Straight-view-field three-dimensional chromatography microscopic imaging device and application thereof

Abstract

The invention discloses a squint field three-dimensional chromatography microscopic imaging device and application thereof. The apparatus comprises: an illumination system for providing mode illumination light having an initial mode illumination surface; the imaging system is used for detecting the fed-back signal light; the light path system is used for projecting the mode illumination light and feeding back and transmitting the signal light, and the initial mode illumination surface forms an inclined excitation surface after passing through the light path system; and the scanning system is used for driving to generate scanning relative motion between the inclined excitation surface and the sample to be observed. According to the invention, optical tomography is carried out on the inclined field of view by means of mode illumination, and three-dimensional scanning is realized by driving the lateral displacement of the inclined field of view relative to an imaging sample; the optical tomography three-dimensional microscopic imaging can be efficiently and quickly implemented without zooming or axially mechanically moving the objective lens or the sample to be observed, the volume imaging rate is remarkably improved, the system design is simplified, the system stability and robustness are enhanced, and an innovative solution is added to the field of three-dimensional microscopic imaging.

Description

Technical Field

[0001] The present invention relates to the technical field of optical three-dimensional microscopic imaging, and in particular to an oblique field three-dimensional tomographic microscopic imaging device and applications thereof. Background Art

[0002] In vivo optical microscopy aims to non-invasively observe (sub)cellular structures in living tissues or model animals and track their functional dynamics. It has wide applications in both life sciences and clinical diagnosis and treatment.

[0003] Compared to thin samples such as ex vivo tissue sections or adherent cultured cells, imaging of living tissues has two major characteristics and also presents technical challenges: first, the need for three-dimensional resolution (also known as optical tomography or depth tomography) to directly explore thick samples without mechanical sectioning; second, sufficient four-dimensional spatiotemporal resolution (three-dimensional space × one-dimensional time) is required to capture dynamic functional information of living organisms. Therefore, a fast three-dimensional volume imaging system is an indispensable tool.

[0004] Optical sectioning is broadly defined as any method that removes signals outside the focal plane. In recent years, the most widely used sectioning techniques include confocal laser scanning microscopy (CLSM), two-photon laser scanning microscopy (2PLSM), light sheet microscopy (LSFM), and structured illumination microscopy (SIM).

[0005] 1) Both laser scanning confocal microscopy and two-photon laser scanning microscopy use point scanning for imaging, resulting in slow imaging speeds and limited three-dimensional volume rates, making them unsuitable for rapid in vivo imaging. To increase the imaging rate, multi-point scanning confocal, line scanning confocal, and spinning disk confocal technologies have been developed based on point scanning confocal microscopy. Although these technologies significantly increase the imaging rate compared to point scanning confocal microscopy, their volumetric imaging rate remains limited.

[0006] 2) Light sheet microscopy (also known as selective plane illumination microscopy) uses two vertically arranged objectives, one of which is responsible for generating the light sheet to excite the sample, and the other is responsible for collecting the excited fluorescence signal. However, the sample space provided by the two vertically placed objectives is limited, resulting in the need for specialized customization of the sample chamber, which limits the types of samples that can be observed. To address the spatial limitation problem, oblique plane microscopy (OPM) or confocal oblique light sheet scanning (SCAPE) microscopy uses the same objective to simultaneously generate an oblique illumination light sheet and collect backscattered fluorescence. However, such a design requires the objective to have a high aperture angle (or numerical aperture, NA). High NA objectives often have a small field of view, making them difficult to apply to large-scale three-dimensional rapid imaging scenarios.

[0007] 3) Optical sectioning structured illumination microscopy (OS-SIM) is a method of defocusing based on the reconstruction of three stripes with different phases. It is very sensitive to the slight movement of the sample, and the method of synthesizing three images into one image greatly reduces the effective frame rate. Subsequently, the HiLo method was invented, which can achieve better defocusing effect by processing a wide-field image and a structured illumination image, effectively improving the effective frame rate. However, the application of OS-SIM or HiLo methods to three-dimensional volume imaging often requires mechanically moving the stage or objective lens. The imaging speed is limited by the inertia, response and stabilization time of the mechanical motion. The final volume imaging rate is often slow and cannot meet the needs of fast in vivo imaging.

[0008] In summary, developing a microscope system capable of rapid volumetric imaging to quickly capture the real-time dynamics of living biological tissues is of great significance for both basic life sciences and clinical diagnosis and treatment applications, which is why the present invention is developed. Summary of the Invention

[0009] In view of the shortcomings of the prior art, the present invention aims to provide an oblique field three-dimensional tomographic microscopy device and its application.

[0010] To achieve the aforementioned object of the invention, the technical solutions adopted by the present invention include:

[0011] In a first aspect, the present invention provides an oblique field three-dimensional tomographic microscopy imaging device, comprising:

[0012] An illumination system for providing patterned illumination light, wherein the patterned illumination light has an initial patterned illumination surface;

[0013] An imaging system is used to detect the signal light fed back after the patterned illumination light is irradiated on the sample to be observed;

[0014] an optical path system for projecting the pattern illumination light onto the sample to be observed and feeding back the signal light to the imaging system, wherein the initial pattern illumination surface forms an oblique excitation surface after passing through the optical path system, and the normal direction of the oblique excitation surface is inclined at an angle to the optical axis of the light projected onto the sample to be observed;

[0015] and a scanning system for driving the oblique excitation surface and the sample to be observed to cause a scanning relative motion, wherein the scanning direction has a component in the normal direction of the oblique excitation surface.

[0016] In a second aspect, the present invention further provides a method for three-dimensional tomographic microscopy with an oblique field of view, comprising:

[0017] Placing the sample to be observed at the position of the oblique excitation surface of the above-mentioned oblique field three-dimensional tomographic microscopy imaging device;

[0018] Driving a scanning relative motion between the oblique excitation surface and the sample to be observed, wherein the scanning direction has a component in the normal direction of the oblique excitation surface;

[0019] The imaging system is continuously used to detect the feedback signal light to obtain a three-dimensional tomographic microscopic image.

[0020] Based on the above technical solution, compared with the prior art, the beneficial effects of the present invention include at least:

[0021] The present invention proposes a universal method and apparatus for three-dimensional tomographic microscopy with an oblique field of view based on patterned illumination. Patterned illumination is used to perform optical tomographic imaging of the oblique field of view, and three-dimensional scanning is achieved by driving the lateral displacement of the oblique field of view relative to the imaging sample. The provided imaging method can efficiently and quickly implement optical tomographic three-dimensional microscopy without the need for zooming or axial mechanical movement of the objective lens or the sample to be observed, significantly improving the volumetric imaging rate, simplifying the system design, and enhancing the system stability and robustness, thus providing an innovative solution for the field of three-dimensional microscopy.

[0022] In addition, the present invention uses an oblique excitation surface for imaging, and its inclination angle is not limited by the aperture angle of the main objective lens. Therefore, it is compatible with the use of a low-NA, large-field-of-view main objective lens, is more suitable for large-scale rapid three-dimensional volume imaging scenarios, and also helps to reduce system costs.

[0023] The above description is only an overview of the technical solution of the present invention. In order to enable those skilled in the art to more clearly understand the technical means of this application and implement them according to the contents of the specification, the following is an explanation of the preferred embodiments of the present invention with detailed drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 A schematic structural diagram of an oblique field three-dimensional tomographic microscopy imaging system provided for some typical implementation cases of the present invention;

[0025] Figure 2 Schematic diagram of the system structure in which the pattern illumination surface generation module adopts a (programmable) array light-emitting device when the pattern illumination and oblique field detection provided in Example 1 of the present invention adopt a discrete optical path design;

[0026] Figure 3 A schematic diagram of the system structure in which the pattern illumination surface generation module adopts an array of light-transmitting devices when the pattern illumination and oblique field detection provided in Example 2 of the present invention adopt a discrete optical path design;

[0027] Figure 4 A schematic diagram of the system structure in which the pattern illumination surface generation module adopts a programmable light intensity modulation device when the pattern illumination and oblique field detection provided in Example 3 of the present invention adopt a discrete optical path design;

[0028] Figure 5 A schematic diagram of a system structure in which a pattern illumination surface generation module uses a phase modulation device when the pattern illumination and oblique field detection provided in the first embodiment of the present invention adopt a discrete optical path design is provided for the fourth embodiment of the present invention;

[0029] Figure 6 A schematic diagram of the system structure in which a pattern illumination surface generation module is introduced into an arrayed waveguide device when a discrete optical path design is adopted for pattern illumination and oblique field detection provided in Example 5 of the present invention;

[0030] Figure 7 Schematic diagram of the system structure in which the pattern illumination surface generation module introduces an arrayed waveguide device with a beveled (exit) end face when the pattern illumination and oblique field detection provided in Example 6 of the present invention adopt a discrete optical path design;

[0031] Figure 8a A schematic diagram of a system structure in which a relay module is added after the initial pattern illumination surface when the pattern illumination and oblique field detection provided in Example 7 of the present invention adopt a discrete optical path design;

[0032] Figure 8b A schematic diagram of another system structure in which a relay module is added after the initial pattern illumination surface when the pattern illumination and oblique field detection provided in Example 7 of the present invention adopt a discrete optical path design;

[0033] Figure 9 A schematic diagram of the system structure when the relay module and the objective lens are coaxial when the pattern illumination and oblique field detection provided in Example 7 of the present invention adopt a discrete optical path design;

[0034] Figure 10 A schematic diagram of the system structure of an array detector using a slant field detection module provided in Example 8 of the present invention;

[0035] Figure 11 A schematic diagram of the system structure of an arrayed waveguide device introduced into the slant field detection module provided in Example 9 of the present invention;

[0036] Figure 12 A schematic diagram of the system structure of an arrayed waveguide device with a beveled (exit) end face introduced into the oblique field detection module provided in Example 9 of the present invention;

[0037] Figure 13 A schematic diagram of the system structure after a relay module is added before the slant field detection module provided in Example 10 of the present invention;

[0038] Figure 14a Another schematic diagram of a system structure when the relay module and the objective lens are tilted is provided in Example 10 of the present invention;

[0039] Figure 14b A schematic diagram of the system structure when the relay module and the objective lens are coaxial, provided in Example 10 of the present invention;

[0040] Figure 15 A schematic diagram of the system structure for generating a secondary pattern illumination surface using an objective lens in a composite optical path for pattern illumination surface generation and oblique field detection provided in Example 11 of the present invention;

[0041] Figure 16a A schematic diagram of another system structure in which the objective lens for generating a secondary pattern illumination surface and the receiving objective lens are tilted in the composite optical path for pattern illumination surface generation and oblique field detection provided in Example 11 of the present invention;

[0042] Figure 16b A schematic diagram of the system structure when the objective lens for generating a secondary pattern illumination surface and the receiving objective lens in the composite optical path of pattern illumination surface generation and oblique field detection provided in Example 11 of the present invention are coaxial;

[0043] Figure 17 A schematic diagram of the system structure for generating a secondary mode illumination surface by introducing an arrayed waveguide device into a composite optical path for mode illumination surface generation and oblique field detection provided in Example 12 of the present invention;

[0044] Figure 18 A schematic diagram of the system structure showing a method for improving the transmittance of illumination light and back-scattered signal light by adding a transmission grating to a composite optical path for pattern illumination surface generation and oblique field detection provided in Example 13 of the present invention;

[0045] Figure 19 A schematic diagram of the system structure of an arrayed waveguide device having a beveled (exit) end facet introduced into a composite optical path for pattern illumination surface generation and oblique field detection provided in Example 14 of the present invention;

[0046] Figure 20 A schematic diagram of the system structure in which pattern illumination and oblique field detection share a composite optical path in a reflective imaging application provided by Example 15 of the present invention;

[0047] Figure 21 A schematic diagram of the system structure when pattern illumination and oblique field detection adopt a discrete optical path design in a reflective imaging application provided by Example 16 of the present invention;

[0048] Figure 22 A schematic diagram of the working principle of the galvanometer scanning mirror provided in Example 16 of the present invention;

[0049] Figure 23 A schematic diagram of the system structure in which pattern illumination and oblique field detection respectively use different reflective surfaces of the scanning prism when the main scanning device provided in Example 17 of the present invention adopts a polygonal scanning prism. DETAILED DESCRIPTION

[0050] In view of the shortcomings of the prior art, the inventors of this case, after long-term research and extensive practice, have proposed the technical solution of the present invention. The following will further explain this technical solution, its implementation process and principles.

[0051] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Therefore, the scope of protection of the present invention is not limited to the specific embodiments disclosed below.

[0052] Moreover, relational terms such as “first” and “second” are merely used to distinguish one component or method step from another with the same name, but do not necessarily require or imply any actual relationship or order between these components or method steps.

[0053] An embodiment of the present invention provides an oblique field three-dimensional tomographic microscopy imaging device, comprising:

[0054] An illumination system for providing patterned illumination light, wherein the patterned illumination light has an initial patterned illumination surface;

[0055] An imaging system is used to detect the signal light fed back after the patterned illumination light is irradiated on the sample to be observed;

[0056] an optical path system for projecting the pattern illumination light onto the sample to be observed and feeding back the signal light to the imaging system, wherein the initial pattern illumination surface forms an oblique excitation surface after passing through the optical path system, and the normal direction of the oblique excitation surface is inclined at an angle to the optical axis of the light projected onto the sample to be observed;

[0057] and a scanning system for driving the oblique excitation surface and the sample to be observed to cause a scanning relative motion, wherein the scanning direction has a component in the normal direction of the oblique excitation surface.

[0058] Specifically regarding the structure and connection relationship of each system, in some embodiments, the lighting system includes a light source and a pattern lighting surface generating module arranged in sequence along the lighting light path; the light source is used to provide original lighting light, and the pattern lighting surface generating module is used to convert the original lighting light into the pattern lighting light.

[0059] Or in some embodiments, the light source and the pattern lighting surface generating module are integrated.

[0060] More specifically, in some embodiments, when the light source and the pattern illumination surface generating module are separately arranged, the light source includes any one or a combination of two or more of a laser light source, a light emitting diode, a supercontinuum light source, and a coherent or incoherent illumination light source transmitted through an optical fiber, and the pattern illumination surface generating module includes any one or a combination of two or more of a fiber light cone, an optical fiber panel, an image transmission optical fiber bundle, an optical fiber array, an optical fiber image guide tube, a light emitting diode array, a digital micromirror array, a spatial light modulator, a diffraction optical element, a microlens array, a microcylindrical lens array, an acousto-optic modulator, and an electro-optic modulator.

[0061] Or in some embodiments, when the light source and the pattern lighting surface generating module are integrated, the lighting system is a programmable array light emitting device.

[0062] Of course, the above-mentioned specific contents about the lighting system are also exemplary. If other alternative functional structures are used to achieve the same function of providing patterned lighting light, it is also within the feasible scope of the present invention.

[0063] Regarding the composition and structure of the imaging system, in some embodiments, the imaging system includes an oblique field detection module, which includes an optically coordinated waveguide imaging device, an image amplification optical path, a filter, and an array detector; the array detector includes any one of a charge coupled device, a complementary metal oxide semiconductor, a single photon avalanche diode array, a photomultiplier tube array, and a silicon photomultiplier tube array.

[0064] Similarly, the specific composition and structure of the imaging system are not limited to the scope of the above examples.

[0065] Regarding the specific optical path, in some embodiments, the optical path system includes a main objective lens and a beam splitter; the pattern illumination light is transmitted to the main objective lens along the illumination system optical path through the first channel of the beam splitter, and is focused by the main objective lens and then projected to form the oblique excitation surface; the signal light is transmitted to the beam splitter after passing through the main objective lens along the imaging system optical path, and is transmitted to the imaging system through the second channel of the beam splitter.

[0066] Specifically, the optical path of the illumination system described in the present invention refers to the complete optical path from the light source through a series of optical elements to the sample to be observed, which includes many sub-optical paths. The parallel, inclined, or angled terms described below are relative to the optical axis of the corresponding sub-optical path. Similarly, the optical path of the imaging system refers to the complete route from the signal light fed back from the object to be observed to the signal light received by the detector, and does not specifically refer to a single straight optical path or a specific direction.

[0067] In some embodiments, the scanning system includes a main scanning device, which is disposed between the beam splitter and the main objective lens. Both the illumination system optical path and the imaging system optical path pass through the main scanning device. In this mode, a scanning mode can be achieved in which the optical path is scanned while the sample to be observed is stationary.

[0068] As for how to couple the illumination system and the imaging system, in some embodiments, the optical path system further includes a first objective lens and a second objective lens, wherein the first objective lens is disposed between the illumination system and the beam splitter, and the second objective lens is disposed between the imaging system and the beam splitter.

[0069] Or in some embodiments, the optical path system further includes a third objective lens, which is disposed in the optical path between the beam splitter and the main objective lens, and the illumination system and the imaging system share the third objective lens.

[0070] In some embodiments, when a light field redistribution device is present in the optical path system, the illumination system and the imaging system share the light field redistribution device.

[0071] More specifically, in some embodiments, when the illumination system and the imaging system share the first objective lens and the second objective lens:

[0072] The optical axis of the first objective lens is arranged parallel and coaxially with the optical path of the illumination system, and the normal direction of the initial mode illumination surface is arranged at an angle to the optical axis of the optical path of the illumination system, or the optical axis of the first objective lens is arranged at an angle to the optical path of the illumination system, and the normal direction of the initial mode illumination surface is arranged parallel to the optical axis of the optical path of the illumination system, or the optical axis of the first objective lens and the optical path of the illumination system are arranged at an angle of a first angle, and the normal direction of the initial mode illumination surface is arranged at an angle of a second angle to the optical axis of the optical path of the illumination system, and the values ​​of the first angle and the second angle are independent of each other;

[0073] Or in some embodiments, the optical axis of the second objective lens is arranged parallel and coaxially with the optical path of the imaging system, and the normal of the imaging plane of the imaging system is arranged obliquely with the optical axis of the optical path of the imaging system, or the optical axis of the second objective lens is arranged obliquely with the optical path of the imaging system, and the normal of the imaging plane of the imaging system is arranged parallel to the optical axis of the optical path of the imaging system, or the optical axis of the second objective lens is arranged obliquely with the optical path of the imaging system at a third angle, and the normal of the imaging plane of the imaging system is arranged obliquely with the optical axis of the optical path of the imaging system at a fourth angle, and the values ​​of the third angle and the fourth angle are independent of each other;

[0074] In some embodiments, when the illumination system and the imaging system share the third objective lens:

[0075] The optical axis of the third objective lens is tilted relative to the optical axis of the second objective lens, and the normal direction of the initial mode illumination plane / imaging plane of the illumination system and the imaging system is parallel to the optical axis of the third objective lens. Alternatively, the optical axis of the third objective lens is tilted relative to the optical path of the illumination system and the optical path of the imaging system at a fifth angle, and the normal direction of the initial mode illumination plane / imaging plane of the illumination system and the imaging system is tilted relative to the optical axis of the third objective lens at a sixth angle, and the values ​​of the fifth angle and the sixth angle are independent of each other.

[0076] Or in some embodiments, the optical axis of the third objective lens is coaxially parallel to the optical axis of the second objective lens, and the normal of the initial mode illumination surface / imaging surface of the illumination system and imaging system is tilted to the optical axis of the third objective lens.

[0077] In addition, an embodiment of the present invention further provides another scanning mode, that is, in some embodiments, the scanning system includes a scanning sample stage having a moving axis, and the axial direction of the moving axis has a component in the normal direction of the oblique excitation surface.

[0078] In this mode, a scanning mode can be realized in which the optical path is stationary and the sample to be observed is active; of course, the embodiments of the present invention do not exclude the use of the two in combination, that is, the optical path and the sample to be observed can move at the same time, and the specific form of movement is based on the ability to perform relative scanning movement.

[0079] In some embodiments, the optical path system includes a relay module, which is used to achieve optical relay and coupling of the illumination system, imaging system, and scanning system. Generally, the beam splitter and various lenses described above should fall under the concept of the relay module. Of course, the relay module is mainly used to construct the optical path and achieve coupling. It may also include some necessary structures such as reflectors, filters, light shields, and base structures. However, this is not a key factor of the present invention and can be adaptively configured by those skilled in the art.

[0080] For some typical examples, see Figure 1 Some specific implementation examples of the present invention provide an oblique field of view three-dimensional rapid volume imaging system comprising the following modules:

[0081] Light source module: can use (including but not limited to) laser light source, light emitting diode, supercontinuum light source, coherent or incoherent illumination light source transmitted through optical fiber, etc.

[0082] Pattern illumination surface generation module: This module is used to control or adjust the light emitted by the light source module to form an initial pattern illumination surface with a specific illumination pattern. It then utilizes optical principles and optomechanical techniques to generate an initial (pattern illumination) oblique excitation surface. Devices that can be used to generate pattern illumination surfaces include (but are not limited to): fiber tapers, fiber faceplates, imaging fiber bundles, fiber arrays, fiber-optic image conduits, light-emitting diode arrays, digital micromirror arrays (DMDs), spatial light modulators (SLMs), diffractive optical elements (DOEs), micro-lens arrays (MLAs), and micro-cylindrical lens arrays.

[0083] Main objective lens module: Contains the main objective lens used to directly face the sample, generate an oblique excitation surface of the sample, and collect backward signal light.

[0084] Main Scanner: This device controls the position and scanning motion of the excitation light at the sample end and descans the collected signal, thereby forming a stationary intermediate image in front of the collection module. This scanner is preferably a galvanometer scanner with a sufficiently large aperture. Other options include (but are not limited to) micro-electro-mechanical system (MEMS) scanners, rotating wedge prism pairs, and polygon scanners.

[0085] Oblique field detection module: This module is used to receive the static intermediate image formed after the (backward) signal light is scanned by the main scanning device. It generally includes a waveguide image transmission device, an image amplification optical path, an array detector, and a filter. Array detectors include (but are not limited to) charge-coupled devices (CCDs), complementary metal oxide semiconductors (CMOSs), single photon avalanche diodes (SPADs) or SPAD arrays, photomultipliers (PMTs) or PMT arrays, and silicon photomultipliers (SiPMs) or SiPM arrays.

[0086] Relay Optical Path Module: This module is used to achieve optical relay and coupling between system modules or key components, such as the pattern illumination surface generation module, main scanning device, main objective lens module, and oblique field detection module. The relay optical path module preferably uses a 4f system with appropriate magnification.

[0087] The above embodiment belongs to the oblique field scanning mode, that is, the sample to be observed is stationary, and the main scanning device is used to change the position of the oblique field illumination light on the sample and drive the scanning motion of the oblique field illumination light.

[0088] In addition, the deformable embodiment of the present invention also provides a sample scanning mode, that is, the oblique field illumination remains stationary, the sample to be observed can be placed on a stage or other device that can move the sample, and volume imaging is achieved by moving the sample position. At this time, the main scanning device can be replaced by optical elements such as reflectors.

[0089] Based on the above technical solution, a second aspect of an embodiment of the present invention further provides an application method of the above device, namely, a method for three-dimensional tomographic microscopy with an oblique field of view, which comprises the following steps:

[0090] Placing the sample to be observed at the position of the oblique excitation surface of the oblique field three-dimensional tomographic microscopy imaging device provided in any of the above embodiments;

[0091] Driving a scanning relative motion between the oblique excitation surface and the sample to be observed, wherein the scanning direction has a component in the normal direction of the oblique excitation surface;

[0092] The imaging system is continuously used to detect the feedback signal light to obtain a three-dimensional tomographic microscopic image.

[0093] The technical solution of the present invention is further described in detail below through several embodiments and in conjunction with the accompanying drawings. However, the selected embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0094] Since the main scanning device simultaneously realizes the scanning of the oblique excitation field and descanning of the back signal light (which can be fluorescence, reflected or scattered light, etc.), the principles and core optical paths of the above two types of scenarios in terms of oblique field illumination and oblique field (tomography) detection are the same. For the sake of simplicity, unless otherwise specified, the present invention will generally not explicitly distinguish and explain these two types of application scenarios when describing various embodiments. However, it should be emphasized that for all embodiments, it is only necessary to keep the main scanning device stationary, or replace it with an ordinary reflector, or directly remove it from the optical path, so that the oblique field scanning mode can be switched to the sample scanning mode; conversely, the scanning device and supporting optical elements can be introduced at the appropriate position of the optical path of the embodiment shown to realize forward illumination light scanning and back signal light descanning, which can be applied to the volume imaging scene where the sample is stationary. The above simple modifications aimed at switching imaging scenes or application modes are all within the scope of protection of the patent of this invention.

[0095] The principle diagram of the following multiple embodiments is as follows Figure 1 As shown, it includes a pattern illumination surface generation module, a relay optical path module, a main scanning device, a main objective lens module and an oblique field detection module. Due to space limitations, the embodiments of the present invention will focus on introducing several typical implementation schemes of the pattern illumination surface generation module or the oblique field detection module to help readers understand the principles and innovations of the present invention; according to the specific implementation schemes of the pattern illumination surface generation module and the oblique field detection module, many different system implementation schemes can be derived through permutations and combinations. Any combination of the introduced illumination and detection schemes is within the scope of protection of this patent.

[0096] [First Example - Separate Design of Illumination and Detection Optical Paths]

[0097] Example 1

[0098] In Example 1, the pattern lighting surface generating module adopts a (programmable) array light-emitting device, whose basic functional feature is that it can flexibly control and adjust the luminous intensity and / or light-emitting direction of different array element positions, thereby generating various required lighting patterns (such as arbitrary periodic or irregular dot arrays, line arrays, stripes, etc.); and then achieve dense sampling coverage of the target inclined illumination field by switching or rotating the light-emitting array elements, which belongs to the implementation method of the integrated setting of the light source and the pattern lighting surface generating module.

[0099] like Figure 2As shown, the array light-emitting device in this embodiment preferably adopts an organic light-emitting diode (OLED) array to achieve higher brightness and programmability. In the optical path, the light-emitting surface of the array light-emitting device is tilted at a certain angle to the focal plane of the first objective lens 233 to achieve an oblique field lighting effect. Specifically, the oblique field lighting is collected by the first objective lens 233 and then split by the first dichroic mirror 235 (as a beam splitter), and then reaches the main scanning device 22 after passing through the relay module composed of the fourth lens 232 and the third lens 231. After being reflected, it passes through the relay module composed of the second lens 213 and the first lens 212 and the main objective lens 211 to reach the sample end.

[0100] After the sample to be observed is irradiated, the back-directed signal light excited passes through the main objective lens 211, the relay module (the first lens 212 and the second lens 213), the main scanning device 22, the relay module (the third lens 231, the fourth lens 232, the first dichroic mirror 235, the second objective lens 234) to form a (tilted) static intermediate image plane, which is collected by the oblique field detection module 25. The imaging surface of the oblique field detection module 25 is also tilted at a corresponding angle. The specific tilt angle may be set to different angles based on clear imaging, which may be affected by different optical paths. Those skilled in the art are fully capable of adjusting the appropriate angle by themselves. Note Figure 2 The solid line and the dotted line are used to distinguish and mark the illumination and detection light paths corresponding to two object points (and corresponding image points) at different positions.

[0101] In corresponding alternative embodiments, the array light-emitting device may also adopt an array light source based on any light-emitting mechanism, such as a common light-emitting diode (LED) array, a super luminescent diode (SLD) array, a micro-LED array, or a laser diode (LD) array.

[0102] Example 2

[0103] In embodiment 2, the pattern illumination surface generation module adopts an array of light-transmitting devices, such as Figure 3 Its basic functional feature is to use a specific arrangement of light-transmitting pinholes to achieve a specific pattern of illumination (such as arbitrary periodic or irregular dot arrays, linear arrays, stripes, etc.); this device generally needs to be combined with an optomechanical actuator to change the spatial position of the transmitted light through movements such as translation, swinging, and rotation, thereby achieving dense sampling coverage of the target oblique illumination field.

[0104] The array light transmission device of this embodiment preferably adopts a cylindrical micro-lens array, which generates oblique field illumination by arranging cylindrical mirrors with different focal lengths. The subsequent remaining light paths are the same as those of the Figure 2 The relevant technical details are not repeated here. Figure 3 The illumination and detection light paths corresponding to two object points (and corresponding image points) at different positions are also marked using real and dotted lines.

[0105] In other alternative implementations, the array light transmission device can also use a Nipkow disk with an Archimedean spiral pinhole, a microlens array, a transmissive or reflective grating, or a mask or reflector with any specific transmission or reflection pattern to change the intensity distribution of the transmitted or reflected light. Depending on the application requirements, the mask or reflector can be combined with an actuator to change the pattern of the output light through translation, rotation, or rotation, thereby achieving oblique field illumination.

[0106] Example 3

[0107] In Example 3, the pattern illumination surface generating module adopts a programmable light intensity modulation device, whose basic functional feature is that it can interact with the incident light, control and adjust the amplitude or intensity of the outgoing light, thereby generating the required illumination pattern (such as arbitrary periodic or irregular dot matrix, line matrix, stripe, etc.); and then achieve dense sampling coverage of the target inclined illumination field of view by modulating the pattern of the outgoing light.

[0108] like Figure 4 As shown, the programmable light intensity control device in this embodiment preferably adopts a digital micromirror array (DMD), and the deflection angle of the DMD lens is usually ±12° (there are also other deflection angles such as ±10°, etc.). In this embodiment, the choice of the direction of the incident light source is related to the size of the tilt angle of the DMD relative to the optical axis. It is necessary to ensure that as much of the outgoing light as possible is collected by the first objective lens 233 to improve the transmittance. By selectively controlling the ±12° rotation of the micro-mirror (i.e., the opening and closing of the lens), any lighting mode can be achieved; and then by rotating or switching the micro-mirrors in different positions on the DMD, the required oblique lighting field is covered. The remaining subsequent optical paths and Figure 2 The relevant technical details are not repeated here. Figure 4 The illumination and detection light paths corresponding to two object points (and corresponding image points) at different positions are also marked using real and dotted lines.

[0109] In corresponding alternative embodiments, the DMD may also be replaced by an amplitude-type spatial light modulator, an acousto-optic modulator, an electro-optic modulator, etc., to achieve dynamic modulation of the amplitude or light intensity of the illumination light field.

[0110] Example 4

[0111] In Example 4, the pattern illumination surface generation module utilizes a phase modulation device. Its fundamental characteristic and function is to modulate the phase and / or wavefront of the outgoing light. This device, combined with a Fourier transform lens, then produces the desired illumination pattern on a specified oblique plane. Through mechanical displacement or electronic control, the applied phase modulation can be adjusted, thereby altering the resulting illumination pattern of the outgoing light, achieving dense sampling coverage of the target oblique illumination field of view.

[0112] like Figure 5 As shown, in this embodiment, the phase modulation device preferably uses a phase-type spatial light modulator. By adjusting the phase distribution of the reflected light and combining it with the fifth lens 241, the desired illumination pattern (such as any periodic or irregular dot matrix, line array, stripe, etc.) can be generated near the focal plane of the latter. Figure 2 The relevant technical details are not repeated here. Figure 5 The illumination and detection light paths corresponding to two object points (and corresponding image points) at different positions are also marked using real and dotted lines.

[0113] In alternative embodiments, the phase-type spatial light modulator can also be replaced by optomechanical elements such as a deformable mirror, an acousto-optic modulator, an electro-optic modulator, or optical elements such as a phase mask, a diffractive optical element (DOE), a metasurface, or a superlens (generally requiring a mechanical actuator to achieve dense sampling coverage of the target oblique excitation field).

[0114] Example 5

[0115] In Example 5, the pattern illumination surface generation module further introduces an arrayed waveguide device, whose basic functional feature is to transmit the illumination light field projected onto its incident end face to the exit end face, and appropriately change the physical properties of the illumination light field during the transmission process, such as applying isotropic or anisotropic size scaling, adjusting the light cone angle shape and direction of the exit light relative to the exit end face, etc., so as to better adapt to the application requirements.

[0116] like Figure 6 As shown, in this embodiment, the arrayed waveguide device preferably uses a fiber light taper. The advantage is that the different core spacings between the large and small ends of the fiber light taper are used to reduce the initial mode illumination pattern of the large end before entering the main optical path system, thereby achieving higher resolution (i.e., finer) light intensity modulation. The subsequent remaining optical paths are similar to Figure 2 The relevant technical details are not repeated here. Figure 6The illumination and detection light paths corresponding to two object points (and corresponding image points) at different positions are also marked using dashed lines. It should be noted that since the fiber light cone is tilted relative to the first objective lens 233, its outgoing light cone cannot be fully collected by the latter. That is, the aperture angles of the first objective lens 233 and subsequent objective lenses are not fully utilized. Figure 6 The optical path distribution characterizes this feature.

[0117] It should be emphasized that the initial pattern illumination projected onto the incident end face of the arrayed waveguide device can be generated by any device and method mentioned in Examples 1-4, including but not limited to (programmable) array light-emitting devices such as organic light-emitting diodes, ordinary light-emitting diodes, superluminescent diode arrays, cylindrical microlens arrays, Nipkow turntables, transmission or reflection gratings, and other array light-transmitting devices (and the required mechanical actuators), digital micromirror arrays (DMDs), amplitude-type spatial light modulators, acousto-optic modulators, and other programmable light intensity modulation devices, phase-type spatial light modulators, diffraction optical elements (DOEs), deformable mirrors, and other phase modulation devices (and the required mechanical actuators).

[0118] In alternative embodiments, the fiber optic taper may be replaced by other types of imaging optical waveguide devices such as a fiber optic faceplate, an imaging fiber bundle, a fiber array, or a fiber-optic image conduit.

[0119] Example 6

[0120] In Example 6, the pattern illumination surface generating module further adopts an arrayed waveguide device with a beveled (exit) end face, whose function is to use the principle of refraction to adjust the direction of the exit light cone relative to the normal of the exit end face, so that it is more biased towards the optical axis of the subsequent optical path, thereby improving the transmittance of the illumination light, the effective numerical aperture and spatial resolution of the system, etc.

[0121] like Figure 7As shown, in this embodiment, the arrayed waveguide device preferably uses an optical fiber faceplate to transmit the initial mode illumination from the incident end face to the exit end face and enter the main optical path system. As previously described, the initial mode illumination projected onto the incident end face of the arrayed waveguide device can be generated by any device and in any manner mentioned in Examples 1-4, including but not limited to (programmable) array light-emitting devices such as organic light-emitting diodes, ordinary light-emitting diodes, and superluminescent diode arrays; array light-transmitting devices such as cylindrical microlens arrays, Nipkow disks, and transmission or reflection gratings (and the required mechanical actuators); programmable light intensity modulation devices such as digital micromirror arrays (DMDs), amplitude-type spatial light modulators, and acousto-optic modulators; and phase modulation devices such as phase-type spatial light modulators, diffractive optical elements (DOEs), and deformable mirrors (and the required mechanical actuators). Figure 7 The rest of the subsequent optical paths and Figure 2 The relevant technical details are not repeated here. Figure 7 The illumination and detection light paths corresponding to two object points (and corresponding image points) at different positions are also marked using real and dotted lines.

[0122] In alternative embodiments, the beveled end fiber panel can also be replaced by other types of beveled end imaging optical waveguide devices such as a beveled end fiber light cone (the scaling ratio from the input to the output end can be flexibly selected), a beveled end fiber-optic image conduit, and a beveled end imaging fiber bungle.

[0123] In this embodiment, the beveled end face is preferably planar beveled. In alternative embodiments, other non-planar end face shapes can be reasonably selected according to the application scenario to better overcome the influence of system aberrations.

[0124] Example 7

[0125] The aforementioned pattern illumination surface generation module can introduce any one or more 4f or relay imaging systems to further modulate the illumination light path and optimize the size, fineness and orientation of the pattern illumination surface.

[0126] like Figure 8a As shown, a 4f system of arbitrary magnification (composed of a fifth lens 241 and a sixth lens 242, used for optical path relay) can be introduced between the initial mode illumination surface and the first objective lens 233 to further modulate the physical properties of the secondary mode illumination surface and the final oblique excitation surface. The initial mode illumination surface can be generated by any of the methods described in Examples 1-6. Figure 8a The illumination and detection light paths of two object points (and corresponding image points) at different positions are also marked using the dashed lines and the real lines.

[0127] In an alternative embodiment, if Figure 8b As shown, when the optical axis of the relay imaging system is tilted with respect to the optical axis of the first objective lens 233 , the normal direction of the initial mode illumination surface can be tilted with respect to the optical axis of the 4f system.

[0128] In alternative embodiments, the relay imaging system may also have other mounting orientations, such as Figure 9 As shown, the relay optical path formed by the fifth lens 241 and the sixth lens 242 can be coaxial with the first objective lens 233 to generate a reduced (or enlarged) secondary pattern illumination surface with a reduced (or increased) tilt, which then enters the main imaging optical path, ultimately modulating the oblique excitation field incident on the imaging sample. The initial pattern illumination surface can be generated by any of the methods described in Examples 1-6. Figure 9 The illumination and detection light paths corresponding to two object points (and corresponding image points) at different positions are also marked using real and dotted lines.

[0129] It is necessary to explain in general with respect to this type of embodiments (including the above-mentioned embodiments 1-7 and alternative embodiments) that there are multiple options for the position where the illumination light path enters the system (i.e., the position of the dichroic mirror in the light path), and is not limited to the light path structure shown in the existing drawings.

[0130] In addition to the aforementioned combinations, the various implementation schemes of the pattern lighting surface generating modules shown in the above embodiments can also have many other combinations. For example, an array light-emitting device (such as OLED) and a light intensity modulation device (such as DMD) can be used in combination to achieve more flexible lighting control or more complex lighting patterns; a light intensity modulation device (such as DMD) and a phase modulation device can also be used in combination, or the same device can be used to simultaneously adjust the amplitude and phase distribution of the output light to achieve more precise lighting control. Due to space limitations, this patent will no longer elaborate on the possible permutations and combinations of pattern lighting surface generating modules (or generation methods); however, these potential permutations and combinations are within the scope of protection of this patent.

[0131] The following several embodiments will focus on demonstrating the implementation of the oblique field detection module.

[0132] In the aforementioned embodiments 1 to 7 and their alternative embodiments, the oblique field detection module may be implemented in any of the following ways.

[0133] Example 8

[0134] In Example 8, the oblique field detection module uses an array detector, whose basic function is to convert optical signals into digital signals and finally record them as discrete digital images.

[0135] like Figure 10As shown, the receiving plane of the array detector is placed on the static intermediate image plane formed by the back-facing signal light, thereby directly capturing the intermediate image. Figure 10 The illumination and detection light paths corresponding to two object points (and corresponding image points) at different positions are also marked using real and dotted lines.

[0136] In this embodiment, the array detector preferably adopts a CCD or CMOS detector. In alternative embodiments, any other type of array detector such as a PMT array, an APD array, a SiPM array, or InGaS (near infrared) may also be used.

[0137] In this embodiment and alternative embodiments, a filter may be installed in front of the array detector to selectively filter out required wavelengths and improve the signal-to-noise ratio or signal-to-background ratio of imaging.

[0138] In this embodiment and alternative embodiments, a rolling shutter mode may be used to implement a virtual detection slit to better suppress the influence of the out-of-focus background.

[0139] Example 9

[0140] In this embodiment, the oblique field detection module introduces an arrayed waveguide device, whose basic functional feature is that it can transmit the signal light projected onto the input end face to the output end face, and appropriately change the physical properties of the illumination light (such as isotropic or anisotropic size scaling, the light cone angle shape and direction of the output light, etc.) during the transmission process to better meet the needs of oblique field detection.

[0141] like Figure 11 As shown, the back-directed signal light forms a stationary intermediate image plane in the focal area of ​​the second objective lens 234, and the incident end face of the arrayed waveguide device is placed to coincide with the stationary intermediate image plane, so that the returned back-directed signal light directly enters the arrayed waveguide device and is transmitted to its output end face, and then passes through the seventh lens 251 and the tube lens 252 to reach the array detector and be received and recorded by it. Figure 11 The (forward) illumination and (backward) detection light paths of an object point (and the corresponding image point) are marked with solid lines.

[0142] In this embodiment, the waveguide image transmission device preferably adopts an optical fiber panel or a fiber light cone; in alternative embodiments, an image transmission optical fiber bundle, an optical fiber array, an optical fiber image guide tube, etc. may also be used.

[0143] In an alternative embodiment, the arrayed waveguide device further adopts an arrayed waveguide device with a beveled incident end face, such as Figure 12As shown, according to the principle of refraction, by changing the bevel angle of the end face, the orientation of the incident light cone angle relative to the end face normal can be adjusted, so that the received light cone angle and the aperture angle of the second objective lens 234 have a higher degree of overlap, thereby improving the collection efficiency of the back-directed signal light. Arrayed waveguide devices with beveled end faces preferably use fiber panels. In addition, other types of waveguide imaging devices such as fiber light cones, imaging fiber bundles, fiber arrays, and fiber image guide tubes can also be used. In alternative embodiments, arrayed waveguide devices can also have other non-planar end face shapes to better meet imaging requirements and compensate for system aberrations.

[0144] Example 10

[0145] One or more 4f or relay optical systems can be introduced into the oblique field detection module to further modulate the detection optical path and optimize the size, precision and orientation of the detection optical path. Figure 13 As shown, a relay optical path consisting of a seventh lens 251 and a tube lens 252 is added between the intermediate stationary image plane and the detection module to modulate the detection optical path. Note that the portion of the light cone that can enter the detection module through the relay optical path is shaded after the second objective lens 234 in this figure.

[0146] In an alternative embodiment, if Figure 14a As shown, the detection module is placed at an angle to the optical axis of the relay system.

[0147] In alternative embodiments, the relay imaging system may have other orientations. Figure 14b As shown, the introduced relay optical path composed of the seventh lens 251 and the tube lens 252 is coaxial with the second objective lens 234; at this time, the detection module may need to be placed at an angle accordingly.

[0148] It needs to be further explained and emphasized again that the various implementation schemes of the pattern illumination surface generating module described in Examples 1-7 (and their alternative embodiments) and the various implementation schemes of the oblique field detection module described in Examples 8-10 (and their alternative embodiments) can be arbitrarily combined; all possible combinations are included in the scope of protection of the patent of this invention.

[0149] [Second embodiment - a composite module for pattern illumination surface generation and oblique field detection based on core device reuse]

[0150] In the aforementioned first type of embodiment, the main optical paths of the pattern illumination surface generating module and the oblique field detection module are independent of each other, and are only merged into the main imaging optical path through a dichroic mirror or other types of spectroscopic devices (such as the spectroscopic prism of Example 16).

[0151] In the second category of embodiments of the present invention, the pattern illumination surface generation module and the oblique field detection module have a higher degree of overlap and reuse, meaning that the optical paths of the two modules share more components. In particular, one or more core components can be reused for both pattern illumination surface generation and oblique field detection. The core advantage of this combined pattern illumination surface generation and oblique field detection module lies in a simpler optical path. In many cases, the pattern illumination surface generation module and oblique field detection module are combined into one, enabling convenient self-calibration and improving the stability and robustness of the entire system.

[0152] Example 11

[0153] In Example 11, a more integrated optical system is used to simultaneously generate the pattern illumination surface and perform oblique field detection. Figure 15 As shown, this embodiment preferentially employs an inclined third objective lens 261 for generating a secondary pattern illumination surface and collecting backlight signal light. The pattern illumination light generated by the pattern illumination surface generation module passes through the eighth lens 264, the second dichroic mirror 262, and the third objective lens 261 to form a secondary pattern illumination surface (which spatially overlaps with the subsequent intermediate static image surface). The pattern illumination light is received by the first objective lens 233, then passes through the relay module composed of the fourth lens 232 and the third lens 231 to reach the main scanning device 22. After being reflected, it passes through the relay module composed of the second lens 213 and the first lens 212 and the main objective lens 211 to reach the sample end, forming the final (pattern illumination) oblique excitation surface. The backlight signal light generated by the sample excitation returns along the original optical path, forms an intermediate static image surface by the first objective lens 233, is then collected by the inclined third objective lens 261, passes through the second dichroic mirror 262 and the tube lens 263, and reaches the array detector, where it is detected and recorded. The pattern lighting surface generating module may adopt any generating method described in embodiments 1-7 (and their alternative embodiments), and the relevant technical details are not repeated here. Figure 15 The solid lines in the figure mark the illumination and detection light paths of an example object point (and the corresponding image point).

[0154] In an alternative embodiment, when the third objective lens 261 and the first objective lens 233 are placed at an angle, the pattern illumination surface generating module and the array detector can also be placed at an angle, such as Figure 16a shown.

[0155] In an alternative embodiment, the third objective lens 261 and the first objective lens 233 can also be placed coaxially, and the pattern illumination surface generating module and the array detector can be placed tilted accordingly, such as Figure 16b shown.

[0156] Example 12

[0157] In Example 12, the mode illumination surface generation module and the oblique field detection integrated module further introduce a multiplexed array waveguide device. Figure 17 As shown, the arrayed waveguide device preferentially utilizes a fiber light cone, utilizing its smaller end to have a larger aperture angle (relative to the larger end) to increase the degree of overlap between the (forward) output light cone angle or (backward) detection aperture angle of the integrated module and the aperture angle of the first objective lens 233. Specifically, the pattern illumination light generated by the pattern illumination surface generation module passes through the eighth lens 264, the second dichroic mirror 262, and the third objective lens 261, and is mapped onto the rear end face of the fiber light cone. It then propagates through the device to the front end face, forming a secondary pattern illumination surface. Light emitted from the front end face is received by the first objective lens 233, then passes through the relay module composed of the fourth lens 232 and the third lens 231 to reach the main scanning device 22. After being reflected, it passes through the relay module composed of the second lens 213 and the first lens 212 to reach the main objective lens 211, and then reaches the sample end to form the final (pattern illumination) oblique excitation surface. The backscattered signal light generated by the sample excitation returns along the original optical path, forming an intermediate static image plane at the front end of the fiber light cone through the first objective lens 233. The light then enters the front end, propagates, and reaches the rear end of the fiber light cone. After exiting, it passes through the second dichroic mirror 262 and lens 263 before being imaged onto the array detector, where it is detected and recorded. The pattern illumination surface generation module can employ any of the generation methods described in Examples 1-7 (and their alternatives), and the relevant technical details are not further elaborated here. Figure 17 The solid lines in the figure mark the illumination and detection light paths of an example object point (and the corresponding image point).

[0158] In alternative embodiments, the arrayed waveguide device may also be other types of image-transmitting optical waveguide devices such as an optical fiber panel, an image-transmitting optical fiber bundle, an optical fiber array, or an optical fiber image guide tube.

[0159] In alternative embodiments, the arrayed waveguide device may also adopt other non-planar end face shapes to better compensate for system aberrations.

[0160] Example 13

[0161] In such Figure 18 In the embodiment 13 shown, a transmission grating is added to the focal plane of the third objective lens 261. By utilizing the deflection effect of its specific diffraction order on the incident light, the output light cone angle or the collection aperture angle of the third objective lens 261 is made to coincide more closely with the aperture angle (or light cone angle) of the first objective lens 233, thereby improving the transmission efficiency of the forward illumination light and the backward signal light. Figure 15 Similarly, the pattern lighting surface generating module can adopt any generating method described in embodiments 1-7 (and their alternative embodiments), and the relevant technical details are not repeated here. Figure 18 The solid lines in the figure mark the illumination and detection light paths of an example object point (and the corresponding image point), and the corresponding relationships between the three pairs of light before and after diffraction are marked with small numbers on both sides of the grating.

[0162] In an alternative embodiment, the transmission grating may be replaced by a reflective grating, and the optical path may be adjusted accordingly according to the grating type and parameters.

[0163] Example 14

[0164] In Example 14, the pattern illumination surface generation module and the detection composite module further use an arrayed waveguide device with a beveled end face. Its function is to use the principle of refraction to adjust the direction of the outgoing light cone relative to the normal line of the outgoing end face so that it is more consistent with the aperture angle of the subsequent optical path, thereby improving the transmittance of the illumination light and the collection rate of the back-scattered signal light. Figure 19 As shown, the beveled end face array waveguide imaging device preferably uses a beveled end face optical fiber panel. The pattern illumination surface generation module can adopt any generation method described in embodiments 1-7 (and their alternative embodiments), and the relevant technical details are not repeated here. Figure 19 The solid lines in the figure mark the illumination and detection light paths of an example object point (and the corresponding image point).

[0165] In alternative embodiments, the beveled end fiber panel can also be replaced by other types of beveled end array waveguide devices such as beveled end fiber light cones (the scaling ratio from input to output can be flexibly selected), beveled end fiber image guide tubes, beveled end image transmission fiber bundles, etc.

[0166] In alternative embodiments, the beveled end facet array waveguide device may also adopt other non-planar end face shapes to better compensate for system aberrations.

[0167] [Third embodiment - reflection imaging mode]

[0168] The aforementioned embodiments are mainly used for fluorescence imaging. When the back-reflected signal light is back-reflected or back-scattered light, embodiments 15-16 can be derived.

[0169] Example 15

[0170] like Figure 20 As shown, in this embodiment, the pattern illumination surface generation module and the oblique field detection module share the same optical path (the same structure as described in the second category of embodiments). Since the (stray) back-reflected light generated on the surface or inside of each device in the optical path will affect the imaging quality, a polarizer 272 (or polarizer), a polarization beam splitter prism 265 (PBS) and a quarter wave plate 271 are used in combination in the optical path to better isolate the (forward) illumination light and the back-reflecting signal light by utilizing the polarization principle. In this embodiment, the polarizer 272 is used as a polarizer and can be placed at any suitable position in the optical path before the polarization beam splitter prism 265 ( Figure 20It is only a schematic representation of the placement). The illumination light (mostly) passing through the polarizer 272 is reflected by the polarization beam splitter prism 265 and enters the third objective lens 261. The 1 / 4 wave plate 271 converts the linearly polarized excitation light into circularly polarized light, which is finally incident on the imaging sample through the main imaging light path; the backscattered or reflected light generated by the sample is still approximately circularly polarized light, but the rotation direction is opposite to that of the excitation light; after passing through the 1 / 4 wave plate 271, it is converted into linearly polarized light, and the polarization direction is perpendicular to the polarization direction of the forward illumination light, so that most of it can be transmitted through the polarization beam splitter prism 265. The 1 / 4 wave plate 271 can be placed in the light path at other suitable positions between the main objective lens 211 and the polarization beam splitter prism 265 ( Figure 20 (This is merely an illustration of one placement.) This design can, to a certain extent, suppress the impact of reflected light from various components in the optical path on imaging. The pattern illumination surface generation module can employ any of the generation methods described in Examples 1-7 (and their alternatives), and the relevant technical details are not further elaborated here.

[0171] In an alternative embodiment, if the illumination light emitted from the pattern illumination surface generating module is linearly polarized light, the polarizer 272 can be omitted (eg, the polarization direction meets the requirements) or replaced with a half-wave plate to adjust the polarization direction.

[0172] Example 16

[0173] This embodiment Figure 21 As shown. The pattern illumination surface generating module and the oblique field detection module adopt a discrete optical path design (the same structure as described in the first category of embodiments). In the application scenario of reflective imaging, the (stray) back-reflected light generated on the surface or inside of each device in the optical path structure has little effect on the imaging quality, so an ordinary beam splitter prism 236 (BS) can be used in the optical path to split the excitation light and the back-reflected signal light. The pattern illumination surface generating module can adopt any of the generation methods described in embodiments 1-7 (and their alternative embodiments), and the relevant technical details will not be repeated here. Figure 21 The illumination and detection light paths corresponding to two object points (and corresponding image points) at different positions are also marked using real and dotted lines.

[0174] In an alternative embodiment, to further reduce signal crosstalk, a polarization beam splitter (PBS), a polarizer, and a quarter wave plate as described in the previous embodiment may be used in combination to fully suppress the impact of back-reflected light from each device on imaging and improve the imaging signal-to-background ratio.

[0175] It should be noted that, as mentioned above, the above embodiments provided by the present invention can be used for sample scanning, and a scanning device can also be introduced to change the position of the oblique illumination light field within the imaging object, thereby achieving three-dimensional imaging. The main scanning device usually uses a galvanometer scanning mirror, such as Figure 22The figure below shows a schematic diagram of the working principle of a galvanometer scanning mirror. Note that the solid and dashed lines represent different scanning positions. The main scanning device can also be a microelectromechanical system (MEMS) scanner, a piezoelectric actuated scanner, a rotating wedge prism, a polygonal prism, or other options.

[0176] Example 17

[0177] This embodiment Figure 23 As shown, the main scanning device uses a regular polygonal scanning prism. The illumination and detection optical paths utilize different reflective surfaces of the polygonal scanning prism, respectively. The optical paths are separated from each other, sharing only the main objective lens and the associated relay optical path module. The polygonal scanning prism can operate in a back-and-forth oscillating manner or in a continuous or step-by-step rotation along a specific direction (clockwise or counterclockwise).

[0178] In this embodiment, the pattern illumination light generated by the pattern illumination surface generation module first strikes a reflective surface of the polygonal scanning prism 28. After reflection, it is relayed by the second lens 213 and the first lens 212 to the main objective lens 211, ultimately striking the imaging sample and forming the desired (pattern illumination) oblique excitation surface. The generated backlight signal light (fluorescence or reflected light) is received by the main objective lens 211, then relayed by the first lens 212 and the second lens 213 to reach another reflective surface of the polygonal scanning prism 27. After reflection, it passes through the third lens 231, the fourth lens 232, and the first objective lens 233 before being received and recorded by the detection module. The pattern illumination surface generation module may employ any of the embodiments described in Examples 1-7 (and corresponding alternative embodiments), and the detection module may employ any of the embodiments described in Examples 8-11 (and corresponding alternative embodiments). The relevant technical details are not further elaborated herein.

[0179] In this embodiment, the polygonal scanning prism 28 has six reflective surfaces. In alternative embodiments, the polygonal scanning prism can have any number of reflective surfaces.

[0180] In this embodiment, the illumination light path and the detection light path use two adjacent reflection surfaces of the polygonal scanning prism. In an alternative embodiment, the illumination light path and the detection light path can use any two (not necessarily adjacent) reflection surfaces of the polygonal scanning prism.

[0181] Finally, it is necessary to explain overall for all embodiments and alternative embodiments of the present invention:

[0182] 1) Each lens system in all the above embodiments may adopt a convex lens, a concave lens, a cylindrical lens, an off-axis parabolic reflector, or any other design familiar to those skilled in the relevant art.

[0183] 2) In all the above embodiments, any one or more relay optical paths or imaging optical paths (such as a 4f system) can be introduced at any position, and the 4f system involved can be composed of a convex lens, a concave lens, a cylindrical lens, or an off-axis parabolic reflector, or any other design familiar to technicians in the relevant field.

[0184] Based on the above embodiments, it is clear that the advantages of the technical solutions provided by the embodiments of the present invention are mainly reflected in:

[0185] (1) Compared with point scanning microscopy modes such as confocal laser scanning microscopy (CLSM) and two-photon laser scanning microscopy (2PLSM): The present invention adopts oblique field illumination to achieve multi-point parallel excitation and parallel optical tomography detection on the oblique excitation surface, significantly improving the imaging frame rate. More importantly, different positions of the oblique excitation surface correspond to different depths in the sample. By driving the oblique excitation surface to translate laterally through the main scanning device, three-dimensional volume imaging can be completed without zooming or axial mechanical movement of the objective lens and sample, resulting in an order of magnitude (10-100 times) improvement in the speed of three-dimensional volume imaging compared to point scanning microscopy modes such as confocal and two-photon laser scanning microscopy.

[0186] (2) Compared with the standard dual-objective light sheet microscope (SPIM): The dual-objective light sheet microscope requires the sample to be placed in the common focal area of ​​the illumination objective and the collection objective (usually placed perpendicular to the former), resulting in a small sample space. The present invention uses a single main objective to simultaneously achieve oblique field mode illumination (excitation) and back signal collection, providing an open sample space that is compatible with imaging samples of any size and shape (such as living animal models such as fruit flies, zebrafish, mice, macaques, organoids, microfluidic organ chips, in vivo or three-dimensional animals or human tissues, etc.), greatly expanding the scope of application and convenience;

[0187] (3) Compared with oblique light sheet microscopy (OPM) and confocal oblique light sheet scanning (SCAPE) microscopy, which are also based on a single main objective lens architecture: OPM and SCAPE usually require a main objective lens with a high numerical aperture (NA) (such as a water immersion objective lens with NA ≥ 0.8) to produce a sufficiently tilted illumination light sheet, thereby increasing system cost, and the imaging field of view of the high NA objective lens is correspondingly limited. The oblique excitation plane adopted by the present invention has an inclination angle that is not limited by the aperture angle of the main objective lens, so it is compatible with the use of a main objective lens with a low NA and a large field of view, is more suitable for large-scale rapid three-dimensional volume imaging scenarios, and also helps to reduce system costs;

[0188] (4) Compared with optical tomography structured light illumination microscopy (OS-SIM): OS-SIM generally uses three-phase stripe structured light illumination, while the present invention can adopt various different illumination and detection modes, and the system design is more flexible and highly scalable; and the present invention does not require zooming, axial mechanical movement of the objective lens or imaging sample, and can efficiently and quickly implement optical tomography three-dimensional microscopic imaging, significantly improving the volume imaging rate.

[0189] It should be understood that the above embodiments are merely illustrative of the technical concepts and features of the present invention. Their purpose is to enable those skilled in the art to understand the contents of the present invention and implement them accordingly. They are not intended to limit the scope of protection of the present invention. Any equivalent variations or modifications made in accordance with the spirit and substance of the present invention are intended to be encompassed within the scope of protection of the present invention.

Claims

1. A three-dimensional tomographic microscopy device with an oblique field of view, characterized in that: include: An illumination system for providing patterned illumination light, wherein the patterned illumination light has an initial patterned illumination surface; An imaging system, configured to detect signal light fed back after the patterned illumination light is irradiated on the sample to be observed; an optical path system for projecting the pattern illumination light onto the sample to be observed and feeding back the signal light to the imaging system, wherein the initial pattern illumination surface forms an oblique excitation surface after passing through the optical path system, and the normal direction of the oblique excitation surface is inclined at an angle to the optical axis of the light projected onto the sample to be observed; and a scanning system for driving the oblique excitation surface and the sample to be observed to cause a scanning relative motion, wherein the scanning direction has a component in the normal direction of the oblique excitation surface.

2. The oblique field three-dimensional tomographic microscopy imaging device according to claim 1, characterized in that: The lighting system includes a light source and a pattern lighting surface generating module arranged in sequence along the lighting light path; The light source is used to provide original illumination light, and the pattern illumination surface generating module is used to convert the original illumination light into the pattern illumination light; Alternatively, the light source and the pattern lighting surface generating module are integrated.

3. The oblique field three-dimensional tomographic microscopy imaging device according to claim 2, characterized in that: When the light source and the pattern illumination surface generating module are separately provided, the light source includes any one or a combination of two or more of a laser light source, a light emitting diode, a supercontinuum light source, and a coherent or incoherent illumination light source emitted through an optical fiber; and the pattern illumination surface generating module includes any one or a combination of two or more of a fiber light cone, an optical fiber panel, an image transmission optical fiber bundle, an optical fiber array, an optical fiber image guide tube, a light emitting diode array, a digital micromirror array, a spatial light modulator, a diffractive optical element, a microlens array, a microcylindrical lens array, an acousto-optic modulator, and an electro-optic modulator; Alternatively, when the light source and the pattern lighting surface generating module are integrated, the lighting system is a programmable array light emitting device; And or, the optical path system is further provided with a light field redistribution device, the light field redistribution device is used to modulate the physical properties of the patterned illumination light and or the signal light, the light field redistribution device includes at least any one of an arrayed waveguide device, a transmission grating, and a beveled arrayed waveguide device; And or, the imaging system includes an oblique field detection module, which includes a waveguide imaging device, an image amplification optical path, a filter and an array detector that are arranged in conjunction with each other; the array detector includes any one of a charge coupled device, a complementary metal oxide semiconductor, a single photon avalanche diode array, a photomultiplier tube array, and a silicon photomultiplier tube array.

4. The oblique field three-dimensional tomographic microscopy imaging device according to claim 1, characterized in that: The optical path system includes a main objective lens and a beam splitter; The pattern illumination light is transmitted along the optical path of the illumination system through the first channel of the beam splitter to the main objective lens, and is focused by the main objective lens and projected to form the oblique excitation surface; The signal light passes through the main objective lens along the optical path of the imaging system and is transmitted to the beam splitter, and is then transmitted to the imaging system through the second channel of the beam splitter.

5. The oblique field three-dimensional tomographic microscopy imaging device according to claim 4, characterized in that: The scanning system includes a main scanning device, which is arranged between the beam splitter and the main objective lens. The light path of the illumination system and the light path of the imaging system both pass through the main scanning device.

6. The oblique field three-dimensional tomographic microscopy imaging device according to claim 4, characterized in that: The optical path system further includes a first objective lens and a second objective lens, wherein the first objective lens is arranged between the illumination system and the beam splitter, and the second objective lens is arranged between the imaging system and the beam splitter; Alternatively, the optical path system further includes a third objective lens, the third objective lens is arranged in the optical path between the beam splitter and the main objective lens, and the illumination system and the imaging system share the third objective lens; And or, when a light field redistribution device exists in the optical path system, the illumination system and the imaging system share the light field redistribution device.

7. The oblique field three-dimensional tomographic microscopy imaging device according to claim 6, characterized in that: When the illumination system and the imaging system use the first objective lens and the second objective lens separately: The optical axis of the first objective lens is arranged parallel and coaxially with the optical path of the illumination system, and the normal direction of the initial mode illumination surface is arranged at an angle to the optical axis of the optical path of the illumination system, or the optical axis of the first objective lens is arranged at an angle to the optical path of the illumination system, and the normal direction of the initial mode illumination surface is arranged parallel to the optical axis of the optical path of the illumination system, or the optical axis of the first objective lens and the optical path of the illumination system are arranged at an angle of a first angle, and the normal direction of the initial mode illumination surface is arranged at an angle of a second angle to the optical axis of the optical path of the illumination system, and the values ​​of the first angle and the second angle are independent of each other; and or, the optical axis of the second objective lens and the optical path of the imaging system are arranged parallel and coaxially, and the normal of the imaging plane of the imaging system is arranged obliquely to the optical axis of the optical path of the imaging system; or, the optical axis of the second objective lens and the optical path of the imaging system are arranged obliquely, and the normal of the imaging plane of the imaging system is arranged parallel to the optical axis of the optical path of the imaging system; or, the optical axis of the second objective lens and the optical path of the imaging system are arranged obliquely at a third angle, and the normal of the imaging plane of the imaging system is arranged obliquely at a fourth angle to the optical axis of the optical path of the imaging system, and the values ​​of the third angle and the fourth angle are independent of each other; When the illumination system and the imaging system share the third objective lens: The optical axis of the third objective lens is tilted to the optical axis of the second objective lens, and the normal of the imaging surface of the initial mode illumination surface of the illumination system and the imaging system is arranged parallel to the optical axis of the third objective lens, or the optical axis of the third objective lens is tilted at a fifth angle to the optical path of the illumination system and the optical path of the imaging system, and the normal of the imaging surface of the initial mode illumination surface of the illumination system and the imaging system is tilted at a sixth angle to the optical axis of the third objective lens, and the values ​​of the fifth angle and the sixth angle are independent of each other.

8. Alternatively, the optical axis of the third objective lens is coaxially parallel to the optical axis of the second objective lens, and the normal direction of the initial mode illumination surface and imaging surface of the illumination system and the imaging system is tilted relative to the optical axis of the third objective lens.

9. The oblique field three-dimensional tomographic microscopy imaging device according to claim 1, characterized in that: The scanning system includes a scanning sample stage having a moving axis, wherein an axial direction of the moving axis has a component in a normal direction of the oblique excitation surface.

10. The oblique field three-dimensional tomographic microscopy imaging device according to claim 1, characterized in that: The optical path system includes a relay module, and the relay module is used to realize optical relay and coupling of the lighting system, imaging system and scanning system.

11. A method for three-dimensional tomographic microscopy with an oblique field of view, characterized in that: include: Placing the sample to be observed at the position of the oblique excitation surface of the oblique field three-dimensional tomographic microscopy imaging device according to any one of claims 1 to 9; Driving a scanning relative motion between the oblique excitation surface and the sample to be observed, wherein the scanning direction has a component in the normal direction of the oblique excitation surface; The imaging system is continuously used to detect the feedback signal light to obtain a three-dimensional tomographic microscopic image.

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