Arbitrary-curved-surface wide-field-of-view turntable confocal tomography microimaging system and method

Abstract

The invention discloses an arbitrary-curved-surface wide-field-of-view turntable confocal tomography microimaging system and method, and belongs to the technical field of optical microimaging, the system comprises an illumination light source and a detection module, and the light output side of the illumination light source is provided with an illumination adaptive modulation module; a first relay lens module is arranged on the light output side of the lighting module; the light output side of the first relay lens module is provided with a zoom pinhole turntable; a second relay lens module is arranged between the zoom pinhole turntable and the sample; a dichroscope is arranged between the zoom pinhole turntable and the first relay lens module; a third relay lens module is arranged between the dichroscope and the detection module; the illumination adaptive modulation module generates a dynamic illumination template according to pre-stored curved surface morphology data; matching the illumination area to the current focal plane imaging area through the first relay lens module and the second relay lens module; and the signal synchronous processing system controls zoom compensation working parameters of the illumination adaptive modulation module, the detection module and the zoom pinhole turntable.

Description

Technical Field

[0001] This application belongs to the field of optical microscopy imaging technology, and particularly relates to an arbitrary-curved-surface wide-field spinning disk confocal tomography microscopy imaging system and method. Background Art

[0002] As an important scientific tool, the microscopy imaging system plays a bridging role in connecting the macroscopic world and the microscopic world. Traditional microscopy imaging systems usually map a planar sample surface onto a planar detector, which requires the sample surface to be detected to have a very strict planar section. However, in nature, the vast majority of samples have complex curved surface shapes. Therefore, in order to observe under a microscope, it is often necessary to slice the sample. However, for those living samples, slicing may cause serious damage to the sample.

[0003] In the fields of biomedical imaging and industrial inspection, the demand for high-speed and high-signal-to-noise-ratio three-dimensional imaging of complex curved surface structures is becoming increasingly urgent. Traditional wide-field microscopy imaging technologies, such as fluorescence microscopes, although capable of achieving large-field-of-view observations, due to their spatial frequency domain aliasing effect, lead to a significant reduction in axial resolution and it is difficult to distinguish the tomography signals of overlapping structures. In recent years, a technology called curved surface selective imaging (such as patent CN20211190828.4) has been developed. It preliminarily realizes the rapid reconstruction of curved surface structures through the spatio-temporal modulation of a digital micromirror device (DMD). However, the wide-field detection mode adopted by this technology limits the signal-to-noise ratio (SNR) of the system. Especially in the imaging of living tissues, the scattered background noise will seriously interfere with the extraction of weak signals.

[0004] The spinning disk confocal microscopy technology significantly improves the imaging signal-to-noise ratio through a physical pinhole array, but its tomography ability depends on mechanical z-axis scanning. Due to the limitation of the response speed of the piezoelectric displacement stage (the typical scanning frequency is less than 10 Hz), it is difficult to meet the real-time requirements for observing dynamic processes. Although in recent years, there have been studies attempting to combine digital scanning technologies (such as light field microscopy or compressive sensing imaging) with it, these methods often require sacrificing spatial resolution or increasing computational complexity. For example, light field microscopy obtains three-dimensional information through single exposure, but its spatial resolution is limited by the sampling of the microlens array.

[0005] At present, the common challenges faced by microscopic imaging technology are mainly reflected in the following three aspects: First, the spatio-temporal bandwidth product limitation of wide-field imaging makes it difficult for us to simultaneously achieve a large field of view, high signal-to-noise ratio, and high-speed tomography. Second, the inertial constraint of mechanical scanning results in the fact that the focal plane switching speed of traditional confocal systems is difficult to break through the millisecond level. Finally, the contradiction between the adaptive imaging requirements of complex curved surfaces and the fixed detection mode leads to a large amount of invalid data acquisition (accounting for up to 40%) when imaging non-planar samples. Especially in scenarios such as in vivo cranial nerve imaging and dynamic monitoring of blood vessel networks, existing systems are difficult to achieve curved surface tracking on the second time scale while maintaining subcellular resolution. These technical bottlenecks severely restrict the application of microscopic imaging technology in dynamic biological systems and industrial on-line detection.

[0006] Application content

[0007] This application provides an arbitrary-curved-surface wide-field spinning disk confocal tomography microscopic imaging system and its imaging method, which realizes high-speed and high signal-to-noise ratio imaging of arbitrary curved surfaces by introducing a selective illumination adaptation modulation module and a zoom pinhole disk. The illumination adaptation modulation module can accurately illuminate different regions at different times, while the zoom pinhole disk realizes multi-layer imaging by dynamically adjusting the focal length and significantly improves the imaging signal-to-noise ratio by using the pinhole design. This innovative imaging system not only maintains high resolution and high sensitivity but also can achieve large field of view and high frame rate imaging, and is suitable for complex application scenarios such as time-series imaging of in vivo deep tissues.

[0008] An embodiment of the first aspect of this application provides an arbitrary-curved-surface wide-field spinning disk confocal tomography microscopic imaging system, including an illumination light source and a detection module;

[0009] A first relay lens module is provided on the light output side of the illumination module;

[0010] A zoom pinhole disk is provided on the light output side of the first relay lens module;

[0011] A second relay lens module is provided between the zoom pinhole disk and the sample;

[0012] A dichroic mirror is provided between the zoom pinhole disk and the first relay lens module;

[0013] A third relay lens module is provided between the dichroic mirror and the detection module;

[0014] The illumination adaptation modulation module generates a dynamic illumination template according to the pre-stored curved surface topography data; and matches the illumination area to the current focal plane imaging area through the first relay lens module and the second relay lens module; the three relay lens modules are used to achieve quadruple conjugation and vignetting compensation;

[0015] The signal synchronization processing system acquires the phase signal of the zoom pinhole turntable in real time, and synchronously controls the zoom compensation working parameters of the illumination module, the detection module and the zoom pinhole turntable according to the phase signal.

[0016] Preferably, the zoom pinhole turntable includes:

[0017] A pinhole component for realizing tomography at different positions on the focal plane;

[0018] A zoom component for changing the microscopic imaging focal plane; the zoom component integrates a stepped variable-thickness zoom glass and a conjugate pinhole array;

[0019] The zoom component and the pinhole component adopt a coaxial laminated structure to realize synchronous rotation scanning.

[0020] Preferably, the pinhole component includes a pinhole turntable and a flat glass zoom turntable; the flat glass zoom turntable switches the position of the pinhole turntable to the position corresponding to the focal plane;

[0021] The first synchronization device receives the timing signal of the signal synchronization processing system and sends the position coding signal of the focusing turntable to the detection module.

[0022] Preferably, the pinhole component includes a pinhole turntable and an optional focusing turntable; the binary array focusing turntable focuses the incident light energy on the light-transmitting position of the pinhole turntable;

[0023] The first synchronization device receives the timing signal of the signal synchronization processing system and sends the position coding signal of the pinhole turntable to the detection module.

[0024] Preferably, the illumination module includes:

[0025] An illumination light source, as the illumination light source of the imaging system, provides energy for the imaging system;

[0026] An illumination adaptation module for adapting the size and incident angle of the illumination light source to the detection numerical aperture;

[0027] An illumination adjustment module that illuminates the imaging area corresponding to the focusing position within the scanning period of the zoom pinhole turntable.

[0028] Preferably, the illumination adjustment module includes:

[0029] A spatio-temporal light field modulation device for modulating the illumination intensity and phase corresponding to the light field at different times;

[0030] A second synchronization device for receiving the timing signal of the signal synchronization processing system and outputting the current illumination state coding and / or detection state coding to the signal synchronization processing system.

[0031] Preferably, the relay lens module includes:

[0032] A relay lens for achieving optical conjugation among the illumination adaptation modulation module, the zoom pinhole turntable, and the imaging focal plane;

[0033] A vignetting lens for eliminating the change of the light angle and removing the vignetting generated by the cascaded relay lenses.

[0034] The second object of the present invention is to provide a confocal tomography microscopy imaging method for an arbitrary curved surface wide field turntable, which uses the above-mentioned confocal tomography microscopy imaging system for an arbitrary curved surface wide field turntable to complete the following steps:

[0035] S1. First, pre-scan and process the sample to obtain curved surface morphology data, where the curved surface morphology data includes the three-dimensional topography data of the sample and the curved surface topology mapping relationship; then calculate the illumination template sequence corresponding to each focal plane in combination with the pre-calibrated vignetting coefficient;

[0036] S2. Dynamic conjugate tomography scanning: Start the zoom pinhole turntable, and determine the current focal plane position in real time according to the turntable encoder signal; synchronously trigger the DMD to load the corresponding illumination template, and only activate the effective imaging area within the depth of field;

[0037] S3. Tomography signal acquisition and synchronous control: Within a single-frame exposure period, control the global exposure window of the detection module to match the phase of the zoom pinhole turntable through the signal synchronization processing system;

[0038] S4. Adopt a time-space multiplexing strategy to encode and stack the tomography signals of different focal planes into a single-frame image in sequence;

[0039] S5. Reconstruct the curved surface image.

[0040] Preferably, a fluorescence thin sheet is used to calibrate the vignetting coefficient of different imaging regions.

[0041] Preferably, S5 includes:

[0042] First, separate the tomography signals, and extract the signals of each focal plane from the stacked image according to the sequential encoding;

[0043] Then perform vignetting compensation, and correct the edge attenuation by applying the pre-calibrated light intensity distribution matrix;

[0044] Finally, perform curved surface fusion, combine the pre-stored three-dimensional topography data, reconstruct a continuous curved surface image, and a first relay lens module is provided on the light output side of the illumination module.

[0045] The advantages and positive effects of the present application are:

[0046] The present invention realizes high-speed and high signal-to-noise ratio imaging of arbitrary curved surfaces by introducing a selective illumination adaptation modulation module and a zoom pinhole turntable. The core of this innovative technology lies in the fact that the illumination adaptation modulation module can accurately illuminate different regions at different times, ensuring the accuracy and efficiency during the imaging process. At the same time, the zoom pinhole turntable can achieve multi-layer imaging by dynamically adjusting the focal length, and this process significantly improves the signal-to-noise ratio of the imaging. This innovative imaging system not only maintains high resolution and high sensitivity, but also can achieve large field of view and high frame rate imaging, and is applicable to complex application scenarios such as time-series imaging of living deep tissues, bringing great progress to the field of biomedical imaging.

[0047] The present invention can achieve quadruple conjugation among the illumination adaptation modulation surface, the zoom pinhole turntable surface, the sample surface, and the imaging surface during the curved surface imaging process; this quadruple conjugation technology ensures the accuracy and stability of the imaging system when dealing with complex curved surfaces, greatly improving the imaging quality.

[0048] The present invention uses a signal synchronization processing system to obtain the phase signal of the zoom pinhole turntable in real time, and synchronously controls the zoom compensation working parameters of the illumination adaptation modulation module, the detection module, and the zoom pinhole turntable according to the phase signal, and then can achieve triple synchronization of the tomography function, the zoom function, and the detection function; the application of this synchronization technology enables the imaging system to more flexibly adapt to different imaging requirements, greatly improving the imaging efficiency and quality.

[0049] The present invention uses a relay lens module to overcome the vignetting phenomenon of a multi-stage relay imaging system. The vignetting phenomenon is a common problem in multi-stage relay imaging systems, which will cause a decrease in the brightness and clarity of the imaging edge area. Through the relay lens module of the present invention, this problem can be effectively solved, ensuring uniform brightness and clarity throughout the imaging area, thereby improving the overall quality of the imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] The above-mentioned and / or additional aspects and advantages of the present application will become apparent and be easily understood from the following description of the embodiments in conjunction with the drawings, where:

[0051] Figure 1 is a system block diagram provided for a preferred embodiment of the present invention;

[0052] Figure 2 is a flowchart provided for a preferred embodiment of the present invention;

[0053] Figure 3 is an imaging system diagram provided for a preferred embodiment of the present invention;

[0054] Figure 4 is a schematic diagram of the conjugate imaging system provided for a preferred embodiment of the present invention;

[0055] Figure 5 Schematic diagram of the vignetting lens provided for the preferred embodiment of the present invention;

[0056] Figure 6 Timing control diagram of the signal synchronization processing system in the preferred embodiment of the present invention.

[0057] Figure 7 Structural diagram of the zoom pinhole turntable provided for the preferred embodiment of the present invention;

[0058] Figure 8 Structural diagram of the flat glass zoom turntable provided for the preferred embodiment of the present invention;

[0059] Figure 9 Overall view of the pinhole turntable provided for the preferred embodiment of the present invention;

[0060] Figure 10 Partial view of the pinhole turntable provided for the preferred embodiment of the present invention. Detailed implementation manners

[0061] The embodiments of the present application will be described in detail below. Examples of the embodiments are shown in the accompanying drawings, where the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to explain the present application and should not be construed as limiting the present application.

[0062] In view of the limitations of the axial scanning tomography technology in the related art mentioned in the above background technology by the axial scanning speed and the camera imaging speed, and the low signal-to-noise ratio of the wide-field fluorescence microscopy imaging of arbitrary curved surfaces, the present invention can, on the basis of the traditional spinning disk confocal microscopy system, adjust the timing relationship among the illumination adaptation modulation module, the detection module and the zoom pinhole turntable through the signal synchronization processing system, and adjust the microscopic imaging focal plane to the corresponding focal plane through the zoom pinhole turntable, and illuminate the imaging area corresponding to the focusing position within the scanning period of the zoom component through the illumination adaptation modulation module, and detect the imaging area through the detection module to collect the focusing plane signals of all planes, so that after the acquisition timing ends, the curved surface image can be obtained based on the three-dimensional shape distribution of the target by combining the focusing plane signals of all planes through the acquisition and image processing component. Thus, high-speed curved surface modeling and imaging are realized by using time division multiplexing, fast zooming and spatial light modulation, the limitation of the axial scanning imaging speed in the traditional spinning disk confocal technology is solved, the high-speed acquisition of curved surface signals is ensured under a certain resolution, and it is applicable to time series imaging of deep living tissues.

[0063] Such as Figure 1 and Figure 3As shown in the figure, an arbitrary surface wide-field turntable confocal tomography microscopy system mainly includes an illumination module, three relay lens modules, a zoom pinhole turntable 3, a detection module 4, and a signal synchronization processing system; among them:

[0064] The illumination module includes a light source 1-1, an illumination adaptation module 1-2, and an illumination adjustment module 1-3;

[0065] The first relay lens module 2-1 is arranged on the light output side of the illumination module;

[0066] The zoom pinhole turntable 3 is arranged on the light output side of the first relay lens module 2-1; the zoom pinhole turntable 3 realizes focal plane switching through a rotational scanning method;

[0067] The second relay lens module 2-2 is arranged between the zoom pinhole turntable 3 and the sample 6;

[0068] The dichroic mirror 5 is arranged between the zoom pinhole turntable 3 and the first relay lens module 2-1;

[0069] The third relay lens module 2-3 is arranged between the dichroic mirror 5 and the detection module 4;

[0070] The illumination adaptation modulation module generates a dynamic illumination template according to the pre-stored surface topography data; and matches the illumination area to the current focal plane imaging area through the first relay lens module and the second relay lens module; as Figure 4 and Figure 5 shown, the three relay lens modules are used to achieve quadruple conjugation and vignetting compensation; the quadruple conjugation is the conjugation among the illumination adaptation modulation plane 100, the zoom pinhole turntable plane 200, the sample plane 300, and the imaging plane 400;

[0071] The signal synchronization processing system acquires the phase signal of the zoom pinhole turntable in real time, and synchronously controls the zoom compensation working parameters of the illumination adaptation modulation module, the detection module, and the zoom pinhole turntable according to the phase signal.

[0072] The light beam generated by the illumination light source sequentially passes through the first relay lens module, the dichroic mirror, the zoom pinhole turntable, the second relay lens module, the sample, the second relay lens module, the zoom pinhole turntable, the dichroic mirror and enters the detection module;

[0073] For a better understanding of the technical concept of the present invention, non-limiting examples are given below:

[0074] The zoom pinhole turntable is an advanced optical device that includes a high-speed rotating stage driving a composite turntable structure. This composite turntable structure integrates stepped variable-thickness zoom glass and a conjugate pinhole array, enabling rapid switching of the focal plane through rotational scanning. The pinhole array is strictly conjugate to the rear focal plane of the microscope objective, meaning they are optically perfectly aligned. Additionally, combined with the refractive index matching design of the stepped variable-thickness zoom glass, this turntable can not only expand the depth of field of imaging but also effectively suppress background noise, thus providing clearer and higher-contrast images.

[0075] The relay lens module uses multiple lens groups to achieve quadruple conjugation of the illumination module, pinhole turntable, camera, and sample plane. At the same time, it corrects the edge light intensity attenuation through a vignetting compensation lens to increase the field uniformity.

[0076] In the present invention, the described zoom pinhole turntable comprises the following components:

[0077] The pinhole component, whose main function is to achieve tomography at different positions on the focal plane, thereby enabling the capture of more detailed and clear image information;

[0078] The zoom component, which is used to change the microscopic imaging focal plane. By gradually adjusting the position of the focal plane, it collects the focal plane signal of all planes to ensure the comprehensiveness and accuracy of imaging. This zoom component integrates stepped variable-thickness zoom glass and a conjugate pinhole array, and the combination of these two technologies makes the zoom process more precise and efficient;

[0079] The zoom component and the pinhole component adopt a coaxial laminated structure. This structural design enables the two to cooperate closely and achieve synchronous rotational scanning through precise alignment. This synchronous rotational scanning mechanism ensures that during the zoom process, the pinhole component can accurately align with each focal plane, thereby collecting high-quality image data.

[0080] In one implementation, the design of the pinhole component includes two main parts, namely the pinhole turntable and the microlens focusing turntable. The function of the pinhole turntable is to control the passage of light through a series of carefully designed pinholes, while the microlens focusing turntable is responsible for focusing the incident light onto the light-passing pinholes of the pinhole turntable. This design enables precise control and focusing of light, thereby improving the performance and efficiency of the imaging system.

[0081] To ensure the synchronous operation of the entire system, the first synchronization device plays a crucial role. It is responsible for receiving timing signals from the signal synchronization processing system and generating position encoding signals for the microlens focusing turntable based on these signals. These encoding signals are then sent to the detection module to ensure that the detection module can accurately know when and at which position to conduct detections. In this way, the first synchronization device ensures the precise synchronization of the entire imaging system, thereby improving the imaging quality and the accuracy of data acquisition.

[0082] In another implementation, the pinhole component not only includes a pinhole turntable but also a binary array focusing turntable; the function of the binary array focusing turntable is to focus the incident light onto the light-transmitting pinhole position of the pinhole turntable, thereby achieving an accurate focusing function.

[0083] The first synchronization device is responsible for receiving the timing signals from the signal synchronization processing system and then sending the position encoding signals of the pinhole turntable to the detection module to ensure that the detection module can accurately obtain the position information of the pinhole turntable, and then conduct effective detections and analyses.

[0084] The illumination adaptation modulation module (illumination adaptation module 1-2 and illumination adjustment module 1-3) provides various options, including but not limited to diaphragms, galvanometric scanners, spatial light modulators (such as digital micromirror device arrays DMD), and digital micromirror device arrays, etc. These components can apply different intensity and phase modulations to the light field at different time periods, thereby achieving precise illumination control. In this embodiment, the illumination adaptation modulation module can generate a dynamic illumination template based on the pre-stored surface topography data. This process ensures that the illumination effect can perfectly adapt to different illumination requirements. Through the optical path relay module, this module realizes the magnification and reduction of the optical path, and its magnification factor ranges from 0.1 times to 100 times, thereby ensuring that the illumination area can accurately match the imaging area of the current focal plane. This precise matching greatly improves the illumination efficiency and significantly reduces the illumination area. Compared with traditional illumination methods, this method not only improves the illumination accuracy but also reduces unnecessary light energy waste, thereby achieving efficient energy utilization while ensuring the imaging quality.

[0085] The illumination module involved in the present invention mainly consists of:

[0086] The main function of the illumination light source is to emit illumination light, and it can choose to use light-emitting diodes or lasers as the light source. In addition, to meet specific excitation wavelength requirements, corresponding filter films can be installed behind the light source to ensure that the emitted light meets the application requirements.

[0087] The illumination adaptation module, whose main function is to adapt the size and incident angle of the illumination light source to the detection numerical aperture, so as to ensure that the illumination light can effectively irradiate the sample 6 (i.e., the object to be measured), thereby improving the illumination effect and the imaging quality of the sample;

[0088] The illumination adjustment module, within the scanning period of the zoom pinhole turntable, is responsible for illuminating the imaging area corresponding to the focusing position. In this way, precise illumination control of different areas can be achieved, thereby enhancing the performance of the imaging system and the accuracy of imaging.

[0089] Furthermore, the illumination adjustment module of the present invention includes the following components:

[0090] The spatio-temporal light field modulation device, whose main function is to perform corresponding illumination intensity and phase modulation on the light field at different time points to achieve precise control of the light field;

[0091] The second synchronization device, which is used to receive the timing signals from the signal synchronization processing system and output the current illumination state code and / or detection state code according to these signals. These coded information will be fed back to the signal synchronization processing system to ensure the synchronous operation of the entire system.

[0092] In the present invention, the described relay lens module is composed of two main parts, namely the relay lens and the vignetting lens. Specifically:

[0093] The relay lens is mainly responsible for realizing the optical conjugate relationship among the illumination adaptation modulation module, the zoom pinhole turntable, and the imaging focal plane. This conjugate relationship ensures that light can be transmitted among these components in an appropriate manner, thereby achieving the expected imaging effect.

[0094] The function of the vignetting lens is to eliminate the vignetting phenomenon that may be caused by changing the light angle. When multiple relay lenses are cascaded, the change in the light angle may cause darkening or blurring at the image edge. The role of the vignetting lens is to eliminate the vignetting caused by the cascaded relay lenses and ensure the consistency and clarity of the image quality.

[0095] The detection module is equipped with a high-sensitivity camera device, such as a scientific-grade CMOS (sCMOS) camera with a global exposure function, whose quantum efficiency exceeds 60%. Such a camera can support real-time acquisition and fusion of multi-layer focusing signals within a wide field of view. Through this process, the detection module can effectively complete the task of curved surface three-dimensional reconstruction;

[0096] The signal synchronization processing system is based on the multi-channel synchronization controller of the digital acquisition card, integrating a rotary encoder to obtain the phase signal of the zoom pinhole turntable in real time, and synchronously triggering the illumination adaptation modulation module, the global exposure of the camera, and the zoom compensation operation.

[0097] The zoom component is used to adjust the microscopic imaging focal plane to the corresponding focal plane; in some embodiments, the zoom component includes a zoom element, a turntable, and a first synchronization device. Among them, the zoom element is used to change the microscopic imaging focal plane until the focusing plane signals of all planes of the focal plane are collected; the turntable is used to switch the position of the zoom element to the position corresponding to the focal plane; the first synchronization device is used to receive the timing signal of the signal synchronization processing system and send the position encoding signal of the turntable to the detection module and the image processing component.

[0098] The illumination adaptation adjustment module is used to illuminate the imaging area corresponding to the focusing position during the scanning period of the zoom component.

[0099] Optionally, in some embodiments, the illumination adaptation adjustment module includes: a spatio-temporal light field modulation device, a lens, and a second synchronization device. Among them, the spatio-temporal light field modulation device is used to modulate the illumination intensity and phase corresponding to the light field at different times; the lens is used to map the emitted light of the target to the focal plane (camera plane) of the zoom pinhole turntable and the detection module; the second synchronization device is used to receive the timing signal of the signal synchronization processing system and send the illumination state encoding and / or detection state encoding to the signal synchronization processing system.

[0100] The zoom pinhole turntable is used to detect the imaging area and collect the focusing plane signals of all planes; the signal synchronization processing system is used to determine the timing relationship between the zoom component, the spatio-temporal illumination modulation module, and the detection module and synchronize the acquisition timing; the detection module is used to obtain a curved surface image based on the three-dimensional shape distribution of the target and combine the focusing plane signals of all planes after the acquisition timing ends.

[0101] Optionally, in some embodiments, the detection module includes: a camera and an imaging module. Among them, the imaging module is used to image the superposition of areas at all heights by using the camera to obtain a curved surface image.

[0102] Optionally, in some embodiments, the detection module further includes: a memory, which is used to store the curved surface image and / or the focusing plane signals of all planes.

[0103] Among them, the zoom element can be glass sheets with different thicknesses, or other optical elements such as a microlens array; the turntable can be an optical chopper, or other optical elements such as a high-speed rotating stage; the first synchronization device can be a turntable position encoding output element, such as a photoelectric position sensor, which is not specifically limited here; the light source can emit illumination light, and the light source can be selected from a light-emitting diode or a laser, and a filter can be placed behind the light source according to the excitation wavelength requirement; the spatio-temporal light field modulation device can be a diaphragm, a galvanometer, a spatial light modulator, or a digital micromirror array, etc. The spatio-temporal light field modulation device can modulate the light field with different intensities and phases at different times; the lens can be a convex lens, a concave lens, a plane mirror, or various lens elements; the second synchronization device can be a single-chip microcomputer processor system; the signal synchronization processing system can be a signal acquisition card with digital-to-analog signal input and output functions; the detection module can include a camera, a computer, and a microcomputer with a peripheral device interface.

[0104] Specifically, the signal synchronization processing system of the embodiment of the present application can determine the timing signals of the zoom component, the spatio-temporal illumination modulation module, and the zoom pinhole turntable, and send the timing signals to the first synchronization device, the second synchronization device, and the zoom pinhole turntable respectively. The zoom element can change the microscopic imaging focal plane, so as to detect different depths of the sample through the zoom element with the corresponding thickness; the embodiment of the present application can receive the timing signal sent by the signal synchronization processing system through the first synchronization device, and the first synchronization device can also output the turntable position encoding signal; the lens can map the spatio-temporal light field modulation device to the sample plane, and the spatio-temporal light field modulation device can modulate the light field with different intensities and phases at different times. The second synchronization device can receive the timing signal sent by the signal synchronization processing system, and the second synchronization device can output the current illumination state encoding; the zoom pinhole turntable can share the spatio-temporal light field modulation device, the lens, and the second synchronization device with the spatio-temporal illumination modulation module. At this time, the second synchronization device can also output the current detection state encoding.

[0105] It should be noted that the zoom pinhole turntable may not share the spatio-temporal light field modulation device, the lens, and the second synchronization device with the spatio-temporal illumination modulation module, that is, the zoom pinhole turntable may also be separately provided with a spatio-temporal light field modulation device, a lens, and a synchronization device. The detection module can include a camera, a memory, and an imaging module, so that after the acquisition timing ends, a curved surface image can be obtained based on the three-dimensional shape distribution of the target and by combining the focusing plane signals of all planes.

[0106] Next, an imaging method of an arbitrary curved surface wide-field turntable confocal tomography microscopy system according to an embodiment of the present application will be described with reference to the accompanying drawings.

[0107] As Figure 2 shown, the process of the imaging method of the arbitrary curved surface wide-field turntable confocal tomography microscopy system includes the following steps:

[0108] S201. First, pre-scan the sample and perform data processing to obtain surface topography data. The three-dimensional topography data includes the three-dimensional topography data of the sample and the surface topology mapping relationship. Then, calculate the illumination template sequence corresponding to each focal plane in combination with the pre-calibrated vignetting coefficient.

[0109] S202. Dynamic conjugate tomography scanning: Start the zoom pinhole turntable, and determine the current focal plane position in real time according to the turntable encoder signal. Synchronously trigger the DMD to load the corresponding illumination template, and only activate the effective imaging area within the depth of field.

[0110] S203. Tomography signal acquisition and synchronous control: During a single-frame exposure period, control the global light window of the detection module to be phase-matched with the zoom pinhole turntable through the signal synchronization processing system.

[0111] S204. Adopt a time-space multiplexing strategy to encode and superimpose the tomography signals of different focal planes onto a single-frame image in sequence.

[0112] S205. Surface image reconstruction and output: After the acquisition time sequence ends, obtain the surface image through the acquisition and image processing component based on the three-dimensional shape distribution of the target and in combination with the focusing plane signals of all planes.

[0113] Optionally, the method according to the embodiment of the present application further includes:

[0114] Change the microscopic imaging focal plane through a zoom element until the focusing plane signals of all planes are collected;

[0115] Use the turntable to switch the position of the zoom element to the position corresponding to the focal plane;

[0116] Receive the timing signal of the signal synchronization component through the first synchronization device, and send the position encoding signal of the turntable to the acquisition and image processing component.

[0117] Optionally, the method according to the embodiment of the present application further includes:

[0118] Modulate the illumination intensity and phase corresponding to the light field at different times through a spatio-temporal light field modulation device;

[0119] Use a lens to map the emitted light of the target to the zoom pinhole turntable and the camera plane;

[0120] Receive the timing signal of the signal synchronization component through the second synchronization device, and output the current illumination state encoding and / or detection state encoding to the signal synchronization component.

[0121] Optionally, the method according to the embodiment of the present application further includes:

[0122] The camera is used to image the areas at all heights after superposition to obtain a curved surface image.

[0123] Optionally, the method according to an embodiment of the present application further includes:

[0124] Storing the curved surface image and / or the focus plane signals of all planes.

[0125] To enable those skilled in the art to further understand the arbitrary curved surface wide-field spinning disk confocal tomography microscopy system according to the embodiments of the present application, the following will be described in detail with reference to specific embodiments.

[0126] As a possible implementation, as Figure 3 shown, Figure 3 is an arbitrary curved surface wide-field spinning disk confocal tomography microscopy system according to a specific embodiment of the present application. The zoom element (such as the flat glass zoom turntable in Figure 8 ) is placed on the turntable (such as a high-speed rotating table) and is placed between the objective lens and the dichroic mirror 5. The laser beam emitted by (such as a laser light source) is expanded by a lens and irradiated on the digital micromirror array through a total reflection prism, and then passes through the first relay lens module 2-1 and the dichroic mirror 5 and is irradiated on the zoom turntable module. The light passing through the zoom pinhole passes through the vignetting correction lens 2-4 and the second relay lens module 2-2 again and is mapped onto the sample 6. The signal light is reflected after passing through the second relay lens module 2-2, the zoom pinhole turntable 3, and the dichroic mirror 5, and is imaged onto the camera 4 through the third relay lens module 2-3. This arbitrary curved surface wide-field spinning disk confocal tomography microscopy system can image an arbitrary-shaped curved surface. Among them, the illumination adaptation modulation module (such as the digital micromirror array), the sample 6, the zoom pinhole turntable, and the camera 4 are conjugate, as Figure 4 shown. The vignetting lens is located near the focal plane and is used to improve the vignetting caused by the cascaded relay system, as Figure 3 shown.

[0127] Optionally, a set of fluorescence filter sheets 7 may be included in the third relay lens module 2-3 to block the incident light.

[0128] Specifically, in combination with Figure 6 it can be seen that before imaging, the sample is first scanned layer by layer to determine the shape of the curved surface, and the part that needs to be illuminated at each depth is determined, and a corresponding illumination template is generated on the digital micromirror array. During imaging, a signal is sent out every circle of the turntable to drive the camera to start exposure. When the turntable rotates to a zoom element (such as a glass sheet) of a certain thickness, the digital micromirror array generates a corresponding template to illuminate the sample. The turntable rotates through all the zoom elements of different thicknesses and selectively illuminates and superimposes the signals at different depths to achieve curved surface imaging, and the imaging speed is consistent with the camera frame rate.

[0129] As a possible implementation, asFigure 7 As shown Figure 7 This is the schematic diagram of the zoom pinhole turntable in a specific embodiment of the present application. The pinhole turntable and the flat glass zoom turntable with variable thickness are coaxially fixed between the pressure plate and the high-speed rotating table. Figure 8 This is the schematic diagram of the flat glass zoom turntable in a specific embodiment of the present application. Glass with different thicknesses is at different angular positions of different turntables, resulting in a shallower imaging depth of the sample under the glass, and the moving distance in the depth direction is x = (n - 1)d / n, where d is the thickness of the glass and n is the refractive index of the glass. Figure 9 This is the schematic diagram of the pinhole turntable in a specific embodiment of the present application. The black represents the light-shielding part, and the white represents the light-transmitting pinholes. Along the diameter direction, the pinholes with different radii are equidistant from each other, and the pinholes in the direction perpendicular to the radius conform to the Archimedean spiral arrangement. An enlarged schematic diagram of a part of the pinhole turntable is as Figure 10 shown.

[0130] According to the arbitrary curved surface wide-field turntable confocal tomography microscopy imaging system proposed in the embodiments of the present application, a relay lens module is used to achieve the four conjugations of the illumination modulator, the pinhole array, the image plane, and the detector, and a vignetting lens is used to correct the aberration of the cascaded imaging system; the three-synchronization timing relationship between the zoom component, the spatio-temporal illumination modulation module, and the detection light modulation component can be determined through the signal synchronization component, and the microscopic imaging focal plane can be adjusted to the corresponding focal plane through the zoom component. During the scanning period of the zoom component, the spatio-temporal illumination modulation module illuminates the imaging area corresponding to the focusing position, and the zoom pinhole turntable is used to detect the imaging area to collect the focusing plane signals of all planes. Thus, after the acquisition timing ends, the curved surface image can be obtained based on the three-dimensional shape distribution of the target by combining the focusing plane signals of all planes through the acquisition and image processing component. Therefore, by adopting time-division multiplexing, fast zooming, and spatial light modulation, high-speed curved surface modeling and imaging are realized, solving the problems in the related art that the axial scanning tomography technology is limited by the axial scanning speed and the camera imaging speed, the multi-plane detection technology is limited by the size of the camera target surface, the spatial resolution of the light field detection technology is poor, and it takes a long time, ensuring the high-speed acquisition of curved surface signals under a certain resolution and being applicable to time-series imaging of deep living tissues.

[0131] It should be noted that the foregoing explanation of the embodiments of the arbitrary curved surface wide-field turntable confocal tomography microscopy imaging system also applies to the arbitrary curved surface wide-field turntable confocal tomography microscopy imaging system and its imaging method of this embodiment, which will not be elaborated here.

[0132] According to the arbitrary curved surface wide-field turntable confocal tomography microscopy system and its imaging method proposed by the embodiments of the present application, the timing relationship between the zoom component, the spatio-temporal illumination modulation module, and the detection light adjustment component can be determined through the signal synchronization component, and the microscopic imaging focal plane can be adjusted to the corresponding focal plane through the zoom component. During the scanning period of the zoom component, the spatio-temporal illumination modulation module illuminates the imaging area corresponding to the focusing position, and the imaging area is detected through the zoom pinhole turntable to collect the focusing plane signals of all planes. Thus, after the acquisition timing ends, the acquisition and image processing component can obtain the curved surface image based on the three-dimensional shape distribution of the target and by combining the focusing plane signals of all planes. Therefore, by adopting time-division multiplexing, fast zooming, and spatial light modulation to achieve high-speed curved surface modeling and imaging, the problems in the related art that the axial scanning tomography technology is limited by the axial scanning speed and the camera imaging speed, the multi-plane detection technology is limited by the size of the camera target surface, the spatial resolution of the light field detection technology is poor, and it takes a long time are solved, ensuring the high-speed acquisition of curved surface signals at a certain resolution and being applicable to time-series imaging of living deep tissues.

[0133] In the description of this specification, the descriptions referring to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or N embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0134] In addition, the terms "first" and "second" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, the features defined with "first" and "second" can explicitly or implicitly include at least one of the features. In the description of the present application, the meaning of "N" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0135] Any process or method description shown in the flowchart or described in other ways herein can be understood as representing a module, segment, or part of code including one or more executable instructions for implementing a customized logical function or process. The scope of the preferred embodiments of the present application includes additional implementations, where the functions can be executed in a substantially simultaneous manner or in the reverse order according to the involved functions, rather than in the order shown or discussed, which should be understood by those skilled in the art of the embodiments of the present application.

[0136] The logic and / or steps represented in the flowchart or otherwise described herein, for example, can be considered as a definable sequence list of executable instructions for implementing logical functions, and can be specifically implemented in any computer-readable medium for use by an instruction execution system, apparatus, or device (such as a computer-based system, a system including a processor, or other systems that can fetch and execute instructions from the instruction execution system, apparatus, or device), or in conjunction with these instruction execution systems, apparatus, or devices. For the purposes of this specification, a "computer-readable medium" can be any device that can contain, store, communicate, propagate, or transport a program for use by or in conjunction with an instruction execution system, apparatus, or device. More specific examples (a non-exhaustive list) of the computer-readable medium include the following: an electrical connection portion (electronic device) having one or N wirings, a portable computer diskette (magnetic device), a random access memory (RAM), a read-only memory (ROM), an erasable programmable read-only memory (EPROM or flash memory), an optical fiber device, and a portable compact disc read-only memory (CDROM). Additionally, the computer-readable medium can even be paper or other suitable media on which the program can be printed, because the program can be obtained electronically, for example, by optically scanning the paper or other media, followed by editing, interpretation, or otherwise processing as appropriate, and then stored in a computer memory.

[0137] It should be understood that various parts of the present application can be implemented by hardware, software, firmware, or a combination thereof. In the above-described embodiments, the N steps or methods can be implemented by software or firmware stored in a memory and executed by a suitable instruction execution system. For example, if implemented in hardware, as in another embodiment, any one or a combination of the following techniques well known in the art can be used: discrete logic circuits having logic gate circuits for implementing logical functions on data signals, application specific integrated circuits having appropriate combinational logic gate circuits, programmable gate arrays (PGAs), field programmable gate arrays (FPGAs), and the like.

[0138] Those of ordinary skill in the art of this technology can understand that all or part of the steps carried by the method of implementing the above embodiments can be completed by instructing relevant hardware through a program, and the program can be stored in a computer-readable storage medium. When the program is executed, it includes one or a combination of the steps of the method embodiments.

[0139] In addition, each functional unit in various embodiments of the present application may be integrated into a processing module, may exist physically alone for each unit, or two or more units may be integrated into one module. The above-mentioned integrated module may be implemented in the form of hardware or in the form of a software functional module. When the integrated module is implemented in the form of a software functional module and sold or used as an independent product, it may also be stored in a computer-readable storage medium.

[0140] The above-mentioned storage medium may be a read-only memory, a magnetic disk or an optical disc, etc. Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present application. Those of ordinary skill in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present application.

Claims

1. An arbitrary curved surface wide field rotating disk confocal tomography microscopy imaging system, comprising an illumination light source and a detection module, characterized in that: A first relay lens module is provided on the light output side of the lighting module; A zoom pinhole dial is provided on the light output side of the first relay lens module; A second relay lens module is provided between the zoom pinhole rotating disk and the sample; A dichroic mirror is provided between the zoom pinhole rotating disk and the first relay lens module; A third relay lens module is provided between the dichroic mirror and the detection module; The illumination adaptation modulation module generates a dynamic illumination template according to the pre-stored curved surface morphology data; and matches the illumination area to the current focal plane imaging area through the first relay lens module and the second relay lens module; Three relay lens modules are used to achieve quadruple conjugation and vignetting compensation; The signal synchronization processing system acquires the phase signal of the zoom pinhole turntable in real time, and synchronously controls the zoom compensation working parameters of the illumination module, the detection module and the zoom pinhole turntable according to the phase signal.

2. The arbitrary curved surface wide field rotating disk confocal tomography microscopy imaging system according to claim 1, characterized in that: The zoom pinhole dial comprises: A pinhole component, used to realize tomographic imaging at different positions on the focal plane; A zoom component, used to change the focal plane of microscopic imaging; the zoom component integrates a stepped variable thickness zoom glass and a conjugate pinhole array; The zoom component and the pinhole component adopt a coaxial stacking structure to achieve synchronous rotation scanning.

3. The arbitrary curved surface wide field rotating disk confocal tomography microscopy imaging system according to claim 2, characterized in that: The pinhole component includes a pinhole rotating disk and a flat glass zoom rotating disk; the flat glass zoom rotating disk switches the position of the pinhole rotating disk to the position corresponding to the focal plane; The first synchronization device receives the timing signal of the signal synchronization processing system, and sends the position coding signal of the focusing turntable to the detection module.

4. The arbitrary curved surface wide field rotating disk confocal tomography microscopy imaging system according to claim 2, characterized in that: The pinhole component includes a pinhole rotating disk and an optional focusing rotating disk; the binary array focusing rotating disk focuses the incident light energy on the light-transmitting position of the pinhole rotating disk; The first synchronization device receives the timing signal of the signal synchronization processing system, and sends the position coding signal of the pinhole turntable to the detection module.

5. The arbitrary curved surface wide field rotating disk confocal tomography microscopy imaging system according to claim 1, characterized in that: The lighting module comprises: An illumination light source element, serving as an illumination light source for an imaging system, provides energy for the imaging system; An illumination adaptation module, used to adapt the size and incident angle of the illumination light source to the detection numerical aperture; The illumination adjustment module illuminates the imaging area corresponding to the focus position during the scanning period of the zoom pinhole dial.

6. The arbitrary curved surface wide field rotating disk confocal tomography microscopy imaging system according to claim 5, characterized in that: The lighting adjustment module comprises: Spatiotemporal light field modulation equipment, used to modulate the illumination intensity and phase of the light field at different times; The second synchronization device is used to receive the timing signal of the signal synchronization processing system and output the current lighting state code and / or detection state code to the signal synchronization processing system.

7. The arbitrary curved surface wide field rotating disk confocal tomography microscopy imaging system according to claim 1, characterized in that: The relay lens module comprises: A relay lens is used to achieve optical conjugation among the illumination adaptation modulation module, the zoom pinhole turntable and the imaging focal plane; Vignetting lenses are used to eliminate the vignetting caused by changing the angle of light and cascade relay lenses.

8. A method for arbitrary curved surface wide field rotating disk confocal tomography microscopy imaging, characterized in that: The arbitrary curved surface wide field rotating disk confocal tomography microscopy imaging system according to any one of claims 1 to 7 is used to complete the following steps: S1. First, the sample is pre-scanned and data processed to obtain surface morphology data, wherein the surface morphology data includes three-dimensional morphology data of the sample and surface topology mapping relationship; then, the illumination template sequence corresponding to each focal plane is calculated in combination with the pre-calibrated vignetting coefficient; S2, dynamic conjugate tomography scanning: start the zoom pinhole turntable, and determine the current focal position in real time according to the turntable encoder signal; synchronously trigger the DMD to load the corresponding illumination template, and only activate the effective imaging area within the depth of field; S3, tomographic signal acquisition and synchronization control: within a single frame exposure cycle, the signal synchronization processing system controls the phase matching between the global exposure window of the detection module and the zoom pinhole turntable; S4, using the time-space multiplexing strategy to encode the tomographic signals of different focal planes into a single frame image in time sequence; S5. Surface image reconstruction.

9. The arbitrary curved surface wide field rotating disk confocal tomography microscopy imaging method according to claim 8, characterized in that: Fluorescent thin slices were used to calibrate the vignetting coefficients of different imaging areas.

10. The arbitrary curved surface wide field rotating disk confocal tomography microscopy imaging method according to claim 8, characterized in that S5 include: Firstly, the tomographic signals are separated and the focal plane signals are extracted from the superimposed images according to the time sequence coding; then the vignetting compensation is performed and the edge attenuation is corrected by using the pre-calibrated intensity distribution matrix; Finally, surface fusion is performed to reconstruct the continuous surface image by combining the pre-stored 3D shape data.

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