Nonlinear optical large-field-of-view imaging system

Abstract

The present invention relates to the field of large-field-of-view scenes. Provided is a nonlinear optical large-field-of-view imaging system, comprising a light source system, a laser scanning system and a laser detection system, wherein the light source system is configured to emit laser; the laser scanning system comprises a focusing lens and a galvanometer, wherein the galvanometer is located between the focusing lens and a sample to be detected, the focusing lens is configured to focus the laser on said sample, and the galvanometer vibrates according to a preset scanning angle so as to realize scanning of a laser focus on said sample; and the laser detection system comprises a compound-eye collection structure and a detector, wherein the compound-eye collection structure comprises a plurality of individual lenses which are closely arranged, optical signals generated by said sample are incident on the compound-eye collection structure, and the detector is configured to detect the optical signals collected by the compound-eye collection structure. The present invention can expand a laser scanning imaging range, can ensure that a laser beam always propagates along the optical axis of the focusing lens, and can comprehensively collect signals excited across the entire scanning field of view, thereby improving the efficiency of large-field-of-view imaging and increasing the signal intensity.

Description

A nonlinear optical large field of view imaging system TECHNICAL FIELD

[0001] The present application relates to the field of large field of view imaging, and in particular to a centimeter-level large field of view nonlinear optical imaging system. BACKGROUND

[0002] Traditional nonlinear optical microscopic imaging, such as Coherent anti-Stokes Raman scattering (CARS), Coherent Stokes Raman scattering (CSRS), Multi-photon emission fluorescence (MPEF), Second harmonic generation (SHG), Third harmonic generation (THG), and Sum frequency generation (SFG), have shown excellent cancer differentiation ability at the microscopic level and helped to determine the boundary of cancer tissue. CARS can image the unique biomarker chemical bonds in cancer tissue, and SHG can microscopically image the abundant collagen fibers in cancer tissue. However, the imaging area of the above-mentioned nonlinear optical microscopic imaging methods is usually only about 300 μm, while cancer surgery navigation often requires a larger imaging field of view. A large imaging field of view means a larger focal spot diameter and a longer working distance at the focal point, which will respectively reduce the generation and detection of nonlinear optical signal light.

[0003] For example, CARS imaging has very strong chemical specificity imaging ability. As a label-free imaging method, CARS usually requires two coherent pulsed lasers as pump light and Stokes light, and requires the photon energy difference of the two lights to be consistent with the vibrational energy level energy of the detected substance molecules. The field of view of traditional CARS imaging is only about several hundred microns. For applications that require a larger imaging range, a stitching method is often used for imaging. That is, multiple images of several hundred microns are stitched together, but this method of realizing large field of view imaging requires a long imaging time, for example, several hours are required for imaging a square centimeter of imaging field of view, and is not suitable for time-critical surgery scenarios.

[0004] SUMMARY

[0005] The present application is used to solve the problem that the large field of view imaging cannot be realized based on the nonlinear optical microscopic imaging in the prior art. Specifically, there are three problems to be solved in the centimeter level large field of view nonlinear optical imaging. One is the problem of large field of view laser scanning, and the other two difficulties exist in the signal collection of large range imaging. One difficulty is that the small detection numerical aperture (NA) leads to weak signal collection ability, resulting in reduced detection signal strength; the other difficulty is that the imaging field of view is large and the detection range of the detector is limited, so that the signals excited in the entire scanning field of view cannot be completely collected, resulting in a smaller imaging field of view.

[0006] In order to solve the above technical problems, the present application provides a nonlinear optical large field of view imaging system in the first aspect, comprising a light source system, a laser scanning system and a laser detection system.

[0007] The light source system is used for emitting laser.

[0008] The laser scanning system comprises a focusing lens and a galvanometer, the galvanometer is located between the focusing lens and the measured sample, the focusing lens is used for focusing the laser on the measured sample, and the galvanometer vibrates according to the preset scanning angle to realize the scanning of the laser focus on the measured sample.

[0009] The laser detection system comprises an ommatidium type collection structure and a detector, the ommatidium type collection structure comprises a plurality of single lenses arranged closely, and the light signal generated by the measured sample is incident on the ommatidium type collection structure; the detector is used for detecting the light signal collected by the ommatidium type collection structure.

[0010] As a further embodiment of the present application, the ommatidium type collection structure is located between the galvanometer and the measured sample.

[0011] As a further embodiment of the present application, the nonlinear optical large field of view imaging system further comprises a dichroic mirror.

[0012] The dichroic mirror is located between the ommatidium type collection structure and the measured sample, the dichroic mirror is used for reflecting the focused laser reflected by the galvanometer to the measured sample, and the light signal passing through the measured sample reaches the ommatidium type collection structure.

[0013] As a further embodiment of the present application, the focusing lens is movable, and the movement of the focusing lens is determined according to the height of the surface of the measured sample.

[0014] As a further embodiment of the present application, the optical axis of the single lens of the ommatidium type collection structure points to the center of the scanning area of the measured sample.

[0015] As a further embodiment of the present application, the probe includes a plurality of filters, a plurality of multi-mode fiber bundles and a plurality of photodetector modules.

[0016] The filters are used to filter the light signals collected by the single lenses.

[0017] The input ends of the multi-mode fiber bundles are used to collect the light signals collected by the single lenses, and the output ends of the multi-mode fiber bundles are connected to the photodetector modules.

[0018] As a further embodiment of the present application, the nonlinear optical large field-of-view imaging system is applied to coherent anti-Stokes Raman scattering, coherent Stokes Raman scattering, second harmonic, third harmonic, sum frequency, two-photon fluorescence, multi-photon fluorescence, single-photon fluorescence, spontaneous Raman, and short-wave infrared Raman large field-of-view imaging.

[0019] As a further embodiment of the present application, the light source system is capable of outputting two coherent femtosecond laser pulses, and the central wavelength energy difference of the two beams matches the molecular bond vibration energy of the measured sample.

[0020] The second aspect of the present application further provides a laser scanning system applied to a nonlinear optical large field-of-view imaging system, including a focusing lens and a galvanometer, wherein the galvanometer is located between the focusing lens and the measured sample.

[0021] The focusing lens is used to focus the laser to the measured sample.

[0022] The galvanometer vibrates according to a preset scanning angle to realize scanning of the laser focal point on the measured sample.

[0023] In a further embodiment of the present application, the focusing lens is movable, and the movement of the focusing lens is determined according to the height of the surface of the measured sample.

[0024] The third aspect of the present application provides a laser probe system applied to a nonlinear optical large field-of-view imaging system, including an ommatidium collecting structure and a probe, wherein the ommatidium collecting structure includes a plurality of single lenses arranged closely.

[0025] The light signals generated by the measured sample are incident to the ommatidium collecting structure.

[0026] The probe is used to probe the light signals collected by the ommatidium collecting structure.

[0027] The nonlinear optical large field-of-view imaging system provided by the application, by setting the laser scanning system to include a focusing lens and a galvanometer, the galvanometer is located between the focusing lens and the measured sample, the focusing lens is used to focus the laser to the measured sample, and the galvanometer vibrates according to the preset scanning angle to realize the scanning of the laser focus on the measured sample, while significantly expanding the imaging range of the laser scanning, the laser beam always propagates along the optical axis of the focusing lens during the laser scanning process, various lens distortions are eliminated, and thus off-axis aberration does not occur. By designing the laser detection system to include an ommatidium type collection structure and a detector, the ommatidium type collection structure includes a plurality of closely arranged single lenses, the detection numerical aperture can be increased, the signals excited in the entire scanning field of view can be comprehensively collected, the signal strength of the large field-of-view imaging is improved, and thus the imaging time of the large field-of-view imaging is reduced.

[0028] In order to make the above and other objects, features and advantages of the present application more apparent, the following will specifically describe a preferred embodiment in combination with the accompanying drawings. BRIEF DESCRIPTION OF DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description are only some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor.

[0030] Fig. 1 shows a schematic diagram of the nonlinear optical large field-of-view imaging system based on point scanning according to an embodiment of the present application;

[0031] Fig. 2 shows a first schematic diagram of the nonlinear optical large field-of-view imaging system according to an embodiment of the present application;

[0032] Fig. 3 shows a second schematic diagram of the nonlinear optical large field-of-view imaging system according to an embodiment of the present application;

[0033] Fig. 4 shows a schematic diagram of the detection system according to an embodiment of the present application;

[0034] Fig. 5 shows an example diagram of the ommatidium type collection structure according to an embodiment of the present application;

[0035] Fig. 6 shows a focal plane diagram according to an embodiment of the present application;

[0036] Fig. 7 shows a schematic diagram of the imaging result according to an embodiment of the present application.

[0037] LIST OF ELEMENTS IN THE DRAWINGS:

[0038] 40, measured sample;

[0039] 401, light source system;

[0040] 402, laser scanning system;

[0041] 403, laser detection system;

[0042] 4021, focusing lens;

[0043] 4022, galvanometer;

[0044] 4031, compound eye collection structure;

[0045] 4032, detector;

[0046] 40311, single lens

[0047] 404, dichroic mirror. DETAILED DESCRIPTION

[0048] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application.

[0049] It should be noted that the terms "first", "second" and the like in the specification and claims of the present application and the above-described drawings are used to distinguish similar objects, and do not necessarily indicate a specific order or sequence. It should be understood that the data thus used can be interchanged under appropriate circumstances. In addition, the terms "comprise" and "have" and any variations thereof are intended to cover non-exclusive inclusion, for example, a process, method, device, product or equipment comprising a series of steps or units does not necessarily limit to those clearly listed steps or units, but can include other steps or units not clearly listed or inherent to these processes, methods, products or equipment.

[0050] The term "large field of view" in the specification and claims of the present application and the above-described drawings is compared with the field of view of nonlinear optical microscopic imaging. The imaging size of the large field of view of the present application is much larger than the microscopic imaging size, with an increase of 2 orders of magnitude. For example, the unit of microscopic imaging range is micrometer (μm), and the unit of large field of view imaging range is centimeter (cm). More specifically, for example, coherent anti-stokes Raman scattering, multiphoton fluorescence, second harmonic, third harmonic and other nonlinear optical microscopic imaging, the imaging field of view is only about 300 μm, while the nonlinear optical large field of view imaging of the present application can reach 3 cm imaging field of view.

[0051] The description and claims of the present application and the above-mentioned similar description of C between A and B in the above-mentioned drawings mean that C is between A and B in terms of relative position relationship of optical path, for example, laser passes through A, C and B in sequence, if there is an optical path between A and B, i.e. laser passes through. If there is no optical path between A and B, C is between A and B in terms of relative position relationship in physical position, for example, C is between A and B in one direction (for example, vertical or horizontal).

[0052] Most of the existing nonlinear optical microscopic imaging relies on the 4f system to provide the optical fixed point of the back surface of the objective lens. This conventional configuration (i.e. 4f system) is usually matched with a large-aperture microscopic objective lens, which has a short working distance and is only suitable for microscopic imaging of a field of view below millimeters, for example, an imaging field of view of about 300 μm, and cannot realize large-field imaging above centimeters. Among them, the 4f system, also known as the 4f scanning system, the 4f point scanning system or the 4f optical system, is a common device in the field of optics, and its specific structure can refer to the prior art, which will not be described here in detail.

[0053] For application scenarios requiring a larger imaging range, a picture assembly method is usually combined with nonlinear optical microscopic imaging to achieve the imaging. Specifically, a nonlinear optical microscopic imaging system is used to image multiple fields of view of hundreds of microns, and then the images of hundreds of microns are assembled. However, this method of realizing large-field imaging requires a long imaging time, for example, several hours are required for imaging of a square centimeter imaging field, and is not suitable for surgical scenarios with high time requirements.

[0054] On the basis of the above problems of the large-field solution combined with the picture assembly method, the inventors propose a nonlinear optical large-field imaging system based on point scanning. Specifically, the objective lens of the nonlinear optical microscopic imaging system is replaced by a long-focus objective lens, and a 4f system is used to realize scanning. The imaging system can expand the imaging field to a certain extent. However, the imaging system has the following problems: first, the structure of the imaging system is complex, which makes the system larger; second, additional off-axis aberrations (including field curvature, coma, spherical aberration, etc.) are introduced. Taking the field curvature as an example, in microscopic imaging, the field area of 300 μm imaging field causes the focal surface to change by about several microns, but if the imaging field is expanded to centimeters, the field curvature causes the focal surface to change to centimeters, as shown by the dashed line in FIG. 1. Therefore, the nonlinear optical microscopic imaging system is not suitable for scenarios requiring a larger imaging field, and the following problems exist: one is the problem of large-field laser scanning, and the other is that after expanding the imaging field, there are two difficulties in signal collection. The signal light generated by the measured object becomes weak, and the existing detection system is still used to collect the signal, which causes the problems of weak signal collection ability leading to reduced detection signal strength and incomplete signal collection leading to smaller imaging field.

[0055] In order to solve the problems of complex structure, introduction of off-axis aberration of the point scanning based nonlinear optical large field of view imaging, and the problems of weak signal collection ability of large field of view imaging, which leads to the reduction of detection signal intensity and the reduction of imaging field of view caused by incomplete signal collection, the application provides a nonlinear optical large field of view imaging system, as shown in Figures 2 and 3, which comprises a light source system 401, a laser scanning system 402 and a laser detection system 403.

[0056] The light source system 401 is used for emitting laser, and the nonlinear optical large field of view imaging system provided by the embodiment can be applied to coherent anti-Stokes Raman scattering, coherent Stokes Raman scattering, second harmonic, third harmonic, sum frequency, two-photon fluorescence, multi-photon fluorescence, single-photon fluorescence, spontaneous Raman, short-wave infrared Raman and the like. When applied to coherent anti-Stokes Raman scattering, the laser is used for exciting the molecular vibration of the measured sample.

[0057] The laser scanning system 402 comprises a focusing lens 4021 and a galvanometer 4022, the galvanometer 4022 is located between the focusing lens 4021 and the measured sample 40, the focusing lens 4021 is used for focusing the laser emitted by the light source system 401 to the measured sample 40, and the galvanometer 4022 vibrates according to the preset scanning angle to realize the scanning of the laser focal point on the measured sample 40.

[0058] The laser detection system 403 comprises an ommatidium type collection structure 4031 and a detector 4032, the ommatidium type collection structure 4031 comprises a plurality of single lenses arranged closely, the light signal generated by the measured sample 40 is incident to the ommatidium type collection structure 4031, and the detector 4032 is used for detecting the light signal collected by the ommatidium type collection structure 4031.

[0059] In detail, the galvanometer 4022 is located between the focusing lens 4021 and the measured sample 40, which means that the laser is focused to the surface of the measured sample 40 after passing through the focusing lens 4021 and the galvanometer 4022 in sequence. The focal surface scanned by the galvanometer 4022 is a spherical surface. The process of detecting and analyzing the light signal by the detector 4032 can refer to the prior art, and the application does not limit the specific analysis process. In specific implementation, the detector 4032 can be connected to a collection card, and the photoelectric signal is collected by the collection card.

[0060] The embodiment discards the existing 4f system, and sets the laser scanning system to include a focusing lens and a galvanometer, the galvanometer is located between the focusing lens and the measured sample, the focusing lens is used for focusing laser on the measured sample, and the galvanometer vibrates according to a preset scanning angle to realize scanning of the laser focus on the measured sample, which can significantly expand the imaging range of laser scanning and ensure that the laser beam always propagates along the optical axis of the focusing lens during the laser scanning process, eliminates various lens distortions, so that off-axis aberration does not occur, and has the advantage of fast imaging speed compared with the large field of view realized by the puzzle, and can be used in real-time large field of view imaging scenes, such as surgical navigation, to facilitate quick determination of a target region (for example, a cancer range). By designing the laser detection system to include an ommatidium type collection structure and a detector, the ommatidium type collection structure includes a plurality of closely arranged single lenses, which can increase the detection numerical aperture and comprehensively collect signals excited in the entire scanning field of view, improve the signal strength of large field of view imaging, and thus reduce the imaging time of large field of view imaging.

[0061] In some embodiments of the application, as shown in FIG. 2, the ommatidium type collection structure 4031 is located between the galvanometer 4022 and the measured sample 40. The ommatidium type collection structure 4031 and the galvanometer 4022 are located above the measured sample 40, the focusing lens 4021 is located above the ommatidium type collection structure 4031 and on one side of the galvanometer 4022. The laser emitted by the light source system 401 is focused by the focusing lens 4021, and then passes through the galvanometer 4022 to focus the laser on the surface of the measured sample 40 and realize scanning of the laser focus on the measured sample 40.

[0062] In some embodiments of the application, considering the limited space between the ommatidium type collection structure 4031 and the detector 4032, another nonlinear optical large field of view imaging system is provided, as shown in FIG. 3, in addition to the light source system 401, the laser scanning system 402 and the laser detection system 403, the nonlinear optical large field of view imaging system also includes a dichroic mirror 404, the dichroic mirror 404 is located between the ommatidium type collection structure 4031 and the measured sample 40, and the dichroic mirror 404 reflects the focused laser reflected by the galvanometer 4022 to the measured sample 40. The dichroic mirror 404 is selected to be a dichroic mirror that can directly transmit the optical signal generated by the measured sample 40, and the dichroic mirror 404 can make the optical signal generated by the measured sample 40 directly pass through the dichroic mirror 404 and then be incident on the ommatidium type collection structure 4031.

[0063] In detail, the dichroic mirror 404 is between the ommatidium type collection structure 4031 and the measured sample 40, that is, the ommatidium type collection structure 4031 is above the dichroic mirror 404, and the measured sample 40 is below the dichroic mirror 404.

[0064] The galvanometer 4022 is located on one side of the dichroic mirror 404, and the laser emitted by the light source system 401 is first focused by the focusing lens 4021 and then incident to the galvanometer 4022, and the galvanometer 4022 rotates according to the preset scanning angle to realize the scanning of the laser focal point on the measured sample 40.

[0065] The layout of the laser scanning system and the laser detection system in the embodiment can avoid the problem that the laser scanning system is inconvenient to be arranged due to the insufficient internal space of the laser detection system.

[0066] In an embodiment of the present application, the measured sample is, for example, a biological tissue, and the specific molecular bond vibration in the tissue (for example, the C-H vibration of the oil in the result shown in FIG. 7) can be detected by coherent anti-Stokes Raman scattering (CARS), the collagen fiber in the tissue can be detected by SHG second harmonic, or the autofluorescence in the tissue can be detected by fluorescence, or the specific tissue structure or molecule can be detected by target fluorescence.

[0067] In an embodiment of the present application, the light source system can output two coherent femtosecond laser pulses, and the central wavelength energy difference of the two beams of light matches the molecular bond vibration energy of the measured sample.

[0068] In an embodiment of the present application, a detection system is further provided, as shown in FIG. 4, the detection system comprises: an ommatidium type collection structure 4031 and a detector 4032.

[0069] The ommatidium type collection structure 4031 comprises a plurality of single lenses 40311 arranged closely, and the light signal generated by the measured sample is incident to the ommatidium type collection structure 4031.

[0070] The detector 4032 is used for detecting the light signal collected by the ommatidium type collection structure 4031. In detail, the detection process of the detector 4032 on the light signal collected by the ommatidium type collection structure 4031 can refer to the prior art, and the present application is not limited thereto, and the detection result is also different according to different application fields. In specific implementation, the detector 4032 comprises a plurality of detection chips and a processing device, as shown in FIG. 3, the number of the detection chips is the same as the number of the single lenses 40311 in the ommatidium type collection structure 4031. The processing device is used for enhancing the light signal detected by the detection chip, so as to improve the collection intensity of the light signal generated by the measured sample and further improve the signal intensity of the large field of view imaging.

[0071] In the embodiment, the ommatidium type collection structure can expand the detection numerical aperture and improve the signal collection capability, at the same time, the ommatidium type collection structure expands the collection range, and can collect the signal excited in the entire scanning field of view, thereby ensuring the accuracy of the detection result.

[0072] For each individual imaging channel, a single lens is used to realize the imaging from object to image, the ratio of the object focal length to the image focal length determines the ratio of the object size to the image size, and selecting appropriate object focal length and image focal length and appropriate size of the detection chip can achieve signal collection of the entire scanning field of view.

[0073] Wherein, the appropriate object focal length and image focal length and the appropriate size of the detection chip satisfy the following relationship:

[0074] (1) 1 / u + 1 / v = 1 / f;

[0075] (2) u / v is greater than the ratio of the imaging field of view size to the detection chip size;

[0076] Wherein, f is the focal length of the single lens 40311, u is the distance between the single lens 40311 and the plane of the measured sample 40, i.e. the object focal length (as shown in FIG. 4), and v is the distance between the detector 4032 and the single lens 40311, i.e. the image focal length (as shown in FIG. 4).

[0077] Wherein, the above relationship (1) is a theoretical formula, and in actual design, the value of 1 / u + 1 / v is approximately equal to the value of 1 / f.

[0078] By determining the distance between the detector 4032 and the single lens 40311, the distance between the single lens 40311 and the measured sample 40, and the size of the detection chip through the above relationships (1) and (2), the light signal generated by the measured sample 40 (i.e. the focal plane) can be focused on the detector 4032. In the process of scanning the galvanometer 4022, the focused light of the laser will scan the focal plane to generate a signal, and the projection of the signal will also change in position. The embodiment can ensure that the projection of the light signal collected by the single lens 40311 in the compound eye type collection structure 4031 on the detector 4032 does not reach outside the detector 4032, i.e. the light signal does not exceed the detection range of the detector 4032.

[0079] In some specific embodiments, the object focal length is 100 mm, the image focal length is 19 mm, the focal length of the single lens in the compound eye type collection structure is 16 mm, the numerical aperture is 0.79, the size of the detection chip is 6 mm, and the imaging field of view is 30x30 mm.

[0080] In some specific embodiments, u / v>4.

[0081] In some embodiments of the application, the plurality of lenses in the compound eye type collection structure 4031 are closely arranged, including a plurality of lenses arranged in a plane. For example, the plurality of lenses are arranged in a circular layer-by-layer manner in the same plane, as shown on the right side of FIG. 5.

[0082] In some embodiments of the present application, as shown in FIG. 5, the multiple lenses in the compound eye collection structure 4031 are closely arranged, including that the lenses are arranged in layers in a circular shape and the lenses are arranged in an arc shape. Specifically, the optical axis of each lens of the compound eye collection structure points to the center of the scanning area of the sample under test, and the center of the scanning area is the center of the focal plane, as shown in FIG. 4.

[0083] In some embodiments of the present application, as shown in FIG. 5, the multiple lenses in the compound eye collection structure 4031 are closely arranged, including that the lenses are arranged in layers in a circular shape and the lenses are arranged in an arc shape. Specifically, the optical axis of each lens of the compound eye collection structure points to the center of the scanning area of the sample under test, and the center of the scanning area is the center of the focal plane, as shown in FIG. 4.

[0084] The arrangement of the lenses in the compound eye collection structure of the present embodiment can make the signal intensity collected by each channel substantially consistent, thereby improving the intensity and accuracy of the detection of the detector 4032.

[0085] In some embodiments of the present application, the focal length of the focusing lens 4021 is in the range of 100-250 mm. The focusing lens with the focusing range can make the focal spot size 15-40 μm, which can ensure the instantaneous power density, thereby improving the signal intensity and having sufficient working distance without affecting the imaging area. The use of the long focal length lens for focusing in the present embodiment results in a longer Rayleigh range, thereby increasing the imaging depth and the ability of z-axis projection.

[0086] For example, when the above nonlinear optical large field imaging system is applied to coherent anti-Stokes Raman scattering imaging (CARS imaging for short), the distance from the focal plane to the focusing lens is fixed. Due to the existence of the galvanometer scanning, the focal plane is a spherical surface, and the curvature radius of the spherical surface depends on the focal length of the focusing lens. In the 3 cm imaging range, there is only about 1 mm deviation, and at this time, the Rayleigh range of the laser used for CARS imaging is about 1 mm, which can basically completely compensate the signal intensity. In addition, the focal point position can be changed during the scanning process by moving the focusing lens in real time or using a focusing lens with variable focal length to realize the scanning focal plane as a plane.

[0087] In some embodiments of the present application, the distance between the focusing lens 4021 and the galvanometer 4022 is within 20 mm.

[0088] In some embodiments of the present application, as shown in FIG. 6, the dotted arc line is the focal plane, and the distance d between the field edge A of the focal plane and the center B of the focal plane is less than the Rayleigh distance of the focused laser at the sample under test 40. Specifically, the distance d between the field edge A of the focal plane and the center B of the focal plane can be determined by the distance R between the galvanometer 4022 and the focal plane and the scanning angle θ of the galvanometer 4022. In some embodiments, the scanning angle θ is in the range of 2-8°.

[0089] The embodiment can realize the limitation of the distance R between the galvanometer and the focal plane and the size of the Rayleigh range by limiting the relationship between the distance between the field of view edge A of the focal plane and the center B of the focal plane and the Rayleigh distance of the laser focused at the measured sample 40, so that the imaging signal is an in-plane signal as much as possible, and compared with the 4f system scanning imaging, the embodiment can make the imaging not exist off-axis aberration.

[0090] In some embodiments of the application, the distance between the galvanometer 4022 and the plane of the measured sample 40, the scanning angle of the galvanometer 4022 and the field of view size are determined by the following formula:

[0091] d<z;

[0092] d=R-R×cos(θ / 2);

[0093] z=πω 2 / λ;

[0094] FOV=R×tanθ;

[0095] Wherein, d is the distance between the field of view edge A of the focal plane and the center B of the focal plane, R is the distance between the galvanometer 4022 and the measured sample 40, θ is the scanning angle of the galvanometer 4022, z is the Rayleigh distance of the focused laser at the measured sample 40, ω is the beam waist radius, λ is the wavelength of the laser, and FOV is the field of view size.

[0096] In some embodiments of the application, the focusing lens 4021 is movable, and the movement of the focusing lens 4021 is determined according to the height of the surface of the measured sample 40, and the movement of the focusing lens 4021 makes the focal point of the focusing lens 4021 on the surface of the measured sample 40.

[0097] In detail, the movement of the focusing lens 4021 includes the movement direction and the movement distance of the focusing lens 4021, wherein the movement direction includes the direction away from the galvanometer 4022 and the direction close to the galvanometer 4022.

[0098] The focal length of the focusing lens 4021 is the distance from the focusing lens 4021 to the focal point on the set surface of the measured sample 40, and the determination of the movement of the focusing lens 4021 according to the height of the surface of the measured sample 40 includes: measuring the distance between the surface of the measured sample 40 at the position of the focal point and the detector 4032; calculating the difference between the distance and the set distance; if the difference is greater than 0, controlling the focusing lens 4021 to move away from the galvanometer 4022 by the difference distance; if the difference is less than 0, controlling the focusing lens 4021 to move close to the galvanometer 4022 by the difference distance; and if the difference is equal to 0, the focusing lens 4021 does not need to be controlled to move.

[0099] In a specific implementation, the distance is, for example, the distance between the surface of the sample 40 at the highest point of the sample 40 and the detector 4032, or the distance between the surface of the sample 40 at the lowest point of the sample 40 and the detector 4032.

[0100] The focusing lens 4021 can be automatically controlled by an automatic control device, which is an intelligent control device, including but not limited to an existing computer device.

[0101] The embodiment can automatically adjust the laser focusing point by the mobility of the focusing lens, effectively correct the focal plane to a plane, and enable the signal detection in a plane.

[0102] In some embodiments of the application, the number of single lenses 40311 in the compound eye collection structure 4031 can be determined according to the intensity of the light signal generated by the sample 40 actually detected by the detector 4032. In a specific implementation, the number of single lenses 40311 is greater than 3, the lens diameter is less than 30 mm, and the numerical aperture is greater than 0.7. The single lens selected in the embodiment can effectively collect signals and improve the signal intensity of large field imaging.

[0103] The numerical aperture of the single lens for collecting signals is small, and the compound eye collection structure composed of multiple lenses effectively increases the numerical aperture and improves the signal collection intensity.

[0104] In a specific implementation, on the basis of sufficient signal intensity, different single lenses 40311 in the compound eye collection structure 4031 can correspond to different imaging channels, thereby realizing multi-modal. The number of single lenses 40311 in the compound eye collection structure 4031 can also be determined according to the number of multi-modal imaging channels. For example, a compound eye collection structure with 7 lenses, 3 lenses for collecting second harmonic waves, and 4 lenses for collecting CARS signals, thereby realizing double-modal signal collection. Specifically, filters can be arranged in the incident direction or the outgoing light direction of the compound eye collection structure, thereby realizing the detection of different nonlinear optical signals. Each filter is used to screen different wavelengths of light, thereby realizing the collection of different types of signals, thereby realizing multi-color imaging.

[0105] In a specific implementation, the object focal length, the image focal length, and the size of the detection chip need to satisfy the following relationships:

[0106] (1) 1 / u + 1 / v = 1 / f;

[0107] (2) u / v is greater than the ratio of the imaging field size to the size of the detection chip;

[0108] Wherein, f is the focal length of the single lens 40311, u is the distance between the single lens 40311 and the plane of the measured sample 40, and v is the distance between the detector 4032 and the single lens 40311, wherein the distances of u and v are shown in Fig. 2.

[0109] In some embodiments, u / v>4.

[0110] By determining the distance between the detector and the single lens and the distance between the single lens and the measured sample through the above relationship, the light signal generated by the measured sample (i.e. the focal plane) can be focused on the detector, and the focused light of the laser during the galvanometer scanning can be scanned on the focal plane to generate a signal, and the projection of the signal can also change in position. The above scheme ensures that the signal does not exceed the detection range of the detector.

[0111] In some embodiments of the application, the gap between the single lenses 40311 in the compound eye collection structure 4031 ranges from 0 to 3 mm.

[0112] The distance between the single lens 40311 and the plane of the measured sample 40 ranges from 100 to 150 mm.

[0113] In some embodiments of the application, the detector 4032 includes a plurality of optical filters, a plurality of multi-mode fiber bundles, and a plurality of photodetection modules.

[0114] The optical filter is used to filter the light signal collected by the single lens 40311 of the compound eye collection structure 4031. In specific implementation, the optical filter is located before the single lens 40311 and / or the optical filter is located after the single lens 40311. In general, in order to achieve better filtering effect, the filter is located before and after the single lens 40311.

[0115] The input end of the multi-mode fiber bundle is used to collect the light signal collected by the single lens 40311 of the compound eye collection structure 4031, and the output end of the multi-mode fiber bundle is connected to the photodetection module.

[0116] Specifically, the photodetection module includes a detection chip, which can be selected according to actual needs, and the application does not make specific limitations thereon.

[0117] In this embodiment, the heat dissipation of the detector is considered, and the detector is placed away from the nonlinear optical large field imaging system, and the multi-mode fiber bundle is used to transmit the light signal, so that the influence of poor heat dissipation of the detector on the measurement result can be avoided.

[0118] The nonlinear optical large field-of-view imaging system provided by the application can be applied to large field-of-view imaging of coherent anti-Stokes Raman scattering, coherent Stokes Raman scattering, second harmonic, third harmonic, sum frequency, two-photon fluorescence, multi-photon fluorescence, single-photon fluorescence, spontaneous Raman, and short-wave infrared Raman.

[0119] When the application is applied to large field-of-view imaging of coherent anti-Stokes Raman scattering (CARS), the light source is used to generate double-channel coherent pulse laser, and the double-channel coherent pulse laser is used to excite molecular vibration in a sample to be measured to generate a CARS signal. By analyzing the intensity and frequency of the CARS signal, vibration information of molecules in the sample to be measured can be obtained. In some specific embodiments, the light source system satisfies the following conditions:

[0120] The double-channel laser output has a central wavelength of 1032 nm and 792 nm, and the two laser beams are phase-stable; the pulse width of the two beams is 300 fs (150-600 fs); and a low-repetition frequency femtosecond laser is used to improve the nonlinear efficiency.

[0121] When the application is applied to large field-of-view imaging of second harmonic, the light source is used to generate monochromatic and coherent femtosecond pulse laser (the above-mentioned 1032 nm pulse laser can be used as the light source), and the laser beam is used to interact with molecules in a sample to be measured, so that the sample to be measured generates a second harmonic signal with a frequency of twice the frequency of the excitation light. According to the image generated by the second harmonic signal, the structure and function of biomolecules in the sample to be measured, such as collagen, etc., can be analyzed.

[0122] When the application is applied to large field-of-view imaging of third harmonic, the light source is used to generate femtosecond pulse laser (the above-mentioned 1032 nm pulse laser can be used as the light source), and the laser pulse is used to interact with a sample to be measured in a nonlinear manner, so that the sample to be measured generates a third harmonic signal with a frequency of three times the frequency of the excitation light. The third harmonic signal corresponds to non-centrosymmetric molecules or structures present in the sample. According to the image generated by the third harmonic signal, specific biomolecular information of the sample to be measured, such as lipid distribution, protein aggregation, etc., can be analyzed.

[0123] When the application is applied to large field-of-view imaging of two-photon fluorescence, the light source is used to generate near-infrared femtosecond pulse laser (the above-mentioned 1032 nm pulse laser can be used as the light source), and the near-infrared laser is used to interact with a sample to be measured in a nonlinear manner, so that the sample to be measured generates fluorescence. According to the image generated by the fluorescence, two-dimensional microstructures of the sample to be measured can be analyzed, and in some cases, specific tissue structures or molecules can be imaged by targeting the fluorescence label.

[0124] The application is applied to the large field of view imaging of multi-photon fluorescence, and a light source is used to generate near-infrared femtosecond pulse laser (the above-mentioned 1032 nm pulse laser can be used as the light source), and the near-infrared laser is used to interact with the measured sample to generate two-photon and three-photon fluorescence effects of the measured sample. The internal structure of the measured sample is analyzed according to the images generated by the two-photon and three-photon.

[0125] The application is applied to the large field of view imaging of spontaneous Raman, and a light source is used to generate continuous laser of specific wavelength or photon energy, and the laser of specific wavelength or photon energy is inelastically scattered when being incident on molecules of a measured sample. The spectrum is analyzed according to the wavelength components of the scattered light, and then the chemical bond information of the measured sample is analyzed by generating images.

[0126] In an example of the application, a nonlinear optical large field of view CARS imaging system is designed according to the above technical solution. Specifically, the CARS imaging system uses pump light power of 55 mW for imaging, the Stokes light power is 130 mW, the number of pixel points for imaging is 3600*3600 pixel points, and the integration time of each pixel point is 20 μs.

[0127] When the CARS imaging system is applied to biological tissue imaging, the cell morphology and fiber state of the biological tissue can be quickly obtained. As shown in FIG. 7, a piece of 2*2 cm pork tissue is imaged by using the CARS imaging system, the imaging optical resolution is 25 μm, and the CARS imaging result of the whole pork tissue is obtained in about five minutes, and the imaging field of view size is 3*3 cm. In the method of combining the existing nonlinear optical microscopic imaging with the puzzle algorithm to realize the large field of view imaging, if the above-mentioned 2*2 cm pork tissue is imaged, at least ten hours are needed to complete the imaging, while the present application only needs five minutes. It can be seen that the present application can improve the efficiency of large field of view imaging.

[0128] Specifically, the left large image in FIG. 7 is a biological tissue imaging image, and the two small images pointed by the arrows are local enlarged images of the biological tissue imaging image. The upper right enlarged image corresponds to the protein fiber in the pork tissue, that is, the lean meat area, and the main signal source is the protein signal in the lean meat tissue. The lower right enlarged image corresponds to the lipid droplet in the pork tissue, that is, the fat meat area, and the main signal source is the oil signal in the fat meat tissue. It can be observed from the biological tissue imaging image shown in FIG. 7 that the morphology and signal intensity of the oil region are different. It can be known from the biological tissue imaging image and the enlarged images that the large field of view imaging image obtained by the present application can observe the obvious lean meat and fat meat tissue, and the local fat cells can be observed. The fiber morphology in the lean meat tissue can be observed by the upper right enlarged image in FIG. 7, and the fat cell morphology in the fat meat tissue can be observed by the lower right enlarged image in FIG. 7.

[0129] The nonlinear optical large field-of-view imaging provided by the application can be applied to surgical navigation, for example, a large field-of-view imaging scene of a tumor surgery on a lesion, in addition to being applied to imaging of biological tissues. The application can overcome the low efficiency limitation of the existing tumor boundary distinguishing through H&E staining, immunohistochemical and molecular pathological methods, and gene sequencing methods, and provide new guidance for surgical operation, facilitating efficient and accurate resection of the lesion.

[0130] In addition, in the existing tumor surgery, a surgeon often identifies the tumor tissue boundary through eyesight and touch. This kind of tumor position determination method has the problems of strong subjectivity and high error rate. During the surgery, the surgeon first cuts the edge part of the cancerous tissue that they think, and then sends the part of the tissue to the intraoperative rapid pathological section. This process often needs more than half an hour to obtain an analysis result. If the section result is cancerous tissue, the surgeon will choose to continue to resect a part, and if it is normal tissue, the surgery can be ended. Whether the cancer in other areas is completely cut is unknown. Therefore, this kind of surgical method has the problems of poor precision and long time consumption.

[0131] The nonlinear optical large field-of-view imaging system provided by the application can be applied to surgical navigation. The nonlinear optical large field-of-view imaging system of the application can quickly obtain a large range (3*3 cm) of image results in a few minutes, and the cancerous region is determined through analysis of the collected single-mode signal or multi-mode signal, so as to assist the surgeon to more quickly and accurately identify the cancerous region. Compared with the subjective identification of the surgeon, the surgical navigation using the application can improve the accuracy of tumor region identification. In addition, since the working distance of the focusing lens of the nonlinear optical imaging system of the application is long, there is enough space for the surgeon to perform the operation when the system is applied, and the surgeon is not disturbed. In summary, the system can effectively improve the surgical efficiency and reduce the postoperative tumor recurrence rate.

[0132] In an embodiment of the application, a laser scanning system is provided, which comprises a focusing lens and a galvanometer. The galvanometer is located between the focusing lens and the measured sample. The focusing lens is used to focus the laser emitted by the light source system to the measured sample. The galvanometer vibrates according to the preset scanning angle to realize the scanning of the laser focal point on the measured sample.

[0133] In detail, the preset scanning angle of the galvanometer can be determined according to the distance between the galvanometer and the measured sample. The specific angle of the application is not limited.

[0134] The laser scanning system provided by the embodiment can be applied to a nonlinear optical large field imaging system, and can also be applied to laser medical and beauty, laser marking and other scenes. When applied to the nonlinear optical large field imaging system, the imaging range of laser scanning can be significantly expanded, and the laser beam can always propagate along the optical axis of the focusing lens during laser scanning, eliminating various lens distortions, so that off-axis aberration does not occur. At the same time, the use of lenses can be reduced, the complexity of the device can be reduced, and the manufacturing cost of the device can be saved.

[0135] In some embodiments, the focal length of the focusing lens ranges from 100 mm to 250 mm. The focusing lens with the focusing range can make the focal spot size range from 15 μm to 40 μm, which can ensure the instantaneous power density at the laser focal point, so as to obtain as high a signal as possible and have a sufficient working distance without affecting the imaging area.

[0136] In some embodiments, the distance between the focusing lens and the galvanometer is within 20 mm.

[0137] In some embodiments, the distance between the field of view edge of the focal plane and the center of the focal plane is less than the Rayleigh distance of the focused laser at the measured sample. In detail, the distance between the field of view edge of the focal plane and the center of the focal plane can be determined by the distance between the galvanometer and the focal plane and the scanning angle of the galvanometer.

[0138] By limiting the relationship between the distance between the field of view edge of the focal plane and the center of the focal plane and the Rayleigh distance of the focused laser at the measured sample, the embodiment can limit the relationship between the distance between the galvanometer and the focal plane and the Rayleigh range, so that the imaging signal is as much as possible in the plane, and compared with the 4f system scanning imaging, the embodiment can make the imaging not have off-axis aberration.

[0139] In some embodiments, the distance between the galvanometer and the measured sample plane, the scanning angle of the galvanometer and the field of view size are determined by the following formula:

[0140] d<z;

[0141] d=R-R×cos(θ / 2);

[0142] z=πω 2 / λ;

[0143] FOV=R×tanθ;

[0144] wherein d is the distance between the field of view edge of the focal plane and the center of the focal plane, R is the distance between the galvanometer and the measured sample, θ is the scanning angle of the galvanometer, z is the Rayleigh distance of the focused laser at the measured sample, ω is the beam waist radius, λ is the laser wavelength, and FOV is the field of view size.

[0145] In some embodiments, the focusing lens is movable, and the movement of the focusing lens is determined according to the height of the surface of the sample to be measured, so that the focal point of the focusing lens is on the surface of the sample to be measured after the movement of the focusing lens.

[0146] The focal length of the focusing lens is the distance from the lens to the focal point on the surface of the sample to be measured, and the movement of the focusing lens determined according to the height of the surface of the sample to be measured includes: measuring the distance between the surface of the sample to be measured at the position of the focal point and the detector; calculating the difference between the distance and the set distance; if the difference is greater than 0, controlling the focusing lens to move away from the galvanometer by the difference; if the difference is less than 0, controlling the focusing lens to move close to the galvanometer by the difference; and if the difference is equal to 0, the focusing lens does not need to be controlled to move.

[0147] In a specific implementation, the set distance is the distance between the surface of the sample to be measured at the position of the highest point of the sample to be measured and the detector, or the distance between the surface of the sample to be measured at the position of the lowest point of the sample to be measured and the detector.

[0148] The embodiment can realize automatic adjustment of the focal point of the laser by the movability of the focusing lens, effectively correct the focal plane to a plane, and enable the signal detection to be in one plane.

[0149] In some embodiments of the application, a laser detection system is also provided, which includes an ommatidium type collection structure and a detector. The ommatidium type collection structure includes a plurality of single lenses arranged closely, and the light signal generated by the sample to be measured is incident on the ommatidium type collection structure. The detector is used to detect the light signal collected by the ommatidium type collection structure.

[0150] The laser detection system provided by the embodiment can be applied to a nonlinear optical large field of view imaging system, and can also be applied to a multi-channel wide field fluorescence scene.

[0151] The embodiment can increase the detection numerical aperture and comprehensively collect the signals excited in the entire scanning field of view by designing the ommatidium type collection structure including a plurality of single lenses arranged closely, and improve the signal strength of the large field of view imaging.

[0152] In some embodiments, the diameter of the single lens in the ommatidium type collection structure is less than 30 mm, and the numerical aperture is greater than 0.7. The single lens selected in the embodiment can effectively collect signals and improve the accuracy of imaging.

[0153] In some embodiments, the gap between the single lenses in the ommatidium type collection structure is in the range of 0-3 mm.

[0154] The distance between the single lens and the plane of the sample to be measured is in the range of 100-150 mm.

[0155] In some embodiments, the object side focal length, the image side focal length, and the size of the detection chip also need to satisfy the following relationship:

[0156] (1) 1 / u + 1 / v = 1 / f;

[0157] (2) u / v is greater than the ratio of the imaging field of view to the size of the detector chip;

[0158] Where f is the focal length of a single lens, u is the distance between a single lens and the plane of the sample being measured, and v is the distance between the detector and the single lens.

[0159] In some specific implementations, u / v > 4.

[0160] By determining the distance between the detector and a single lens and the distance between the single lens and the sample under test through the above relationship, the light signal generated by the sample under test (i.e., the focal plane) can be focused on the detector. During the galvanometer scanning process, the laser-focused light will scan the focal plane to generate a signal, and the projection of the signal will also change position. The above scheme ensures that the signal will not exceed the detection range of the detector.

[0161] In some embodiments, the multiple lenses in the compound eye collection structure are closely arranged, including multiple lenses arranged in a planar manner.

[0162] In some embodiments, the multiple lenses in the compound-eye collection structure are closely arranged, including a circular arrangement of the lenses with an arc-shaped bottom surface. Specifically, the optical axis of a single lens in the compound-eye collection structure points to the center of the scanning area of ​​the sample being measured. Compared to a planar arrangement, the lens arrangement in this embodiment of the compound-eye collection structure ensures that the signal intensity acquired by each channel is basically consistent, improving the signal scanning intensity in the edge scanning area and thus improving the accuracy of the detector.

[0163] In some specific implementations, the detector includes multiple filters, multiple multimode fiber bundles, and multiple photoelectric detection modules.

[0164] The filter is used to filter the light signal collected by the individual lens of the compound eye collection structure. In practice, the filter is located before and / or after the individual lens.

[0165] The input end of the multimode fiber bundle is used to collect the optical signal collected by a single lens of the compound eye collection structure, and the output end of the multimode fiber bundle is connected to the photoelectric detection module.

[0166] In practice, the detector can be selected according to actual needs, and this invention does not impose any specific limitations on it.

[0167] In this embodiment, considering the heat dissipation of the detector, the detector is placed far away from the nonlinear optical large field-of-view imaging system, and a multimode fiber bundle is used to transmit the optical signal, which can avoid the poor heat dissipation effect of the detector affecting the measurement results.

[0168] It should also be understood that, in the embodiments of the application, the term "and / or" merely describes an associated relationship with the associated objects, and indicates that there can be three relationships. For example, A and / or B can represent three cases: A exists alone, A and B exist together, and B exists alone. In addition, the character " / " in the application generally represents an "or" relationship between the preceding and following associated objects.

[0169] The principles and implementation manners of the application are described by using specific embodiments in the application. The above embodiment descriptions are only used to help understand the method of the application and its core idea. Meanwhile, for those skilled in the art, according to the idea of the application, the specific implementation manners and application ranges will be changed. In summary, the content of the specification should not be understood as a limitation on the application.

Claims

1. A nonlinear optical large field-of-view imaging system, characterized in that, The system comprises a light source system, a laser scanning system and a laser detection system. The light source system is configured to emit laser light. The laser scanning system comprises a focusing lens and a galvanometer, the galvanometer is located between the focusing lens and a sample to be measured, the focusing lens is configured to focus the laser light on the sample to be measured, and the galvanometer vibrates at a preset scanning angle to realize scanning of a laser focal point on the sample to be measured. The laser detection system comprises an eye-shaped collection structure and a detector, the eye-shaped collection structure comprises a plurality of single lenses arranged closely, and light signals generated by the sample to be measured are incident on the eye-shaped collection structure; and the detector is configured to detect the light signals collected by the eye-shaped collection structure.

2. The nonlinear optical large field-of-view imaging system of claim 1, wherein, The eye-shaped collection structure is located between the galvanometer and the sample to be measured.

3. The nonlinear optical large field-of-view imaging system of claim 1, wherein, Further comprising: a dichroic mirror; The dichroic mirror is located between the eye-shaped collection structure and the sample to be measured, and is configured to reflect the focused laser light reflected by the galvanometer to the sample to be measured, and transmit the light signals passing through the sample to be measured to the eye-shaped collection structure.

4. The nonlinear optical large field-of-view imaging system of claim 1, wherein, The focusing lens is movable, and the movement of the focusing lens is determined according to the height of the surface of the sample to be measured.

5. The nonlinear optical large field-of-view imaging system of claim 1, wherein, The optical axis of the single lens of the eye-shaped collection structure points to the center of the scanning area of the sample to be measured.

6. The nonlinear optical large field-of-view imaging system of claim 1, wherein, The detector comprises a plurality of filters, a plurality of multimode fiber bundles and a plurality of photodetector modules. The filters are configured to filter the light signals collected by the single lenses. The input ends of the multimode fiber bundles are configured to collect the light signals collected by the single lenses, and the output ends of the multimode fiber bundles are connected to the photodetector modules.

7. The nonlinear optical large field-of-view imaging system of claim 1, wherein, The nonlinear optical large field-of-view imaging system is applied to coherent anti-Stokes Raman scattering, coherent Stokes Raman scattering, second harmonic, third harmonic, sum frequency, two-photon fluorescence, multi-photon fluorescence, single-photon fluorescence, spontaneous Raman, and short-wave infrared Raman large field-of-view imaging.

8. The nonlinear optical large field-of-view imaging system of claim 1, wherein, The light source system is capable of outputting two coherent femtosecond laser pulses, and the central wavelength energy difference of the two beams of light matches the molecular bond vibration energy of the sample to be measured.

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