WO2024078183A1 - Dark-field reflection ultraviolet optical microscopic imaging method and system - Google Patents

Abstract

The present invention relates to the technical field of rapid pathological testing. Disclosed is a dark-field reflection ultraviolet optical microscopic imaging method, comprising the following steps: S10, acquiring a tissue sample; S20, placing the tissue sample on an objective stage, and ultraviolet light generated by an ultraviolet light source being obliquely incident 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 by means of a bright signal which is generated by means of diffuse reflection light on a shallow-surface layer of the tissue sample, and a dark signal which is generated by means of a cell nucleus absorbing incident ultraviolet light. On the basis of dark-field illumination of ultraviolet light, when the interference of specular reflection light and scattered light is eliminated, an image contrast is provided by using the dark signal which is generated by means of a cell nucleus absorbing ultraviolet incident light, and the bright signal which is generated by means of diffuse reflection light on the shallow-surface layer of the tissue sample. The method requires no fluorescent staining, and a sample requires no special slicing treatment, such that a pathological tissue imaging result can be rapidly and accurately provided at a high signal-to-background ratio, and a rapid and accurate reference basis is provided for intraoperative incisal-margin evaluation.

Description

A dark field reflection ultraviolet optical microscopy imaging method and system Technical Field

The patent of this invention relates to the technical field of rapid pathology detection, specifically, to a dark-field reflection ultraviolet optical microscopy imaging method and system.

Background technique

Histopathological examination is an important means of biomedical diagnosis. The conventional histopathological examination process includes: first taking a piece of tissue removed during surgery, then embedding it in paraffin blocks, cutting it into thin slices, then staining it with hematoxylin-eosin (HE), and finally observing it under a microscope to obtain the corresponding pathological diagnosis results. However, conventional paraffin section pathological examination takes a long time, usually 3-4 days, and cannot provide help for intraoperative margin assessment that requires rapid diagnosis results.

technical problem

At present, frozen sections are often used in clinical surgery instead of paraffin-embedded sections. The process includes sampling, embedding, freezing, sectioning, staining and observation diagnosis, which usually takes 30 minutes or more. However, the freezing process often causes broken holes in tissue samples, which affects the judgment and diagnosis of pathologists and greatly limits the use of frozen section technology in intraoperative margin assessment.

Intraoperative margin assessment: Evaluation of frozen sections of biopsies obtained during surgery can be a routine part of clinical care, but it is fraught with difficulties. These difficulties include the time involved in embedding, freezing, cutting, staining, and viewing the resulting stained sections. This process can take 10 minutes or longer for each sample. In addition, the quality of most frozen samples may not be optimal and is often 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. If a margin assessment is not performed during surgery, additional surgery may be required. For example, 20% to 40% of breast cancer surgeries must be re-performed to remove residual cancer present at or near the surgical margin; ideally, a rapid and accurate pathological assessment can be performed during the initial surgical procedure to ensure that the cancer deposits are completely removed, reducing the pain and risk of reoperation.

Patent document CN201580061933.4 discloses a system and method for controlling the imaging depth in tissues using a fluorescence microscope under ultraviolet excitation after staining with a fluorescent agent. The method uses a variety of exogenous fluorophores to label biological tissues, then illuminates the biological tissues with ultraviolet light, and then detects a variety of fluorescent signals that can characterize tissue information. Although this method can also image thick tissues that do not require thin sectioning, the fluorescent labeling process is not only time-consuming, but may also contaminate biological tissues, affecting subsequent molecular analysis of tissue samples.

Technical Solutions

The object of the present invention is to provide a dark-field reflected ultraviolet optical microscopy imaging method and system, aiming to solve the problem in the prior art that there is a lack of a method and system for quickly and accurately obtaining pathological images for intraoperative resection margin assessment.

The present invention is implemented as follows: a dark field reflection ultraviolet optical microscopic imaging method comprises the following steps:

S10: Obtain tissue samples;

S20: placing the tissue sample on the stage, and causing the ultraviolet light generated by the ultraviolet light source to be incident obliquely on the tissue sample;

The ultraviolet reflected light from the tissue sample is collected by an ultraviolet optical system, and an ultraviolet dark field reflection image is obtained through the bright signal of the diffuse reflected light of the superficial layer of the tissue sample and the dark signal of the cell nucleus absorbing the ultraviolet incident light.

Optionally, in step S20, an ultraviolet light source having a wavelength selected from a light wavelength having a center 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 includes at least one of the following: LED; laser; tunable laser; or a continuous source, wherein the continuous source includes a continuous laser light source, an arc lamp, a laser ignition arc lamp, a krypton-bromine excimer lamp or at least one of other sources with sufficient brightness in the desired spectral range.

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 image formed by the ultraviolet reflected light is normalized, and the normalization process includes the following steps:

S21: At each collection wavelength, take an image of a blank area without structural features on the tissue sample, which serves as a reference image for normalization;

S22: Divide 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 then allocate the obtained pixel coefficient to each image actually captured.

Optionally, in step S10, the cleaned tissue sample is placed on a glass slide, a certain amount of refractive index matching liquid is slowly dripped on the tissue sample, and then a cover glass is placed on the surface of the tissue sample so that the space between the surface of the tissue sample and the cover glass is filled with the refractive index matching liquid; wherein the cover glass has a high transmittance to ultraviolet light.

Optionally, in step S20, the angle of oblique incidence of the ultraviolet light is: the angle between the oblique incidence of the ultraviolet light and the plane of the tissue sample is between 30° and 70°.

Optionally, in S20, ultraviolet light excites an autofluorescence reaction of a tissue sample, and the ultraviolet optical system is used to collect autofluorescence signals from the tissue sample 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 has a high transmittance to ultraviolet light, and the second filter has a high transmittance to autofluorescence; by switching the filter wheel, an ultraviolet dark field reflection image and an autofluorescence image of the tissue sample are obtained respectively.

Optionally, performing virtual HE staining on the UV dark field reflectance image of the tissue sample comprises the following steps:

1) Invert the signal of the collected UV dark field reflection image to obtain an inverted image, and use the inverted image as the H channel in the virtual HE staining;

2) The collected autofluorescence image is used as the E channel in virtual HE staining;

3) The signal values of the H channel and the E channel are allocated to the R, G, and B channels in the virtual staining image according to a certain ratio, and then the signals of the R, G, and B channels are combined 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 allocated to the R, G, and B channels in the virtual staining map according to the following formula:

A dark field reflection ultraviolet optical microscopic imaging system, comprising:

An ultraviolet light source, the ultraviolet light source is used to generate ultraviolet light in the ultraviolet band, 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 is used to generate one or more images based on the optical information provided by the microscope.

Optionally, it also includes an image processing system and a display system, wherein the image processing system is used to process the image acquired by the image acquisition system, and the display system is used to display the processed image for analysis.

A dark-field reflective ultraviolet optical microscopic imaging system for rapid pathological detection is characterized in that it comprises an ultraviolet light source and an ultraviolet optical system, wherein the ultraviolet optical system comprises a stage, an objective lens, a tube lens and an image sensor, wherein the stage is used to carry a tissue sample to be detected; the ultraviolet light source is arranged away from the receiving optical path of the ultraviolet optical system, and the ultraviolet light emitted by the ultraviolet light source is obliquely incident on the tissue sample, and the ultraviolet reflected light reflected by the tissue sample passes through the objective lens and the tube lens in sequence, and is received by the image sensor to obtain an ultraviolet dark-field reflection image of the tissue sample.

Optionally, the objective lens and the tube lens have high transmittance to ultraviolet light.

Optionally, the ultraviolet dark field reflectance image of the tissue sample includes bright signals of diffusely reflected light from the superficial layer of the tissue sample and dark signals of ultraviolet incident light absorbed by the cell nucleus.

Optionally, the ultraviolet light source uses a wavelength selected from a wavelength of light with a center located at or near the following wavelengths: 290nm; 280nm; 270nm; 260nm; 250nm; 245nm; 240nm; 235nm; 230nm; 225nm; 220nm; 210nm; 200nm;

Wherein, the ultraviolet light source includes at least one of the following: LED; laser; tunable laser; or a continuous source, wherein the continuous source includes a continuous laser light source, an arc lamp, a laser ignition arc lamp, a krypton-bromine excimer lamp or at least one of other sources with sufficient brightness in the desired spectral range.

Optionally, the angle at which the ultraviolet light is obliquely incident on the tissue sample is: the angle between the obliquely incident ultraviolet light and the plane of the tissue sample is between 30° and 70°.

Optionally, the ultraviolet optical system also includes a filter wheel, which is arranged between the tube lens and the image sensor, and includes a first filter and a second filter, the first filter has a high transmittance to ultraviolet light, and the second filter has a high transmittance to autofluorescence; when the filter wheel is switched to the first filter or the second filter, an ultraviolet dark field reflection image or an autofluorescence image of the tissue sample is obtained respectively.

Optionally, it also includes an image processing system and a display system, wherein the image processing system is used to process the image obtained by the image sensor, and the display system is used to display the processed image for analysis.

Optionally, the tissue sample is placed on a glass slide, the glass slide is placed on the stage, a cover glass is provided above the tissue sample, and the cover glass has a high transmittance to ultraviolet light; a refractive index matching liquid is filled between the tissue sample and the cover glass, and the refractive index matching liquid is a liquid with a refractive index close to that of the tissue sample and with a high transmittance to ultraviolet light.

Optionally, at least two protective plates are vertically arranged on the slide glass, the tissue sample is located between the two protective plates, and the cover glass is abutted against the top of the protective plates.

Optionally, multiple protective plates of equal height are connected end to end to form a cavity with the glass slide for placing tissue samples, and the cover glass is covered on the top of the cavity. Compared with the prior art, the present invention provides a dark field reflection ultraviolet optical microscopy imaging method, which obliquely incidents ultraviolet light onto tissue samples, and is based on dark field illumination (oblique incidence) of ultraviolet light (mainly 260 nm, which wavelength is the absorption peak of the cell nucleus), while eliminating the interference of mirror reflection light and scattered light, and uses the dark signal generated by the absorption of ultraviolet light by the cell nucleus and the bright signal generated by the diffuse reflection of the superficial layer of biological tissue to provide image contrast. This method does not require fluorescent staining, and the sample does not require special sectioning processing (such as frozen sectioning), and can provide pathological tissue imaging results quickly, accurately, and with a high signal-to-background ratio, providing a fast and accurate reference for intraoperative resection margin assessment.

Beneficial Effects

The dark field reflection ultraviolet optical microscopic imaging system for rapid pathological detection provided by the present invention forms dark field illumination (oblique incidence) of ultraviolet light by obliquely incident ultraviolet light emitted by an ultraviolet light source to tissue samples, and provides image contrast by using dark signals generated by the absorption of ultraviolet light by the cell nucleus and bright signals generated by diffuse reflection of the superficial layer of biological tissue while eliminating interference from mirror reflection light and scattered light. Therefore, during the detection of tissue samples, there is no need for fluorescent staining, and the samples do not need special sectioning (such as frozen sections), and can provide pathological tissue imaging results quickly, accurately, and with high signal-to-background ratio, providing a fast and accurate reference basis for intraoperative margin assessment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic flow chart of a specific embodiment of a dark field reflection ultraviolet optical microscopic imaging method provided by the present invention;

FIG2 is a schematic diagram of the principle of a dark field reflection ultraviolet optical microscopy imaging method provided by the present invention;

FIG3 is a schematic diagram of an optical path of a dark field reflective ultraviolet optical microscopic imaging system provided by the present invention;

FIG4 is a processing flow chart of virtual staining of a dark field reflection ultraviolet optical microscopy imaging method provided by the present invention;

FIG5 is a comparison of an ultraviolet dark field reflection image, an autofluorescence image, and a fluorescence image after DAPI staining of a dark field reflection ultraviolet optical microscopy imaging method provided by the present invention;

FIG6 is a signal-to-background ratio imaging comparison diagram of an ultraviolet dark field reflection image, an autofluorescence image, and a fluorescence image after DAPI staining of a dark field reflection ultraviolet optical microscopy imaging method provided by the present invention.

Description of reference numerals:

100-tissue sample; 200-ultraviolet light, 210-ultraviolet light source, 220-filter, 300-ultraviolet optical system, 310-stage, 311-slide, 312-protective plate, 313-cover glass, 314-refractive index matching liquid, 320-objective lens, 330-image sensor, 340-filter wheel, 350-tube lens.

Specific embodiments of the present invention

In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

The implementation of the present invention is described in detail below in conjunction with specific embodiments.

The same or similar numbers in the drawings of this embodiment correspond to the same or similar parts; in the description of the present invention, it should be understood that if the terms "upper", "lower", "left", "right" and the like indicate directions or positional relationships based on the directions or positional relationships shown in the drawings, it is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific direction, be constructed and operated in a specific direction. Therefore, the terms describing the positional relationship in the drawings are only used for illustrative purposes and cannot be understood as limitations on this patent. For ordinary technicians in this field, the specific meanings of the above terms can be understood according to specific circumstances.

1-6 , which are preferred embodiments of the present invention.

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, and the ultraviolet light 200 generated by the ultraviolet light source 210 is incident obliquely on the tissue sample 100;

The ultraviolet reflected light from the tissue sample 100 is collected by the ultraviolet optical system 300, and an ultraviolet dark field reflection image is obtained through the bright signal of the diffuse reflection light of the superficial layer of the tissue sample 100 and the dark signal of the cell nucleus absorbing the ultraviolet incident light.

The present embodiment provides a dark field reflection ultraviolet optical microscopic imaging method, which obliquely incidents ultraviolet light 200 onto a tissue sample 100, and based on dark field illumination (oblique incidence) of ultraviolet light (mainly 260 nm, which is the absorption peak of the cell nucleus), eliminates the interference of mirror reflection light and scattered light, and uses the dark signal generated by the absorption of ultraviolet light by the cell nucleus and the bright signal generated by the diffuse reflection of the superficial layer of the biological tissue to provide image contrast. This method does not require fluorescent staining, and the sample does not require special sectioning (such as frozen sectioning), and can quickly, accurately, and provide pathological tissue imaging results with a high signal-to-background ratio, providing a fast and accurate reference for intraoperative margin assessment.

In the prior art, it is difficult to quickly and accurately evaluate the tissue sample 100. If the ultraviolet light 200 is simply directed directly to the tissue sample 100, the dark signal of the cell nucleus of the tissue sample 100 absorbing the incident ultraviolet light cannot be obtained due to the strong interference of the mirror reflected light and the scattered light.

The ultraviolet optical system 300 of this embodiment minimizes the loss and absorption of ultraviolet light during the transmission process. The optical elements involved in the ultraviolet optical system 300 include a transmission element (mainly playing a transmission role in the optical path), which has a high transmittance for ultraviolet light to avoid excessive loss of ultraviolet light during the transmission process; or, the ultraviolet optical system 300 may also include a reflection element (mainly playing a reflection role in the optical path), which has a high reflectivity for ultraviolet light to avoid excessive loss of ultraviolet light during reflection.

The ultraviolet optical system 300 includes an objective lens 320 and an image sensor 330 . The ultraviolet reflected light is transmitted through the objective lens 320 and is received by the image sensor 330 .

The ultraviolet optical system 300 in this embodiment is a dark-field reflective wide-field microscope. 1) All components in the system have high transmittance to ultraviolet light; 2) Light source part: 260nm LED light or laser, 200-400nm LED light or laser, etc. can be used.

The ultraviolet light 200 generated by the ultraviolet light source 210 is incident obliquely on the tissue sample 100 , and is reflected by the tissue sample 100 to produce ultraviolet reflected light, which is received by the image sensor after passing through the ultraviolet optical system 300 to obtain an ultraviolet dark field reflected image.

Optionally, the oblique incident angle of the ultraviolet light 200 is: the angle between the oblique incident ultraviolet light 200 and the plane of the tissue sample 100 is between 30° and 70°, so as to better eliminate the adverse effects of specular reflection light.

As shown in FIG. 2 , FIG. 2 ( a ) shows the ultraviolet reflected light component from the tissue sample 100 under the bright field illumination condition of the prior art; FIG. 2 ( b ) shows the principle of the dark field reflected ultraviolet optical microscopy imaging of the present invention.

In Figure 2 (a), under the traditional bright field illumination condition, the incident ultraviolet light is vertically 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 reflection light ①, diffuse reflection light of the superficial layer of the tissue sample 100 ②, and scattered light ③ caused by the uneven refractive index of the interface of the tissue sample 100. The light signal received by the ultraviolet optical system 300 is I _detect =① + ② + ③ -④, where ④ represents the dark signal of the cell nucleus absorbing the incident ultraviolet light, which characterizes the distribution of the cell nucleus in the tissue sample 100 and is also the key information in pathological detection.

However, under traditional bright field illumination, the cellular structure of biological tissues cannot be observed in general bright field and dark field reflected light microscopes because the mirror reflected light ① and scattered light ③ are too strong and will drown out the dark signal of the cell nucleus' absorption of ultraviolet incident light ④.

As shown in FIG2(b), under the condition of dark field illumination, that is, the incident ultraviolet light is obliquely incident on the cover glass 313 above the tissue sample 100. In this embodiment, on the basis of dark field illumination, the influence of specular reflection can be eliminated as much as possible, because the ultraviolet light 200 is obliquely incident, and its specular reflection light ① is also obliquely emitted, so its specular reflection light ① is not received by the ultraviolet optical system 300.

Furthermore, in this embodiment, the tissue sample 100 is flattened as much as possible by using a cover glass 313, and a layer of refractive index matching liquid 314 is filled between the cover glass 313 and the tissue sample 100 to eliminate the influence of scattered light ③, and the image contrast is mainly obtained based on the dark signal ④ of the ultraviolet light absorption of the cell nucleus and the diffuse reflection light ② of the superficial layer of the tissue sample 100. At this time, the light signal received by the ultraviolet optical system 300: I _detect =② -④, eliminating the influence of the dark signal being overwhelmed by the mirror reflection light and the scattered light.

Specifically, in step S10, the obtained tissue sample 100 is cleaned and then placed on the glass slide 311. For example, the surface to be observed and the opposite surface of the biological tissue sample 100 can be slightly smoothed with a scalpel, and then washed with phosphate-buffered saline (PBS) for 5 seconds, and then the tissue is placed on the glass slide 311. After the cleaning process, stains can be prevented from affecting the subsequent ultraviolet microscopic imaging effect.

Specifically, in step S10, the cleaned tissue sample 100 is placed on a glass slide 311, a certain amount of refractive index matching liquid 314 is slowly dripped on the tissue sample 100, and then a cover glass 313 is placed on the surface of the tissue sample 100, so that the surface of the tissue sample 100 and the cover glass 313 are filled with the refractive index matching liquid 314; wherein the cover glass 313 has a high transmittance to ultraviolet light. The refractive index matching liquid 314 can eliminate the influence of the material change on the optical detection result caused by the interface of the joint, so that the cover glass 313 and the tissue sample 100 have better adhesion and light transmittance, greatly reduce the reflection loss related to the contact surface between the tissue sample 100 and the air, greatly improve the ultraviolet microscopic imaging effect of the tissue sample 100, and make the ultraviolet dark field reflection image of the tissue sample 100 clearer.

For example, a certain amount of PBS solution or glycerol is slowly dripped onto the biological tissue to avoid bubbles, and then a cover glass 313 is placed on the surface of the tissue to ensure that the space between the biological tissue and the cover glass 313 is filled with PBS solution or glycerol and free of bubbles. It is worth noting that the slide glass 311 and cover glass 313 used here are both quartz glass slides, which have high transmittance to ultraviolet light; if the cover glass 313 has low transmittance to ultraviolet light, it will greatly affect the acquisition of ultraviolet dark field reflection images.

Preferably, two protective plates 312 of equal height are arranged in parallel on the slide, the tissue sample 100 is placed between the two protective plates 312, and the two sides of the cover glass 313 are abutted against the top of the two protective plates 312. For example, two additional small slides can be cut as protective plates 312, which are fixed (by bonding) on the original slide 311 and located on both sides of the tissue sample 100 (forming a groove structure in which the tissue sample 100 is placed), and then the two sides of the cover glass 313 are attached to the two small slides to obtain a relatively flat observation surface.

Furthermore, four protective plates 312 may be arranged on the glass slide 311, the four protective plates 312 are fixed on the carrier, the four protective plates 312 are arranged in a square shape, and the four protective plates 312 and the glass slide 311 together form an open square cavity structure. The tissue sample 100 is placed in the square cavity structure, and then the cover glass 313 is covered at the opening of the square cavity, so that a relatively flat observation surface can be obtained, and the refractive index matching liquid 314 is easily filled without being easily lost.

Optionally, in step S20, an ultraviolet light source 210 having a wavelength selected from a light wavelength having a center at or near the following wavelengths is used: 290 nm; 280 nm; 270 nm; 260 nm; 250 nm; 245 nm; 240 nm; 235 nm; 230 nm; 225 nm; 220 nm; 210 nm; 200 nm;

The ultraviolet light source 210 includes at least one of the following: an LED; a laser; a tunable laser; or a continuous source, the continuous source including a continuous laser light source, an arc lamp, a laser ignition arc lamp, a krypton-bromine excimer lamp, or at least one of other sources with sufficient brightness within the desired spectral range.

Specifically, in step S20, the image formed by the ultraviolet reflected light is normalized, and the normalization process includes the following steps:

S21: taking an image of a blank area without structural features on the tissue sample 100 at each collection wavelength, and using it as a normalized reference image;

S22: Divide 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 then allocate the obtained pixel coefficient to each image actually captured.

Through the above normalization process, the influence of non-uniform illumination is eliminated, so that the contrast of the image is optimized.

Furthermore, the normalization process may include the following steps:

i) First, take an image of a blank area without structural features on the sample at each collection wavelength, and use it as a normalized reference image (I _ref (x, y), where I is the measured intensity value and (x, y) is the corresponding pixel coordinate);

ii) Then the intensity value of each pixel on the reference image is divided by the maximum intensity value of the reference image to obtain the calibration matrix C (where );

iii) Multiply the obtained calibration matrix C by each picture actually taken to eliminate the influence of non-uniform illumination;

iv) The image at the set central collection wavelength is the normalized UV dark field reflectance image obtained (for example, the central collection wavelength is 260 nm), in which the dark negative signal represents the cell nucleus and the bright positive signal comes from the diffuse reflection of the superficial layer of the tissue sample 100. The normalized UV dark field reflectance image can clearly identify the cell nucleus structure.

Optionally, in S20, ultraviolet light excites the autofluorescence reaction of the tissue sample 100, and the ultraviolet optical system 300 collects the autofluorescence signal from the tissue sample 100 to obtain an autofluorescence image of the tissue sample 100. Autofluorescence is the light naturally emitted by biological structures (such as mitochondria and lysosomes) when they absorb light, and is used to distinguish light from artificially added fluorescent markers (fluorophores). Autofluorescence images also do not require fluorescent staining, but autofluorescence is usually weak and cannot provide sufficient light and dark signal contrast, and can be used as an auxiliary detection method.

The ultraviolet optical system 300 includes a filter wheel 340, which includes a first filter and a second filter. The first filter has a high transmittance to ultraviolet light, and the second filter has a high transmittance to autofluorescence. By switching the filter wheel 340, an ultraviolet dark field reflection image and an autofluorescence image of the tissue sample 100 are obtained respectively.

For example, after the reflected light and autofluorescence signal from the superficial layer of the tissue sample 100 are collected by the objective lens 320 of the ultraviolet optical system 300, they pass through an electric wheel (filter wheel 340) equipped with filters with central wavelengths of 260 nm (first filter) and 357 nm (second filter). The filter wheel 340 will distinguish the signals in actual image acquisition, where the 260 nm channel corresponds to the ultraviolet dark field reflection image, and the 357 nm channel corresponds to the autofluorescence image.

Optionally, performing virtual HE staining on the ultraviolet dark field reflection image of the tissue sample 100 includes the following steps:

1) Invert the signal of the collected UV dark field reflection image to obtain an inverted image, and use the inverted image as the H channel in the virtual HE staining;

2) The collected autofluorescence image is used as the E channel in virtual HE staining;

3) The signal values of the H channel and the E channel are allocated to the R, G, and B channels in the virtual staining image according to a certain ratio, and then the signals of the R, G, and B channels are merged to obtain a virtual HE image corresponding to the UV dark field reflectance image.

Among them, the cell nucleus detected by the UV dark field reflection image is a black negative signal. When doing virtual staining, the reflection image needs to be signal inverted (that is, the dark cell nucleus becomes brighter, and the bright signal of diffuse reflection becomes darker; this can be directly achieved through imagej. The algorithm is as follows: taking an 8-bit image as an example, the original reflection image is I ₁ , and the signal after 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, and B channels in the virtual staining map according to the following formula: .

Virtual HE staining, that is, virtual hematoxylin-eosin (HE) staining, is used to transform the black and white image detected by reflection (ultraviolet dark field reflection image) into the effect of virtual HE staining, which is convenient for pathologists to make judgments. Virtual HE staining can achieve high signal-to-background ratio cell nucleus imaging (similar to the effect of DAPI staining), which is much stronger than the contrast brought by autofluorescence. This virtual result can be compared with the actual HE staining image.

A dark field reflection ultraviolet optical microscopy imaging system based on a dark field reflection ultraviolet optical microscopy imaging method comprises: an ultraviolet light source 210, the ultraviolet light source 210 is used to generate ultraviolet light 200 in an ultraviolet band, and the ultraviolet light 200 is obliquely incident on a tissue sample 100;

a microscope for providing optical information about the tissue sample 100;

An image acquisition system is used to generate one or more images based on the optical information provided by the microscope.

The ultraviolet light source 210 generates ultraviolet light 200, which passes through the filter 220 and the short-focus lens and is incident obliquely on the cover glass 313. The ultraviolet light 200 is transmitted through the cover glass 313, passes through the refractive index matching liquid 314, and is irradiated on the surface of the tissue sample 100. Part of the light is reflected by the cover glass 313, and the reflected light is emitted obliquely and is not received by the ultraviolet optical system 300; part of the light is diffusely reflected by the tissue sample 100 and is received by the ultraviolet optical system 300; and the dark signal of the cell nucleus absorbing the incident ultraviolet light can be detected by the ultraviolet optical system 300.

Optionally, it also includes an image processing system and a display system, the image processing system is used to process the image obtained by the image acquisition system, and the display system is used to display the processed image for analysis.

A dark-field reflective ultraviolet optical microscopic imaging system for rapid pathological detection, comprising an ultraviolet light source 210 and an ultraviolet optical system 300, wherein the ultraviolet optical system 300 comprises an object stage 310, an objective lens 320, a tube lens 350 and an image sensor 330, and the object stage 310 is used to carry a tissue sample 100 to be detected;

The ultraviolet light source 210 deviates from the receiving light path arrangement of the ultraviolet optical system 300. The ultraviolet light 200 emitted by the ultraviolet light source 210 is obliquely incident on the tissue sample 100. 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 to obtain an ultraviolet dark field reflection image of the tissue sample 100.

The dark field reflection ultraviolet optical microscopic imaging system for rapid pathological detection provided in this embodiment obliquely incidents the ultraviolet light 200 emitted by the ultraviolet light source 210 to the tissue sample 100, forming dark field illumination (oblique incidence) of the ultraviolet light 200, and using the dark signal generated by the absorption of the ultraviolet light 200 by the cell nucleus and the bright signal generated by the diffuse reflection of the superficial layer of the biological tissue to provide image contrast while eliminating the interference of the mirror reflection light and the scattered light. Therefore, during the detection of the tissue sample 100, there is no need for fluorescent staining, and the sample does not need special sectioning (such as frozen sectioning), and the pathological tissue imaging results can be provided quickly, accurately, and with a high signal-to-background ratio, providing a fast and accurate reference basis for intraoperative resection margin assessment.

The objective lens 320 and the tube lens 350 have high transmittance to ultraviolet light, thereby preventing excessive loss of ultraviolet light during transmission. The image sensor 330 can receive ultraviolet light signals.

The ultraviolet dark field reflection image of the tissue sample 100 includes bright signals of diffuse reflection light from the superficial layer of the tissue sample 100 and dark signals of ultraviolet incident light absorbed by the cell nucleus.

The central wavelength of the ultraviolet light 200 emitted by the ultraviolet light source 210 may be 260 nm, which is the absorption peak of the cell nucleus and is beneficial for obtaining a high-quality ultraviolet dark-field reflectance image of a tissue sample.

The ultraviolet light source 210 uses a wavelength band selected from the following wavelengths with the center located at or near the following wavelengths: 290nm; 280nm; 270nm; 260nm; 250nm; 245nm; 240nm; 235nm; 230nm; 225nm; 220nm; 210nm; 200nm.

The currently used 260nm ultraviolet LED light source can be expanded to an LED light source or laser in the 200-400 nm band, and the collected reflected light band is not limited to 260 nm, but can also be expanded to the 200-400 nm band accordingly.

The angle at which the ultraviolet light 200 is obliquely incident on the tissue sample 100 is: the angle between the obliquely incident ultraviolet light 200 and the plane of the tissue sample 100 is between 30° and 70°.

In a specific embodiment, a 260 nm ultraviolet LED light source passes through a filter 220 with a central wavelength of 260 nm and one or two short-focus optical lenses, and then is incident obliquely on the sample surface. The oblique incident angle is between 30° and 70° with the sample plane, the illumination area on the sample surface is 5-10 mm ² , and the illumination intensity of a single light source is about 20 mW.

Specifically, the tissue sample 100 is placed on a glass slide 311, which is placed on a stage 310. A cover glass 313 is provided above the tissue sample 100, and the cover glass 313 has a high transmittance to ultraviolet light. A refractive index matching liquid 314 is filled between the tissue sample 100 and the cover glass 313, and the refractive index matching liquid 314 is a liquid having a refractive index close to that of the tissue sample 100 and having a high transmittance to ultraviolet light. For example, the refractive index matching liquid 314 includes but is not limited to the currently used PBS (phosphate-buffered saline, PBS) solution or glycerol, and these solutions can be replaced with a liquid having a refractive index close to that of the biological tissue to be tested and which is transparent to ultraviolet light.

The dark-field reflected ultraviolet optical microscopy imaging system for rapid pathological detection can also collect autofluorescence images of the tissue sample 100 .

Specifically, at least two protective plates 312 are vertically arranged on the slide glass 311 , the tissue sample is located between the two protective plates 312 , and the cover glass 313 abuts against the top of the protective plates 312 .

Preferably, two protective plates 312 of equal height are arranged in parallel on the carrier slide, the tissue sample 100 is placed between the two protective plates 312, and the two sides of the cover glass 313 are abutted against the top of the two protective plates 312. For example, two additional small glass slides can be cut as protective plates, which are fixed (by bonding) on the original glass slide 311 and located on both sides of the tissue sample 100 (forming a groove structure in which the tissue sample 100 is placed), and then the 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 protective plates 312 of the same height are connected end to end to form a cavity with the glass slide 311 for placing the tissue sample 100 , and a cover glass 313 is covered on the top of the cavity.

For example, four protective plates 312 may be arranged on the glass slide 311, the four protective plates 312 are fixed on the carrier, the four protective plates 312 are arranged in a square shape, and the four protective plates 312 and the glass slide 311 together form an open square cavity. The tissue sample 100 is placed in the square cavity, and then the cover glass 313 is covered at the opening of the square cavity, so that a relatively flat observation surface can be obtained, and the refractive index matching liquid 314 is easily filled without being easily lost.

In the above embodiment, the ultraviolet light source 210 generates ultraviolet light 200, which is incident obliquely to the cover glass 313 after passing through the filter 220 and the short-focus lens. The ultraviolet light 200 is transmitted through the cover glass 313, passes through the refractive index matching liquid 314, and is irradiated on the surface of the tissue sample 100. Part of the light is reflected by the cover glass 313, and the reflected light is emitted obliquely and is not received by the ultraviolet optical system 300; part of the light is diffusely reflected by the tissue sample 100 and is received by the ultraviolet optical system 300; and the dark signal of the cell nucleus absorbing the incident ultraviolet light can be detected by the ultraviolet optical system 300.

In the following specific embodiment, a dark field reflected ultraviolet optical microscopic imaging method and system are provided, which can perform rapid, label-free imaging of thick pathological tissue samples 100 without sectioning, so as to facilitate rapid and accurate pathological diagnosis and provide a rapid and accurate basis for intraoperative resection margin assessment. As shown in FIG2 , the specific method flow is as follows:

1. Take a freshly excised or formalin-fixed biological tissue sample 100, first use a scalpel to slightly smooth the surface to be observed and its opposite surface (to facilitate detection), then wash it with phosphate-buffered saline (PBS) for 5 seconds, and then place the tissue on a slide 311.

2. Take a certain amount of PBS solution or glycerol and slowly drip it on the biological tissue to avoid bubbles, then cover the tissue surface with a cover glass 313 to ensure that the space between the biological tissue and the cover glass 313 is filled with PBS solution or glycerol and there are no bubbles. It is worth noting that the slide glass 311 and cover glass 313 used here are both quartz glass slides, which have high transmittance to ultraviolet light.

3. Fix the prepared biological sample on a stage 310, such as a three-dimensional translation stage, and then use 260 nm ultraviolet light for dark field illumination, and then use the ultraviolet optical system 300 to collect the reflected light signal with a central wavelength of 260 nm and the autofluorescence signal with a central wavelength of 357 nm.

The 260 nm UV LED light source passes through a filter 220 with a central wavelength of 260 nm and one or two short-focus optical lenses before being incident obliquely on the sample surface. The oblique incident angle is between 30° and 70° with the sample plane, the illumination area on the sample surface is 5-10 mm ² , and the illumination intensity of a single light source is about 20 mW.

4. Normalize the images captured by the camera to eliminate the influence of non-uniform illumination: First, take an image of a blank area without structural features on the sample at each collection wavelength as a normalized reference image; then divide the intensity value of each pixel on the reference image by the maximum intensity value of the reference image, and then assign the obtained pixel coefficients to each image actually captured. Among them, the image with a central collection wavelength of 260 nm is the obtained UV dark field reflection image, which can clearly identify the cell nuclear structure.

5. As shown in Figure 4, the collected black-and-white ultraviolet dark field reflectance image can be further subjected to virtual HE staining. The specific process includes: i) inverting the signal of the ultraviolet dark field reflectance image corresponding to the central collection wavelength of 260 nm, and using the inverted image as the cell nucleus channel (H channel) in HE staining; ii) using the autofluorescence image corresponding to the central collection wavelength of 357 nm as the cytoplasm channel (E channel) in HE staining; iii) allocating the signal values of the H channel and the E channel to the R, G, and B channels in the virtual staining image according to a certain ratio (Formula (1)), and then merging the three RGB channels to obtain a virtual HE image corresponding to the black-and-white ultraviolet dark field reflectance image.

(1)

Among them, the reflected light and autofluorescence signal from the sample surface are collected by the objective lens 320 and then pass through an electric wheel (filter wheel 340) equipped with filters with central wavelengths of 260 nm and 357 nm respectively. The filter wheel 340 will distinguish the signals in the actual image acquisition, where the 260 nm channel corresponds to the ultraviolet dark field reflection image, and the 357 nm channel corresponds to the autofluorescence image.

The optical signal then passes through a tube lens 350 matched with the objective lens 320 and is then collected by a camera that is sensitive to ultraviolet light.

The currently used 260nm UV LED light source can be expanded to a 200-400nm LED light source or laser, and the collected reflected light band is not limited to 260nm, but can be expanded to 200-400nm accordingly. At the same time, the refractive index matching liquid 314 is not limited to the currently used PBS solution or glycerol, and these solutions can be replaced by liquids that are close to the refractive index of the biological tissue to be tested and can transmit UV light.

Compared with the prior art, the greatest advantages of the present invention are its fast imaging speed and high signal-to-background ratio of the image.

1) Fast imaging speed: Based on the method of the present invention, the time from sample preparation to actual image acquisition is about 2-3 minutes, and the algorithms for image normalization and virtual staining are also extremely simple (the calculation time is less than 1 second). The existing MUSE (Microscopy with ultraviolet surface excitation) technology requires staining, which may not only contaminate the sample, but also the staining process is time-consuming, requiring 10 minutes or even longer to obtain an image. Although the CHAMP (high-throughput autofluorescence microscopy by pattern illumination) technology does not require staining, it requires the acquisition of 36 frames of images and computational reconstruction to obtain the final image. The entire process is also relatively time-consuming, and the image quality is poor, making it difficult to serve as a reliable basis for intraoperative margin assessment.

2) High signal-to-background ratio of imaging: As shown in Figure 5, the method proposed in the present invention can perform high signal-to-background ratio imaging of cell nuclei, wherein Figure 5-a corresponds to the UV dark field reflection image obtained by the method proposed in the present invention, Figure 5-b corresponds to the autofluorescence image of the tissue sample, and Figure 5-c corresponds to the fluorescence image after DAPI staining. In comparison, the signal contrast of the UV dark field reflection image shown in Figure 5-a is much higher than that of the autofluorescence image shown in Figure 5-b, because the autofluorescence signal of biological tissue samples is usually weak, while the bright signal of the UV reflection light of the tissue sample and the dark signal absorbed by the cell nucleus have a higher signal contrast.

In the actual comparative experiment, the imaging verification was carried out using the mouse brain tissue sample 100, and the ultraviolet dark field reflection image (Figure 5-a) and the autofluorescence image (Figure 5-b) were measured respectively; then the cell nucleus of the sample was stained with DAPI (4',6-diamidino-2-phenylindole), a common fluorescent staining method, and a filter with a central wavelength of 447 nm was added to the filter wheel 340 of the ultraviolet optical system, and then the sample image was collected again under the filter (Figure 5-c). Referring to Figure 6, it can be found through the comparison of the experimental results that, without the need for staining, the cell nucleus signal contrast of the ultraviolet dark field reflection image obtained by the method proposed in the present invention is close to the level of fluorescent staining with extremely high specificity and sensitivity, which is much higher than the signal-to-background ratio in the autofluorescence image, and can be used as a reference for intraoperative margin assessment. And because the implementation process of the method provided by the present invention is very fast (the whole process only takes 2-3 minutes), and no staining, freezing, or damage to tissue samples are required, it is very suitable for intraoperative margin assessment.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims (22)

1. A dark field reflection ultraviolet optical microscopic imaging method, characterized in that it comprises the following steps:

   S10: Obtain tissue samples;

   S20: placing the tissue sample on the stage, and causing the ultraviolet light generated by the ultraviolet light source to be incident obliquely on the tissue sample;

   The ultraviolet reflected light from the tissue sample is collected by an ultraviolet optical system, and an ultraviolet dark field reflection image is obtained through the bright signal of the diffuse reflected light of the superficial layer of the tissue sample and the dark signal of the cell nucleus absorbing the ultraviolet incident light.
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 selected from a wavelength of light centered 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 includes at least one of the following: LED; laser; tunable laser; or a continuous source, wherein the continuous source includes a continuous laser light source, an arc lamp, a laser ignition arc lamp, a krypton-bromine excimer lamp or at least one of other sources with sufficient brightness in the desired spectral range.
3. A dark-field reflective ultraviolet optical microscopy imaging method as described in claim 2, characterized in that 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.
4. The dark field reflected ultraviolet optical microscopy imaging method according to claim 1 is characterized in that, in step S20, the image formed by the ultraviolet reflected light is normalized, and the normalization processing comprises the following steps:

   S21: At each collection wavelength, take an image of a blank area without structural features on the tissue sample, which serves as a reference image for normalization;

   S22: Divide 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 then allocate the obtained pixel coefficient to each image actually captured.
5. A dark-field reflective ultraviolet optical microscopy imaging method as described in claim 1, characterized in that, in step S10, the cleaned tissue sample is placed on a glass slide, a certain amount of refractive index matching liquid is slowly dripped on the tissue sample, and then a cover glass is covered on the surface of the tissue sample, so that the surface of the tissue sample and the cover glass are filled with the refractive index matching liquid; wherein the cover glass has a high transmittance to ultraviolet light, and the refractive index matching liquid is a liquid having a refractive index close to that of the tissue sample and having a high transmittance to ultraviolet light.
6. A dark field reflection ultraviolet optical microscopy imaging method as described in claim 1, characterized in that, in step S20, the angle of oblique incidence of the ultraviolet light is: the angle between the oblique incident ultraviolet light and the plane of the tissue sample is between 30° and 70°.
7. A dark-field reflection ultraviolet optical microscopy imaging method as described in any one of claims 1 to 6, characterized in that in S20, ultraviolet light excites the autofluorescence reaction of the tissue sample, and the ultraviolet optical system is used to collect the autofluorescence signal from the tissue sample to obtain an autofluorescence image of the tissue sample.
8. A dark-field reflective ultraviolet optical microscopy imaging method as described in claim 7, characterized in that the ultraviolet optical system includes a filter wheel, the filter wheel includes a first filter and a second filter, the first filter has a high transmittance to ultraviolet light, and the second filter has a high transmittance to autofluorescence; by switching the filter wheel, an ultraviolet dark-field reflective image and an autofluorescence image of the tissue sample are obtained respectively.
9. A dark field reflection ultraviolet optical microscopic imaging method as claimed in claim 7, characterized in that virtual HE staining is performed on the ultraviolet dark field reflection image of the tissue sample, comprising the following steps:

   1) Invert the signal of the collected UV dark field reflection image to obtain an inverted image, and use the inverted image as the H channel in the virtual HE staining;

   2) The collected autofluorescence image is used as the E channel in virtual HE staining;

   3) The signal values of the H channel and the E channel are allocated to the R, G, and B channels in the virtual staining image according to a certain ratio, and then the signals of the R, G, and B channels are combined to obtain a virtual HE image corresponding to the ultraviolet dark field reflection image.
10. A dark field reflection ultraviolet optical microscopy imaging method according to claim 9, characterized in that, in step 3), the signal values of the H channel and the E channel are allocated to the R, G, and B channels in the virtual staining map according to the following formula: .
11. A dark field reflection ultraviolet optical microscopy imaging system based on a dark field reflection ultraviolet optical microscopy imaging method according to any one of claims 1 to 10, characterized in that it comprises:

    An ultraviolet light source, the ultraviolet light source is used to generate ultraviolet light in the ultraviolet band, 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 is used to generate one or more images based on the optical information provided by the microscope.
12. The dark-field reflected ultraviolet optical microscopy imaging system as described in claim 11 is characterized in that it also includes an image processing system and a display system, wherein the image processing system is used to process the image obtained by the image acquisition system, and the display system is used to display the processed image for analysis.
13. A dark-field reflective ultraviolet optical microscopic imaging system for rapid pathological detection, characterized in that it comprises an ultraviolet light source and an ultraviolet optical system, wherein the ultraviolet optical system comprises an objective stage, an objective lens, a tube lens and an image sensor, and the objective stage is used to carry a tissue sample to be detected;

    The ultraviolet light source is arranged away from the receiving light path of the ultraviolet optical system. The ultraviolet light emitted by the ultraviolet light source is incident obliquely on the tissue sample. The ultraviolet reflected light reflected by the tissue sample passes through the objective lens and the tube lens in sequence and is received by the image sensor to obtain an ultraviolet dark field reflection image of the tissue sample.
14. The dark-field reflective ultraviolet optical microscopy imaging system for rapid pathological detection as described in claim 13 is characterized in that the objective lens and the tube lens have high transmittance to ultraviolet light.
15. The dark-field reflective ultraviolet optical microscopy imaging system for rapid pathological detection as described in claim 14 is characterized in that the ultraviolet dark-field reflective image of the tissue sample includes a bright signal of diffusely reflected light from the superficial layer of the tissue sample and a dark signal of ultraviolet incident light absorbed by the cell nucleus.
16. The dark field reflection ultraviolet optical microscopic imaging system for rapid pathological detection as claimed in claim 15, characterized in that the ultraviolet light source adopts a wavelength band selected from the following wavelengths with the center located at or near the following wavelengths: 290nm; 280nm; 270nm; 260nm; 250nm; 245nm; 240nm; 235nm; 230nm; 225nm; 220nm; 210nm; 200nm;

    Wherein, the ultraviolet light source includes at least one of the following: LED; laser; tunable laser; or a continuous source, wherein the continuous source includes a continuous laser light source, an arc lamp, a laser ignition arc lamp, a krypton-bromine excimer lamp or at least one of other sources with sufficient brightness in the desired spectral range.
17. The dark-field reflective ultraviolet optical microscopy imaging system for rapid pathological detection as described in claim 15 is characterized in that the angle of oblique incidence of ultraviolet light to the tissue sample is: the angle between the oblique incident ultraviolet light and the plane of the tissue sample is between 30° and 70°.
18. The dark-field reflective ultraviolet optical microscopy imaging system for rapid pathological detection as described in claim 15 is characterized in that the ultraviolet optical system also includes a filter wheel, the filter wheel is arranged between the tube lens and the image sensor, the filter wheel includes a first filter and a second filter, the first filter has a high transmittance to ultraviolet light, and the second filter has a high transmittance to autofluorescence; when the filter wheel is switched to the first filter or the second filter, an ultraviolet dark-field reflective image or an autofluorescence image of the tissue sample is obtained respectively.
19. The dark-field reflective ultraviolet optical microscopy imaging system for rapid pathological detection as described in any one of claims 13 to 18 is characterized in that it also includes an image processing system and a display system, wherein the image processing system is used to process the image obtained by the image sensor, and the display system is used to display the processed image for analysis.
20. The dark-field reflective ultraviolet optical microscopy system for rapid pathological detection as described in any one of claims 13 to 18 is characterized in that the tissue sample is placed on a glass slide, the glass slide is placed on the stage, a cover glass is provided above the tissue sample, and the cover glass has a high transmittance to ultraviolet light; a refractive index matching liquid is filled between the tissue sample and the cover glass, and the refractive index matching liquid is a liquid having a refractive index close to that of the tissue sample and having a high transmittance to ultraviolet light.
21. The dark-field reflective ultraviolet optical microscopy imaging system for rapid pathological detection as described in claim 20 is characterized in that at least two protective plates are vertically arranged on the slide, the tissue sample is located between the two protective plates, and the cover glass is abutted against the top of the protective plates.
22. The dark-field reflective ultraviolet optical microscopy imaging system for rapid pathological detection as described in claim 21 is characterized in that a plurality of guard plates of equal height are connected end to end to form a cavity with the glass slide for placing tissue samples, and the cover glass is covered on the top of the cavity.