass fluorescence light; and respectively obtaining an ultraviolet dark field reflection image and an auto-fluorescence image of the tissue sample by switching the filter wheel.
9. The dark-field reflection ultraviolet optical microscopy imaging method as set forth in claim 7, wherein the virtual HE staining is performed on the ultraviolet dark-field reflection image of the tissue sample, including the steps of:
1) Carrying out signal inversion on the collected ultraviolet dark field reflection image to obtain an inversion image, and using the inversion image as an H channel in virtual HE dyeing;
2) Using the collected autofluorescence image as an E channel in virtual HE staining;
3) And distributing the signal values of the H channel and the E channel to R, G, B channels in a virtual dyeing map according to a certain proportion, and then combining signals of R, G, B channels to obtain a virtual HE image corresponding to the ultraviolet dark field reflection image.
10. The dark-field reflection ultraviolet optical microscopy imaging method as claimed in claim 9, characterized in that in step 3), the signal values of the H channel and the E channel are assigned to R, G, B channel in the virtual staining map according to the following formula,
11. the dark-field reflection ultraviolet optical microscopy imaging system based on the dark-field reflection ultraviolet optical microscopy imaging method as set forth in any one of claims 1 to 10, characterized by comprising:
the ultraviolet light source is used for generating ultraviolet light in an ultraviolet waveband, and the ultraviolet light is obliquely incident on the tissue sample;
a microscope for providing optical information about the tissue sample;
an image acquisition system for generating one or more images from optical information provided by the microscope.
12. The dark-field reflectance ultraviolet light microscopy imaging system of claim 11, further comprising an image processing system for processing the image obtained by the image acquisition system and a display system for displaying the processed image for analysis.
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