CN111603140B

CN111603140B - In-situ visual positioning method and system for biological target

Info

Publication number: CN111603140B
Application number: CN202010500135.XA
Authority: CN
Inventors: 王子兰
Original assignee: Beijing Hancheng Medical Equipment Co ltd
Current assignee: Beijing Hancheng Medical Equipment Co ltd
Priority date: 2020-06-04
Filing date: 2020-06-04
Publication date: 2021-12-21
Anticipated expiration: 2040-06-04
Also published as: CN111603140A

Abstract

The invention belongs to the field of biological target positioning, and provides a positioning method and a positioning system for in-situ visualization of a biological target, which can be used for efficiently and accurately positioning, identifying, protecting and judging the boundary of the biological target in real time within the visual field range of naked eyes in order to solve the problems that the boundary of the biological target cannot be accurately identified and the biological target cannot be marked in situ. The in-situ visible positioning method for the biological target comprises the steps of collecting fluorescence emitted by a fluorophore of the biological target in real time to form a fluorescence image of the biological target; processing the fluorescence image of the biological target to obtain an image which only contains the biological target and has a clear boundary; and adjusting a projection light beam light path projected to the biological target to be coaxial with a fluorescence light path emitted by the biological target, and projecting an image which only contains the biological target and has a clear boundary to a biological target area emitting fluorescence in situ in real time in an equal proportion.

Description

In-situ visual positioning method and system for biological target

Technical Field

The invention belongs to the field of high-end medical optical instruments, and particularly relates to a method and a system for positioning a biological target in situ visually.

Background

The statements in this section merely provide background information related to the present disclosure and may not necessarily constitute prior art.

In the biomedical field, some normal or pathological biological targets (such as tumors, microorganisms, lymph nodes, soft tissue sarcomas or other tissues or organs) are integrated with the surrounding environment background, and professionals cannot accurately judge the position of the biological target, and even if the position of the biological target is clearly judged, the complete shape and the accurate boundary of the biological target are difficult to judge. This can cause difficulties in later procedures on the biological target (such as in situ protection, in situ removal, surgical resection or puncture) and does not allow for precise protection or removal. For example: when the in-situ protection is carried out on a normal extremely important target in the surgical operation, due to inaccurate judgment, the target is often cut off by mistake, so that the loss and the complications which are difficult to recover are brought, and even the life is threatened. When a diseased biological target is surgically removed, since the complete form and boundary of the target cannot be accurately determined, the removed area may be enlarged or insufficient during the operation, which may cause damage to normal tissues or organs or leave diseased tissues, such as: in the surgical resection process of malignant tumors, the judgment of the tumor position and the boundary is extremely important, many malignant tumors cannot be distinguished from surrounding normal tissues under the observation of visual field, the boundary is more difficult to judge accurately, and an operator is extremely difficult to resect under the condition of inaccurate judgment. The following steps are repeated: skin fungal infection, the accurate fungal site, morphology and boundaries cannot be judged by naked eyes, and pathological sampling and treatment are difficult to carry out.

In addition, in the actual operation process of the biological target, the optical image display device is usually used to display the image of the biological target, and the image of the biological target cannot be displayed in situ on the living body.

Moreover, the existing biological target imaging includes ultrasound, X-ray, nuclear magnetic resonance, CT imaging, etc., but the inventor finds that the ultrasound, X-ray, nuclear magnetic resonance, and CT imaging all have radiation, the organism can not be in the radiation environment for a long time, otherwise, the health of the organism is damaged, and for some biological targets, the imaging device has poor imaging effect, and the device is complex and heavy and is difficult to use flexibly in the operation environment.

The existing biological target imaging also comprises imaging technologies such as infrared thermal imaging, however, images formed by infrared thermal imaging also comprise images of organs or tissues around the biological target besides the biological target, and the inventor finds that the infrared thermal imaging has more interference factors and influences the identification of the biological target and the accurate positioning of the boundary thereof.

In the aspect of researching the in-situ visual positioning of a biological target, the inventor finds application No. 201810298713.9 entitled handheld fluorescence image navigation positioning device, which comprises a handheld shell, a power supply module, a micro projection module, an image acquisition module, an image processing module, an excitation light source module and a distance measurement module, wherein the device adopts ICG (indocyanine green) as a contrast agent, directly projects the acquired fluorescence image to a corresponding lesion part in a real-time projection manner, and is added with the distance measurement module to calibrate the projection position and size in real time. The inventor finds that the distance between the working equipment and the target is an important parameter for spatial positioning, and the invention considers the parameter, but has the following defects: although there is the distance detection unit in this patent of hand-held type fluorescence image navigation positioner, can real-time detection distance parameter then bring into the operation of space algorithm, but the distance detection calculation can be more consuming time, and the distance detection unit is the distance of gauge point to face, can cause the parameter confusion when the face is the curved surface, actual working scene is just not the plane, but the fluctuation of height changes, the distance parameter that the distance detection unit obtained is different with the distance of actual target to imaging device (for example, the distance detection unit obtains the distance data and is 20cm certainly, but the target is located 25 cm's hole), cause after the operation of space algorithm, obtain chaotic result, the projection is skew with the target position, form and boundary are also not identical, can not the normal position cover. In addition, the angle between the working equipment and the target is another important parameter of space positioning, the change of the angle can also cause the projection to deviate from the target position, and the shape and the boundary are not consistent. The handheld fluorescent image navigation and positioning device disclosed by application number 201810298713.9 needs to fix a working distance before each use, needs to mark an anchoring position point in a target range, is calibrated, is very inconvenient to use, wastes time and labor, needs to be recalibrated when the working distance is changed, and cannot achieve the working purpose of being real-time, variable in distance and variable in angle.

The application number is 201611048852.3, the name is a many probes compatible formula normal position projection imaging processing system, it comprises optical system module and image acquisition processing module, the optical system module integration has multiunit imaging probe, every group imaging probe produces the laser emission respectively and shines the operation field, and handle and convey to image processing module from the reflected light of operation field reflection, obtain the visual image of operation field after the processing of image acquisition processing module rethread, through projection technique with the visual image of operation field in the operation field. The inventor finds that the multi-probe compatible in-situ projection imaging processing system finally projects the whole visible light image, not the image of the fluorescence target, but the visible light image of the whole visual field is projected back in situ. It collects reflected light rather than fluorescence. Since the near-infrared camera in the patent (which receives visible light in the described band) obtains two full-view undifferentiated images of the reflected light, the target is not displayed separately, and the visible color camera obtains a visible full-view undifferentiated image, which still does not display the target separately. And it plans to realize the normal position projection of a plurality of probes and projection arrangement through the dichroic mirror, can't design the light path as coaxial, is difficult to realize 1: 1 in-situ projection.

The application number is 20191030279.5, the name is a teleoperation navigation method and device based on normal position projection technology, it includes the operation table, image device, the connecting rod, operation end processor, internet and remote end processor, the camera is located the operation table top, the camera still includes the beam splitter, band elimination filter, camera lens and color camera, it realizes the normal position projection though having adopted optical element, the on-the-spot video data of its collection, and the projected is the image after the distal end doctor marks according to the experience, because doctor's experience belongs to subjective factor, can appear the operation target positioning accuracy poor and can not clearly determine the problem of target boundary like this.

In summary, the inventors found that the following problems are prevalent in the current biological target localization technology: the image projected into the biological target area contains much stray light, so that the biological target cannot be accurately identified, and further the biological target cannot be displayed or labeled on an organism in situ so as to accurately identify the position, the form and the boundary of the biological target.

Disclosure of Invention

In order to solve the above-mentioned problem that the biological target cannot be displayed or labeled in situ on the living body for accurately identifying the position, shape and boundary of the biological target, a first aspect of the present invention provides a positioning method for in-situ visualization of the biological target by using an optical projection technique, which can accurately realize the optical in-situ positioning of the biological target and its boundary.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides a method for positioning a biological target in situ by visual observation, which comprises the following steps:

collecting fluorescence emitted by a fluorophore of the biological target in real time to form a fluorescence image of the biological target;

processing the fluorescence image of the biological target to obtain an image which only contains the biological target and has a clear boundary;

and adjusting a projection light beam light path projected to the biological target to be coaxial with a fluorescence light path emitted by the biological target, and projecting an image which only contains the biological target and has a clear boundary to a biological target area emitting fluorescence in situ in real time in an equal proportion.

Further, the optical path between the biological target and the device for generating the projection beam is set as a first optical path, the optical path between the biological target and the device for collecting the fluorescence emitted by the fluorophore of the biological target is set as a second optical path, and the first optical path and the second optical path are equal.

Further, the process of processing the fluorescence image of the biological target is:

and sequentially performing enhancement, noise reduction and rendering on the fluorescence image of the biological target to enhance the contrast of the biological target and the surrounding background so that only the biological target is contained in the image and the boundary is clear.

Further, the process of sequentially enhancing and denoising the fluorescence image of the biological target comprises:

carrying out gray scale conversion and gray scale range calibration on the fluorescence image of the biological target to obtain a gray scale value range of a foreground signal and a gray scale value range of background noise; converting the fluorescence image of the biological target into an n-bit GRAY image, wherein the GRAY value of any pixel (x, y) is GRAY (x, y);

comparing the image gray values converted from the fluorescence images of the biological targets with the calibration gray range one by one, dividing the image gray values into a foreground area and a background area, and simultaneously obtaining segmentation values DIV1 of the foreground area and the background area;

finding out the average value AVERB of the partition value DIV1 and the gray value of the brightest point, and the partition value DIV1 and the gray value AVERD of the darkest point;

calculating the image between AVERD and AVERB based on the maximum inter-class variance method to obtain the optimal segmentation threshold DIVT of the corresponding region and the corresponding enhancement factor ENFA thereof;

respectively carrying out enhancement and noise reduction treatment on the two divided regions, wherein the treatment formula is as follows:

in order to solve the above-mentioned problem that the biological target cannot be displayed or labeled on the living body in situ for accurately identifying the boundary of the biological target, the second aspect of the present invention provides a positioning system for in situ visualization of the biological target by using an optical projection technology, which is capable of accurately positioning the biological target and the boundary thereof in situ.

the invention provides a positioning system for in-situ visualization of a biological target, which comprises:

a spectroscope for reflecting fluorescence emitted from a fluorophore of a biological target to an imaging system;

an imaging system for acquiring fluorescence emitted by fluorophores of the biological target in real time to form a fluorescence image of the biological target;

the image processing and analyzing unit is used for processing the fluorescence image of the biological target to obtain an image which only contains the biological target and has a clear boundary;

the image positioning projection unit is used for projecting an image which only contains a biological target and has a clear boundary to a biological target area emitting fluorescence in situ in real time in an equal proportion after the projection light beam is transmitted by the spectroscope; wherein, between the biological target and the beam splitter, the optical path of the projection beam is coaxial with the optical path of the fluorescence emitted by the biological target.

Further, the optical path from the biological target to the lens in the image positioning projection unit is set as a first optical path, the optical path from the biological target to the spectroscope is set as a second optical path, the optical path from the spectroscope to the lens in the imaging system is set as a third optical path, and the first optical path is equal to the sum of the second optical path and the third optical path.

The invention provides another positioning system for in-situ visualization of a biological target, comprising:

a spectroscope for transmitting fluorescence emitted from a fluorophore of a biological target to an imaging system;

the image positioning projection unit is used for projecting an image which only contains a biological target and has a clear boundary to a biological target area emitting fluorescence in situ in real time in equal proportion after the projection light beam is reflected by the spectroscope; wherein, between the biological target and the beam splitter, the optical path of the projection beam is coaxial with the optical path of the fluorescence emitted by the biological target.

Further, the optical path from the biological target to the lens in the imaging system is set as a first optical path, the optical path from the biological target to the spectroscope is set as a second optical path, the optical path from the spectroscope to the lens in the image positioning projection unit is set as a third optical path, and the first optical path is equal to the sum of the second optical path and the third optical path.

Further, the image processing analysis unit is configured to:

furthermore, the positioning system for in situ visualization of the biological target further comprises an excitation light source for illuminating the biological target region to excite the fluorescent group of the biological target to emit fluorescence.

Further, the positioning system for in situ visualization of biological targets also has supplemental light sources.

Compared with the prior art, the invention has the beneficial effects that:

(1) the invention combines the fluorescence image with real-time equal proportion in-situ projection, solves the problems that the imaging of the biological target can not be displayed on the organism in situ, and the operator can not directly and accurately judge the complete form and the boundary of the biological target on the organism.

(2)1: 1 real-time in-situ projection is realized in a non-coaxial light path design through a space positioning algorithm, the distance between a working device and a target in the space positioning algorithm is an important parameter, the angle between the working device and the target is another important parameter, the change of the distance and the angle can cause the deviation of the projection and the target position, the form and the boundary are not matched, in the space positioning algorithm, the two problems are difficult to solve, and the real-time change of the working distance and the angle is difficult to realize, and still can be 1: 1 real-time in-situ projection. The invention adopts coaxial light path design, the light path of the projection light beam is coaxial with the light path of the fluorescence emitted by the biological target between the biological target and the beam splitter, the coaxial design ensures that the original view field of the collected image is completely coincided with the center position of the view field covered by the projection image under the condition of changing the working distance and the angle in real time, even if the two are different in size, the straight lines of the diagonals of the two are also completely coincided, the design with equal light path ensures that the original view field of the collected image is completely equal to and coincided with the view field covered by the projection image under the condition of changing the working distance and the angle in real time, therefore, the real-time equal-proportion in-situ projection of the image which only contains the biological target and has clear boundary to the biological target area emitting the fluorescence is realized, and the real-time in-situ accurate projection is still realized under the condition of changing the working distance and the angle in real time, and after the calibration is delivered out of a factory, the device does not need to be calibrated before working every time, and the working angle and the working distance between the device and a target can be changed in real time, so that great convenience is brought to operation.

(3) The invention forms the fluorescence image of the biological target by collecting the fluorescence emitted by the fluorescent group of the biological target in real time, and utilizes the transition between specific energy levels in the fluorescent molecules in the fluorescent group of the biological target to generate corresponding fluorescence for imaging, thereby avoiding the radiation problem of X-ray and CT scanning, causing no health damage to the organism, having small equipment volume and occupying small space.

(4) Compared with the problem of a plurality of interference factors such as infrared thermal imaging, the method disclosed by the invention can be used for forming the fluorescence image of the biological target in real time, reducing the interference of other tissues or organs in the biological target imaging image, and easily identifying the biological target and the boundary thereof, thereby improving the biological target identification and boundary positioning effects.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 is a flow chart of a method for locating a biological target visually in situ according to example 1 of the present invention;

FIG. 2(a) is a field of view of a biological target area according to an embodiment of the present invention;

FIG. 2(b) is a fluorescence image obtained by the imaging system of an embodiment of the present invention;

FIG. 2(c) is an image of a fluorescence image of a biological target after sequential enhancement, noise reduction, and rendering according to an embodiment of the present invention;

FIG. 2(d) is an image of a fluorescence image of an embodiment of the present invention projected in situ, in equal scale, onto a biological target area;

FIG. 3 is a flow chart of sequential enhancement and noise reduction of a fluorescence image of a biological target according to an embodiment of the present invention;

FIG. 4 is a schematic structural diagram of a positioning system for in situ visualization of a biological target according to example 2 of the present invention;

FIG. 5 is a schematic structural diagram of an imaging system of an embodiment of the invention;

FIG. 6 is a schematic structural diagram of a positioning system for in situ visualization of a biological target according to embodiment 3 of the present invention;

FIG. 7 is a schematic structural diagram of a positioning system for in situ visualization of a biological target according to example 4 of the present invention;

FIG. 8 is a schematic structural diagram of a positioning system for in situ visualization of biological targets according to example 5 of the present invention;

FIG. 9 is a schematic structural diagram of a positioning system for in situ visualization of a biological target according to example 6 of the present invention;

FIG. 10 is a schematic structural diagram of a positioning system for in situ visualization of a biological target according to example 7 of the present invention;

FIG. 11 is a schematic structural diagram of a positioning system for in situ visualization of a biological target according to example 8 of the present invention;

FIG. 12 is a schematic structural diagram of a positioning system for in situ visualization of a biological target according to example 9 of the present invention;

FIG. 13 is a schematic structural diagram of a positioning system for in situ visualization of a biological target according to embodiment 10 of the present invention;

FIG. 14 is a schematic structural view of a positioning system for in situ visualization of a biological target according to example 11 of the present invention;

FIG. 15 is a schematic diagram of optical path equality for an embodiment of the present invention;

FIG. 16 is a schematic diagram of optical path equality including a lens structure according to an embodiment of the present invention.

Detailed Description

The invention is further described with reference to the following figures and examples.

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

In the present invention, terms such as "upper", "lower", "left", "right", "front", "rear", "vertical", "horizontal", "side", "bottom", and the like indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, and are only terms of relationships determined for convenience of describing structural relationships of the parts or elements of the present invention, and are not intended to refer to any parts or elements of the present invention, and are not to be construed as limiting the present invention.

In the present invention, terms such as "fixedly connected", "connected", and the like are to be understood in a broad sense, and mean either a fixed connection or an integrally connected or detachable connection; may be directly connected or indirectly connected through an intermediate. The specific meanings of the above terms in the present invention can be determined according to specific situations by persons skilled in the relevant scientific or technical field, and are not to be construed as limiting the present invention.

To address the biological target localization mentioned in the background art there are: the invention provides a method and a system for in-situ visual positioning of a biological target, which can not accurately identify the boundary of the biological target without damage and can not carry out in-situ display or labeling on the biological target, so that the problems that the later operation time of the biological target is prolonged, the operation precision is low and the life threat to the organism is possibly caused are caused.

The technical solution of the in-situ visual positioning method and system of the present invention will be described in detail with reference to the accompanying drawings and specific embodiments.

Example 1

Fig. 1 is a flow chart of a positioning method for in-situ visualization of a biological target according to the present embodiment. As shown in fig. 1, the in-situ visual positioning method for a biological target of the embodiment includes:

s101: and collecting fluorescence emitted by the fluorescent group of the biological target in real time to form a fluorescence image of the biological target.

In step S101, the fluorophore of the biological target is a fluorophore inherent to the biological target or an exogenous fluorophore received by the biological target.

When the fluorophore of the biological target is a fluorophore intrinsic to the biological target, the fluorescence emitted by the fluorophore of the biological target is: fluorescence generated by the inherent fluorophore of the biological target or fluorescence generated by the inherent fluorophore of the biological target excited by an external light source. The inherent fluorescent group of the biological target is inherent, harmless and non-invasive to organisms, and safe and reliable. Intrinsic fluorophores include: flavin adenine dinucleotide, porphyrin, chlorophyll, nicotinamide adenine dinucleotide, etc., which are widely present in various tissues and organs of the human body (e.g., soft tissue sarcoma).

When the fluorophore of the biological target is an exogenous fluorophore received by the biological target, the fluorescence emitted by the fluorophore of the biological target is: the exogenous fluorescent group received by the biological target spontaneously generates fluorescence or is excited by an external light source to generate fluorescence. The exogenous fluorescent group comprises fluorescein sodium, indocyanine green, aminolevulinic acid hydrochloride, hypericin, protoporphyrin IX, neoindocyanine green, various fluorescent probes and the like.

Biological targets contemplated herein include normal or diseased biological targets (e.g., tumors, microorganisms, lymph nodes, soft tissue sarcomas, or other tissues or organs).

Here, if fluorescence emitted from a fluorophore of a biological target needs to be excited to generate fluorescence, the fluorescence needs to be irradiated with an excitation light source. Wherein: the excitation light source may be a light source having a specific emission wavelength range, such as an LD laser diode or an LED light emitting diode. The power of the excitation light source can be continuously adjustable, and the size of a light spot irradiated on a biological target area, the optical power density and the energy distribution can be kept stable and unchanged under the condition that the distance between the excitation light source and the biological target is variable under a certain specific power, so that the system can generate a stable and unchanged fluorescence excitation effect within a certain space distance from the biological target part. The irradiation range of the excitation light source is always larger than the biological target area to be shot by the imaging system. Wherein the wavelength of the light emitted by the excitation light source matches the biological target characteristics.

It should be noted that the power of the excitation light sources herein may also be set to be adjustable, and the number of the excitation light sources may be one or more.

To increase the illumination intensity or to improve the visualization of the biological target area, the biological target area may also be illuminated with an additional light source.

It should be noted that the power of the supplemental light source can also be set to be adjustable, and the number of the supplemental light sources can be one or more.

For example: for exogenous fluorophores:

in the surgical operation, the lymph node is often difficult to distinguish from surrounding fat and other tissues, indocyanine green can be injected into a patient in advance for searching the lymph node position of the patient, the indocyanine green can be effectively distributed in the lymph node as a typical exogenous fluorescent group, the absorption peak value of the exogenous fluorescent group to excitation light is about 785nm, fluorescence of about 830nm can be generated after the excitation light with 770-790nm waveband is irradiated, 820nm long-pass filter or 820-850nm band-pass filter needs to be loaded at the front end of an infrared camera to filter the light of the excitation light source and stray light in the environment, after the fluorescence image of the lymph node is obtained, the fluorescence image is subjected to noise reduction and enhancement by an image processing unit, a green image only containing the lymph node shape and a clear boundary is obtained, and the image is projected in situ to the lymph node position by a projector, the system helps the operator to accurately locate and determine the boundaries of lymph nodes in the direct vision field and further accurately protect or excise lymph nodes through operation.

For intrinsic fluorophores:

in the treatment of tinea (Dermatomycosis) caused by microsporidia, it is difficult to visually recognize the range of tinea caused by microsporidia, which contains a fluorophore inherent to itself, the absorption peak value of the inherent fluorophore to the excitation light is about 320-400nm, the excitation light in the wavelength band of 320-400nm can generate the fluorescence of about 490-510nm after being irradiated, the front end of the camera needs to be loaded with a 480nm long-pass filter or a 480-520nm band-pass filter, to filter the light of the excitation light source and the stray light in the environment, after the fluorescence image of the tinea is obtained, the image processing unit is used for reducing noise and enhancing the fluorescence image, a green image which only contains the range and the clear boundary of the tinea is obtained, the projector is used for projecting the image in situ to cover the position of the tinea, and a doctor is helped to accurately position the position of the tinea in the direct vision field, judge the boundary of the tinea and further sample and treat pathology.

The embodiment forms a fluorescence image of the biological target by collecting fluorescence emitted by the fluorescent group of the biological target in real time, generates corresponding fluorescence by utilizing transition between specific energy levels in the fluorescent group of the biological target, does not cause health damage to the organism, has energy of an excitation light source far lower than biological safety standard, has small equipment volume and small occupied space, can reduce interference in the imaging image on the other hand, is easier to identify the biological target and the boundary thereof, and further improves the positioning and boundary judgment effects of the biological target.

S102: and processing the fluorescence image of the biological target to obtain an image which only contains the biological target and has a clear boundary.

In step S102, the process of processing the fluorescence image of the biological target is:

Specifically, some images of interfering objects and stray light inevitably exist in a fluorescence image of the biological target formed from fluorescence emitted from fluorophores of the biological target, such as: when the biological target is a diseased tissue, the fluorescence image of the diseased tissue may contain image interference of surrounding blood vessels, muscles, etc. In order to obtain a clean fluorescence image containing only biological targets, the fluorescence image of the original biological target needs to be subjected to noise reduction processing.

In specific implementation, the fluorescence image of the original biological target may be denoised by any one or a combination of filtering methods, such as mean filtering, adaptive wiener filtering, or wavelet denoising.

In a specific implementation, the enhancement process is performed on the fluorescence map of the original biological target.

Enhancing useful information in an image, which may be a process of distortion, is aimed at improving the visual impact of the image for a given image application. The method aims to emphasize the overall or local characteristics of the image, changes the original unclear image into clear or emphasizes certain interesting characteristics, enlarges the difference between different object characteristics in the image, inhibits the uninteresting characteristics, improves the image quality, enriches the information content, enhances the image interpretation and identification effects, and meets the requirements of certain special analysis.

Image enhancement is achieved by adding some information or transformation data to the original image by some means to selectively highlight features of interest in the image or to suppress (mask) some unwanted features in the image to match the image to the visual response characteristics. In the image enhancement process, the reason of image degradation is not analyzed, and the processed image is not necessarily close to the original image. The image enhancement technology can be divided into two categories, namely an algorithm based on a space domain and an algorithm based on a frequency domain according to different spaces of the enhancement processing process.

It is understood that the method for enhancing the biological target in the noise-reduced fluorescence image in the present embodiment can be implemented by using methods such as histogram equalization, direct gray scale transformation, smoothing and sharpening in various time-frequency domains.

The purpose of rendering after the enhancement processing is to enhance the visual contrast between the biological target and the surrounding background and to improve the identifiability of the biological target.

It should be noted that the rendering process can be implemented by using an existing method, for example, rendering the biological target to highlight the boundary of the biological target, so as to achieve the purpose of accurately positioning the boundary of the biological target.

In this embodiment, as shown in fig. 3, the procedure of enhancing and denoising the fluorescence image of the biological target in sequence is as follows:

carrying out gray scale conversion and gray scale range calibration on the fluorescence image of the biological target to obtain a gray scale value range of a foreground signal and a gray scale value range of background noise; converting the fluorescence image of the biological target into an n-bit GRAY image (for example, n is 8), wherein the GRAY value of any pixel (x, y) is GRAY (x, y);

wherein, the process of calculating the corresponding enhancement factor ENFA by using the optimal segmentation threshold DIVT of the corresponding region is as follows:

where α is an enhancement factor, and is a known value less than 1, such as 0.01. The specific setting value of alpha can be set according to actual conditions.

The method for enhancing and reducing noise of the embodiment reduces half of the calculated amount by combining the experimental result and the theoretical calculation, and simultaneously improves the reliability.

As shown in fig. 2(a) -2 (d), assuming that the biological target is F, taking as an example that the naked eye cannot distinguish F and its boundary from the surrounding background:

the field of view of the biological target area is shown in FIG. 2(a), wherein the dashed box is the biological target area to be imaged by the imaging system; the area illuminated by the excitation light source is within the dashed circle and is larger than the dashed box.

Fig. 2(b) shows a fluorescence image obtained by the imaging system, wherein the dotted line F is an image which is displayed by the imaging display unit after being photographed by the imaging system when the biological target emits fluorescence; within the dashed box is a biological target area captured by the imaging system.

The image after enhancement, noise reduction and rendering of the fluorescence image of the biological object is given in fig. 2 (c). As can be seen in fig. 2(c), the enhancement, noise reduction and rendering process enhances the visual contrast of the biological object F with the surrounding background.

The fluorescence image in fig. 2(d) is projected in situ to the image of the biological target area in equal scale. Wherein, fig. 2(c) and fig. 2(b) are overlapped with the dotted square frame of fig. 2(a), and the biological target F which is difficult to distinguish by naked eyes is covered in situ by the green or other specific color image F which is visible by naked eyes in the biological target area, and the position, the shape and the boundary of the biological target are marked clearly in situ.

S103: and adjusting a projection light beam light path projected to the biological target to be coaxial with a fluorescence light path emitted by the biological target, and projecting an image which only contains the biological target and has a clear boundary to a biological target area emitting fluorescence in situ in real time in an equal proportion.

Specifically, the optical path between the biological target and the device for generating the projection light beam is set as a first optical path, the optical path between the biological target and the device for collecting fluorescence emitted by the fluorophore of the biological target is set as a second optical path, and the first optical path and the second optical path are equal.

The coaxial optical path design of the embodiment is that a projection light beam optical path is coaxial with a fluorescence optical path emitted by a biological target between the biological target and a beam splitter, the coaxial design ensures that the original view field of the acquired image is completely coincided with the center position of the view field covered by the projection image under the condition of changing the working distance and the angle in real time, even if the two are different in size, the straight lines of the diagonals of the acquired image and the projected image are also completely coincided, and the design with the same optical path ensures that the original view field of the acquired image is completely equal to and coincided with the view field covered by the projection image under the condition of changing the working distance and the angle in real time, so that the real-time equal-proportion in-situ projection of the image which only contains the biological target and has a clear boundary is realized to the biological target area emitting fluorescence, and under the condition of really changing the working distance and the working angle in real time, the real-time in-situ accurate projection can still be realized, and after the factory calibration, the device does not need to be calibrated before working every time, and the working angle and the working distance between the device and a target can be changed in real time, so that great convenience is brought to operation.

The working distance does not need to be fixed before each use, the in-situ projection is realized by using physical hardware, the working distance and the angle are not influenced by random change during working, and almost no time delay exists; the embodiment combines the fluorescence image with real-time equal-proportion in-situ projection, solves the problems that the biological target imaging can not be displayed in situ on the organism, and an operator can not directly and accurately judge the complete form and the boundary of the biological target on the organism, and utilizes the projection light beam to project the image which only contains the biological target and has clear boundary to the biological target area emitting fluorescence in real time equal-proportion in situ, thereby realizing the accurate in-situ display of the biological target and the boundary thereof on the organism, greatly shortening the time for identifying and positioning the boundary of the biological target, and improving the efficiency for identifying and positioning the boundary of the biological target.

Example 2

Fig. 4 is a schematic diagram illustrating a positioning system for in-situ visualization of a biological target according to an embodiment of the present invention.

The in-situ visual positioning system for the biological target comprises an imaging system, an image processing and analyzing unit, an image positioning and projecting unit and a spectroscope.

The present embodiment provides a positioning system for in situ visualization of a biological target, including:

(1) and the imaging system is used for acquiring fluorescence emitted by the fluorescent group of the biological target in real time to form a fluorescence image of the biological target.

The fluorophore of the biological target is a fluorophore that is intrinsic to the biological target or an exogenous fluorophore that the biological target receives.

To increase or decrease the illumination intensity of the biological target area and to improve the visualization of the biological target, supplemental light sources may also be utilized to assist the excitation light source in illuminating the biological target area.

For example: for exogenous fluorophores:

For intrinsic fluorophores:

(2) And the image processing and analyzing unit is used for processing the fluorescence image of the biological target to obtain an image which only contains the biological target and has a clear boundary.

The process of processing the fluorescence image of the biological target is as follows:

Specifically, images of some interfering substances inevitably exist in the fluorescence image of the biological target formed from fluorescence emitted from the fluorophore of the biological target, such as: when the biological target is diseased tissue, the fluorescence image of the diseased tissue may contain blood vessel image interference. In order to obtain a clean fluorescence image containing only biological targets, the fluorescence image of the original biological target needs to be subjected to noise reduction processing.

(3) And the image positioning projection unit is used for projecting the image which only contains the biological target and has a clear boundary to the biological target area emitting fluorescence in situ in real time in an equal proportion by utilizing the projection light beam.

In this embodiment, a beam splitter is adopted and is denoted as a first beam splitter; the first spectroscope is used for reflecting fluorescence emitted by the fluorescent group of the biological target to the imaging system; the first beam splitter is also configured to transmit the projection beam such that the projection beam is counter-current and coaxial to the fluorescence light path.

The optical path from the biological target to the lens in the image positioning projection unit is set as a first optical path, the optical path from the biological target to the spectroscope is set as a second optical path, the optical path from the spectroscope to the lens in the imaging system is set as a third optical path, and the first optical path is equal to the sum of the second optical path and the third optical path, as shown in fig. 15 and 16.

In another embodiment, the first beam splitter is configured to transmit fluorescence emitted by fluorophores of the biological target to the imaging system; the first beam splitter is also configured to reflect the projection beam such that the projection beam is counter-current and coaxial to the fluorescence light path. The optical path from the biological target to the lens in the imaging system is set as a first optical path, the optical path from the biological target to the spectroscope is set as a second optical path, the optical path from the spectroscope to the lens in the image positioning projection unit is set as a third optical path, and the first optical path is equal to the sum of the second optical path and the third optical path.

The embodiment adopts a coaxial light path design, a projection light beam light path is coaxial with a fluorescence light path emitted by a biological target between the biological target and a beam splitter, the coaxial design ensures that the original view field of the acquired image is completely coincided with the center position of the view field covered by the projection image under the condition of changing the working distance and the angle in real time, even if the two are different in size, the straight lines of the diagonals of the acquired image and the projected image are also completely coincided, and the design with the same light path ensures that the original view field of the acquired image is completely equal to and coincided with the view field covered by the projection image under the condition of changing the working distance and the angle in real time, so that the real-time equal-proportion in-situ projection of the image which only contains the biological target and has a clear boundary is realized to the biological target area emitting fluorescence, and under the condition of really changing the working distance and the working angle in real time, the real-time in-situ accurate projection can still be realized, and after the factory calibration, the device does not need to be calibrated before working every time, and the working angle and the working distance between the device and a target can be changed in real time, so that great convenience is brought to operation.

In this embodiment, as shown in fig. 5, the imaging system is composed of a filter, an optical coupling unit, and an imaging unit, which are integrated into a whole; the optical coupling unit is positioned between the optical filter and the imaging unit, and the optical paths of the optical coupling unit, the optical filter and the imaging unit are coaxial. The filter plate is used for filtering stray light of non-target fluorescence bands and improving the fluorescence imaging effect. The optical coupling unit may be implemented using an optical lens, and the imaging unit may be implemented using a CMOS camera or a CCD camera.

The embodiment combines the fluorescence image with real-time equal-proportion in-situ projection, solves the problems that the biological target imaging can not be displayed in situ on the organism, and an operator can not directly and accurately judge the complete form and the boundary of the biological target on the organism, and utilizes the projection light beam to project the image which only contains the biological target and has clear boundary to the biological target area emitting fluorescence in real time equal-proportion in situ, thereby realizing the accurate in-situ display of the biological target and the boundary thereof on the organism, greatly shortening the time for identifying and positioning the boundary of the biological target, and improving the efficiency for identifying and positioning the boundary of the biological target.

The following is a detailed description of the positioning system for in-situ visualization of biological targets, which includes an imaging system, an image positioning projection unit, an image processing and analysis unit, an excitation light source, an supplemental light source, and at least one spectroscope as an example:

example 3

As shown in fig. 6, the positioning system for in-situ visualization of biological targets of this embodiment includes an imaging system, an image positioning projection unit, an image processing and analyzing unit, an excitation light source, an additional light source, and a spectroscope. And the imaging system, the image positioning projection unit, the image processing and analyzing unit and the spectroscope are integrated into a core whole to form a complete positioning device. The imaging system, the image positioning projection unit, the image processing and analyzing unit and the spectroscope are all arranged in the positioning equipment. The positioning device is further provided with a window for transmitting the fluorescent light and the projection light beam.

In this embodiment, the optical path from the biological target to the lens in the image positioning projection unit is set as a first optical path, the optical path from the biological target to the spectroscope is set as a second optical path, the optical path from the spectroscope to the lens in the imaging system is set as a third optical path, and the first optical path is equal to the sum of the second optical path and the third optical path, as shown in fig. 6.

In this embodiment, the imaging system is composed of three of a filter, an optical coupling unit, and an imaging unit. Furthermore, the optical path of the projection beam is opposite and coaxial to the optical path of the fluorescence emitted by the fluorophores of the biological target.

The fluorescence emitted by the fluorophore of the biological target is reflected to an imaging system through a spectroscope and is used for collecting the fluorescence in real time to form a fluorescence image of the biological target, and the fluorescence image is transmitted to an image processing and analyzing unit for pretreatment; the image processing and analyzing unit transmits the fluorescent image which contains the biological target and is clear in boundary and obtained after the preprocessing to the image positioning and projecting unit; the image positioning projection unit utilizes the projection light beam to project an image which only contains a biological target and has a clear boundary to a biological target area emitting fluorescence in real time in an equal proportion in situ after being projected by the spectroscope.

Wherein, the spectroscope is plated with a high-reflection film for a fluorescence wave band and a high-transmission film for a projection light wave band. The included angle between the spectroscope and the projection light beam light path and the fluorescence light path emitted by the fluorescent group of the biological target can be set to be any acute angle, and the optimal angle is 45 degrees. The angle is beneficial to the setting of the position of the device and the accuracy of optical path transmission.

In this embodiment, the excitation light source and the supplemental light source are provided outside the device. The excitation light source can be selected as a laser with a specific wavelength or an LED light source with a specific emission wavelength range, the output light wavelength of the excitation light source is positioned at the characteristic absorption peak of a fluorescent substance contained in the biological target, the output power can be continuously adjusted, the size of a light spot irradiated to a target area, the optical power density and the energy distribution can be kept stable and unchanged under the condition that the distance between the excitation light source and the target is variable under a certain specific power, and the system can be ensured to generate a stable and unchanged fluorescence excitation effect within a certain spatial distance from the target part.

The front end of the excitation light source can also be additionally provided with a filter plate with a specific wavelength, so that the filter plate can highly transmit light with corresponding wavelength of the excitation light source and can filter stray light with other wavelengths.

In this embodiment, the excitation light source is a handheld structure, and the supplemental light source is installed at the front end of the window of the positioning device.

In the embodiment, the fluorescence image generated by the imaging system can be transmitted to the image display unit for displaying; the image generated after the preprocessing of the image processing and analyzing unit can also be transmitted to the image display unit for displaying.

Example 4

As shown in fig. 7, the positioning system for in-situ visualization of biological targets of this embodiment includes an imaging system, an image positioning projection unit, an image processing and analyzing unit, an excitation light source, an additional light source, and a beam splitter. Unlike embodiment 3, the excitation light source is also provided at the front end of the window of the positioning apparatus.

Example 5

As shown in fig. 8, the positioning system for in-situ visualization of biological targets of this embodiment includes an imaging system, an image positioning projection unit, an image processing and analyzing unit, an excitation light source, an supplemental light source, and two beam splitters.

This embodiment is different from embodiment 3 in that the excitation light source is located inside the positioning apparatus.

The exciting light generated by the exciting light source is reflected by the second spectroscope and then is transmitted by the first spectroscope to irradiate the biological target area, the light path of the exciting light in the light path between the second spectroscope and the biological target is coaxial with the projection light path, and the exciting light path, the fluorescence light path and the projection light path between the first spectroscope and the biological target are coaxial.

The angles between the first spectroscope and the second spectroscope and the light paths of the fluorescence, the projection light and the excitation light are all 45 degrees. Two spectroscopes are placed in parallel. The second spectroscope is plated with a high-transmittance film for the wavelength range of projection light and a high-reflection film for the wavelength range of excitation light. The first spectroscope is coated with a highly reflective film for the fluorescent wavelength range and a highly transmissive film for the projection light wavelength range.

Example 6

As shown in fig. 9, the positioning system for in-situ visualization of biological targets of the present embodiment includes an imaging system, an image positioning projection unit, an image processing and analyzing unit, an excitation light source, an supplemental light source, and three beam splitters.

This embodiment differs from embodiment 3 in that both the excitation light source and the supplemental light source are located inside the positioning device.

The exciting light generated by the exciting light source is reflected by the second spectroscope and then is transmitted by the first spectroscope to irradiate the target area. The supplementary light generated by the supplementary light source is reflected by the third spectroscope, and then sequentially transmitted by the second spectroscope and the first spectroscope to irradiate the target area.

The included angles between the three beam splitters and the light paths of the fluorescence light, the projection light, the exciting light and the supplementary light are all 45 degrees. The three beam splitters are arranged in parallel. The third spectroscope is plated with a projection light waveband high-transmittance film and a supplementary light waveband high-reflection film. The second side spectroscope is plated with a high-transmittance film for a projection light waveband and an additional light waveband and a high-reflectance film for an excitation light waveband. The first spectroscope is plated with a high-transmittance film for a projection light waveband, a supplementary light waveband, an excitation light waveband and a high-reflectance film for a fluorescence waveband.

The projection light and the supplementary light optical path are coaxial between the third spectroscope and the biological target, the projection light, the supplementary light and the exciting light optical path are coaxial between the second spectroscope and the biological target, and the projection light, the supplementary light, the exciting light and the fluorescence optical path of the image positioning projection unit are coaxial between the first spectroscope and the biological target.

Example 7

As shown in fig. 10, the positioning system for in-situ visualization of biological targets of this embodiment includes an imaging system, an image positioning projection unit, an image processing and analyzing unit, an excitation light source, an supplemental light source, and two beam splitters.

The excitation light source is positioned in the device, the excitation light generated by the excitation light source is reflected by the second spectroscope and then reflected by the first spectroscope to irradiate the target area, the excitation light and the fluorescence light path are coaxial between the second spectroscope and the biological target, and the excitation light, the fluorescence and the projection light path are coaxial between the first spectroscope and the biological target.

The included angles between the two spectroscopes and the light paths of the fluorescence, the projection light and the exciting light are all 45 degrees. Two spectroscopes are placed in parallel. The first spectroscope is plated with a projection light waveband high-transmittance film and an excitation light and fluorescence waveband high-reflectance film. The second spectroscope is plated with a fluorescent wave band high-transmittance film and an exciting light wave band high-reflectance film.

Example 8

As shown in fig. 11, the positioning system for in-situ visualization of biological targets of this embodiment includes an imaging system, an image positioning projection unit, an image processing and analyzing unit, an excitation light source, an supplemental light source, and three beam splitters.

The exciting light generated by the exciting light source is reflected by the second spectroscope and then is reflected by the first spectroscope to irradiate the target area. The augmented light generated by the augmented light source is reflected by the third spectroscope, transmitted by the second spectroscope and reflected by the first spectroscope to irradiate the target area.

The included angles between the three beam splitters and the light paths of the fluorescence light, the projection light, the exciting light and the supplementary light are all 45 degrees. The three beam splitters are arranged in parallel. The third spectroscope is plated with a fluorescent wave band high-transmittance film and a supplementary light wave band high-reflectance film. The second spectroscope is plated with a fluorescent wave band, a supplementary light wave band high-transmittance film and an exciting light wave band high-reflection film. The first spectroscope is plated with a fluorescent wave band, a supplementary light wave band, an exciting light wave band high-reflection film and a projection light wave band high-transmission film.

The light paths of the fluorescence and the supplementary light are coaxial between the third spectroscope and the biological target, the light paths of the fluorescence, the supplementary light and the exciting light are coaxial between the second spectroscope and the biological target, and the light paths of the projection light, the supplementary light, the exciting light and the fluorescence of the image positioning projection unit are coaxial between the first spectroscope and the biological target.

Example 9

As shown in fig. 12, the positioning system for in-situ visualization of biological targets of the present embodiment includes an imaging system, an image positioning projection unit, an image processing and analyzing unit, an excitation light source, an supplemental light source, and two beam splitters.

This embodiment differs from embodiment 3 in that the fluorescence enters the imaging system after being reflected by two beam splitters.

The included angles between the two spectroscopes and the light paths of the fluorescence light and the projection light are both 45 degrees. Two spectroscopes are placed in parallel. The first spectroscope is plated with a fluorescent wave band high-reflection film and a projection light wave band high-transmission film. The second spectroscope can be only plated with a fluorescent band high-reflection film. The optical paths of the projected light and the fluorescent light are coaxial between the first beam splitter and the biological target.

Example 10

As shown in fig. 13, the positioning system for in-situ visualization of biological targets of this embodiment comprises an imaging system, an image positioning projection unit, an image processing and analyzing unit, an excitation light source, an additional light source and a spectroscope.

The difference between this embodiment and embodiment 3 is that the direct fluorescence of the biological target enters the imaging system after passing through the spectroscope, and the projection light of the image positioning projection unit is projected to the biological target after being reflected by the spectroscope.

The included angles between the spectroscope and the light paths of the fluorescence light and the projection light are both 45 degrees.

The spectroscope is plated with a projection light wave band high-reflection film and a fluorescence wave band high-transmission film.

The optical paths of the projected light and the fluorescence are coaxial between the beam splitter and the biological target.

Example 11

As shown in fig. 14, the positioning system for in-situ visualization of biological targets of this embodiment includes an imaging system, an image positioning projection unit, an image processing and analyzing unit, an excitation light source, an supplemental light source, and two beam splitters.

The difference between this embodiment and embodiment 5 is that the direct fluorescence of the biological target passes through the spectroscope and enters the imaging system, and the projection light of the image positioning projection unit is reflected by the two spectroscopes in sequence and then projected to the biological target.

The included angles between the two spectroscopes and the light paths of the fluorescence light and the projection light are both 45 degrees. Two spectroscopes are placed in parallel. The first spectroscope is plated with a fluorescent wave band high-transmittance film and a projection light wave band high-reflectance film. The second beam splitter may be coated with only a high reflection film for the projection light band. Between the first beam splitter and the biological target, the optical path of the projected light and the fluorescence are coaxial.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for locating a biological target visually in situ, comprising:

processing the fluorescence image of the biological target to obtain an image which only contains the biological target and has a clear boundary; specifically, enhancement, noise reduction and rendering processing are sequentially carried out on the fluorescence image of the biological target to enhance the contrast between the biological target and the surrounding background, so that the image only contains the biological target and has a clear boundary;

adjusting a projection light beam light path projected to the biological target to be coaxial with a fluorescence light path emitted by the biological target, and projecting an image which only contains the biological target and has a clear boundary to a biological target area emitting fluorescence in situ in real time in equal proportion;

setting the optical path between the biological target and the device for generating the projection light beam as a first optical path, setting the optical path between the biological target and the device for collecting the fluorescence emitted by the fluorophore of the biological target as a second optical path, wherein the first optical path is equal to the second optical path;

if the fluorescence emitted by the fluorescent group of the biological target needs to be excited to generate fluorescence, the fluorescence needs to be irradiated by an excitation light source; the excitation light source is positioned in the positioning equipment; wherein, between biological target and beam splitter, excitation light path, projection beam light path and the fluorescence light path that biological target sent are coaxial, specific:

the excitation light generated by the excitation light source is reflected by the second spectroscope and then is transmitted by the first spectroscope to irradiate the biological target area, the light path of the excitation light in the light path between the second spectroscope and the biological target is coaxial with the projection light path, and the excitation light path, the fluorescence light path and the projection light path between the first spectroscope and the biological target are coaxial;

alternatively, the first and second electrodes may be,

the exciting light generated by the exciting light source is reflected by the second spectroscope and then reflected by the first spectroscope to irradiate a target area, the exciting light and the fluorescence light path are coaxial between the second spectroscope and the biological target, and the exciting light, the fluorescence and the projection light path are coaxial between the first spectroscope and the biological target.

2. The method of claim 1, wherein the enhancing and de-noising the fluorescence image of the biological target comprises:

carrying out gray scale conversion and gray scale range calibration on the fluorescence image of the biological target to obtain a gray scale value range of a foreground signal and a gray scale value range of background noise; the fluorescence image of the biological target is converted into an n-bit grayscale image, any pixel (x,y) The GRAY values of (a) are GRAY (x,y)；

3. the method of claim 1, wherein the fluorescent moiety of the biological target emits fluorescence that: fluorescence that is spontaneously formed by fluorophores inherent to the biological target or by exogenous fluorophores received by the biological target.

4. The method of claim 1, wherein the fluorescent moiety of the biological target emits fluorescence that: the inherent fluorescent group of the biological target generates fluorescence when being excited by an external light source.

5. The method of claim 1, wherein the fluorescent moiety of the biological target emits fluorescence that: the exogenous fluorescent group received by the biological target is excited by an external light source to generate fluorescence.

6. A localization system for in situ visualization of a biological target, comprising:

the image processing and analyzing unit is used for processing the fluorescence image of the biological target to obtain an image which only contains the biological target and has a clear boundary; specifically, enhancement, noise reduction and rendering processing are sequentially carried out on the fluorescence image of the biological target to enhance the contrast between the biological target and the surrounding background, so that the image only contains the biological target and has a clear boundary;

the image positioning projection unit is used for projecting an image which only contains a biological target and has a clear boundary to a biological target area emitting fluorescence in situ in real time in an equal proportion after the projection light beam is transmitted by the spectroscope;

the excitation light source is positioned inside the positioning equipment;

wherein, between biological target and beam splitter, excitation light path, projection beam light path and the fluorescence light path that biological target sent are coaxial, specific:

alternatively, the first and second electrodes may be,

the exciting light generated by the exciting light source is reflected by the second spectroscope and then reflected by the first spectroscope to irradiate a target area, the exciting light and the fluorescence light path are coaxial between the second spectroscope and the biological target, and the exciting light, the fluorescence and the projection light path are coaxial between the first spectroscope and the biological target;

the optical path from the biological target to the lens in the image positioning projection unit is set as a first optical path, the optical path from the biological target to the spectroscope is set as a second optical path, the optical path from the spectroscope to the lens in the imaging system is set as a third optical path, and the first optical path is equal to the sum of the second optical path and the third optical path.

7. The in situ visual localization system of a biological target of claim 6, wherein the image processing and analysis unit is configured to:

8. the localization system of claim 6, wherein the fluorophore of the biological target emits fluorescence of: fluorescence that is spontaneously formed by fluorophores inherent to the biological target or by exogenous fluorophores received by the biological target.

9. The localization system of claim 6, wherein the fluorophore of the biological target emits fluorescence of: the inherent fluorescent group of the biological target generates fluorescence when being excited by an external light source.

10. The localization system of claim 6, wherein the fluorophore of the biological target emits fluorescence of: the exogenous fluorescent group received by the biological target is excited by an external light source to generate fluorescence.

11. The system of claim 6, wherein the excitation light source is configured to illuminate a region of the biological target to excite fluorophores of the biological target to fluoresce.

12. The localization system of claim 11, wherein the localization system for in situ visualization of biological targets further comprises an additional light source.