High-speed wide-field multi-plane tomography method and device based on Bessel beam

Abstract

The invention discloses a high-speed wide-field multi-plane tomography method and a high-speed wide-field multi-plane tomography device based on Bessel beams. The high-speed wide-field multi-plane tomography method comprises the following steps of excitation beam adjustment, Bessel beam modulation, illumination selection and multi-plane imaging. The surface scanning Bessel beam and the multi-plane imaging technology are adopted, tomography is achieved, meanwhile, objects at different axial positions are mapped to different areas of the image sensor, high-speed three-dimensional imaging is achieved, and the volume imaging collection rate is increased. Meanwhile, an illumination technology is selected, an illumination area can be adjusted in real time according to different sample structures and work, background light and light damage to the samples are further reduced, and high-speed wide-field multi-plane tomography is achieved.

Description

Technical Field

[0001] The invention relates to the technical field of optical microscopy design, and in particular to a high-speed wide-field multi-plane tomography method and device based on Bessel beams. Background Art

[0002] Optical microscopy is a technology that uses optical methods and optical devices to capture static or dynamic high-resolution images of target objects. It is mainly used for structural construction and functional signal analysis of microscopic objects. It has become an indispensable optical instrument in many research fields such as neurobiology, oncology, pathology, and behavior. Among the existing conventional microscopic imaging technologies, single-photon wide-field imaging technology cannot image deep biological scattering tissues and does not have tomography capabilities. The background signal in the collected images is serious, and it is difficult to directly analyze the functional signals in the images; light field imaging technology uses multi-view imaging technology to reconstruct three-dimensional structures through a single image to achieve high-speed three-dimensional imaging, but the multi-view solution causes resolution loss, and the background light can easily lead to three-dimensional reconstruction errors, thereby obtaining erroneous images; single-photon confocal microscopes and two-photon scanning microscopes can both have tomography capabilities and greatly eliminate background signals, but they are both based on the principle of point scanning, and the imaging field of view, spatial resolution, and temporal resolution are mutually restricted, making it difficult to meet some practical needs.

[0003] In order to simultaneously meet the requirements of large field of view, high resolution, and tomography capability in imaging, relevant literature has proposed line scanning confocal technology. Compared with point scanning confocal technology, this technology has two main technical improvements: first, a linear excitation spot is formed at the excitation end through a cylindrical lens and a galvanometer is used for one-dimensional scanning, thereby realizing the excitation of the two-dimensional sample surface; correspondingly, in order to cooperate with the linear excitation spot, the detection end changes the original small hole into a slit to shield the background light at the non-focal surface. Thanks to the development of image sensors based on rolling shutter technology, the commonly used method of shielding background light is to use the rolling shutter of the image sensor as a movable slit to achieve background light shielding. Although the line scanning confocal technology is slightly weaker than the point scanning solution in tomography capability, it has a higher imaging rate under the requirements of large field of view and high spatial resolution.

[0004] In actual biological optical imaging, in order to study the structure and function in three-dimensional space, three-dimensional high-speed imaging is required. To use the line scanning confocal method for three-dimensional high-speed imaging, it is necessary to perform high-speed layer-by-layer scanning of the target three-dimensionally and realize synchronous imaging accordingly. Although many optoelectronic devices can currently realize axial scanning functions, such as high-speed zoom lenses, diffractive optical devices, piezoelectric displacement stages, etc., they are limited by the frame rate of the image sensor. The acquisition of three-dimensional images through layer-by-layer scanning will inevitably lead to a significant decrease in the imaging rate, making it difficult to achieve high-speed and large-field three-dimensional acquisition.

[0005] The existing technologies mainly include single-photon wide-field microscopy, two-photon microscopy, line scanning confocal microscopy, and light-field microscopy.

[0006] The main disadvantages of single-photon wide-field microscopy are: 1) weak anti-scattering ability, unable to image deep in scattering tissue; 2) no tomography capability, causing the target signal to be submerged by the background signal; 3) layer-by-layer scanning is required in three-dimensional imaging, and the imaging rate is low due to the performance limitations of existing image sensors.

[0007] The main disadvantages of two-photon microscopy are: 1) the point scanning scheme limits the data throughput and the imaging rate in large-field three-dimensional imaging; 2) higher-power light excitation is required, causing thermal damage to the tissue.

[0008] The main disadvantages of line scanning confocal microscopy are: 1) It requires layer-by-layer scanning in three-dimensional imaging, which is limited by the performance of existing image sensors and has a low imaging rate.

[0009] The main disadvantages of light field microscopy are: 1) there is no tomography capability, and background light can easily cause image reconstruction errors; 2) the collected images require time to be reconstructed in three dimensions, making real-time observation impossible. Summary of the invention

[0010] The object of the present invention is to provide a high-speed wide-field multi-plane tomography method and device based on Bessel beams to overcome the deficiencies in the prior art.

[0011] To achieve the above object, the present invention provides the following technical solutions: The present application discloses a high-speed wide-field multi-plane tomography method based on Bessel beam, comprising the following steps: S1, excitation beam adjustment step: generating, collimating and amplifying the excitation beam through a beam generating device to obtain an initial beam; S2, Bessel beam modulation step: phase modulating the initial beam obtained in step S1 by a Bessel beam modulation device to obtain a one-dimensional surface Bessel beam; and scanning the one-dimensional Bessel beam by a scanning device to extend the axial illumination range while maintaining the lateral resolution of the one-dimensional surface Bessel beam; S3, a selective illumination step: generating a selective illumination pattern by selecting an illumination device, performing binary modulation on the one-dimensional Bessel light beam, and then transmitting the modulated one-dimensional Bessel light beam to the sample position through a relay optical path, thereby achieving selective illumination of the biological sample; S4, multi-plane imaging step: through the multi-plane imaging device, the four specific axial positions of the biological sample are mapped to different areas of the image sensor, and the rolling shutter exposure position of the image sensor is ensured to be consistent with the position of the sample illuminated area mapped on the image sensor through the synchronization control circuit, so as to realize multi-plane tomographic imaging.

[0012] Preferably, the light beam generating device comprises a light source, a first plane mirror, a second plane mirror and a beam expander; in step S1, the light source emits a light beam, which is expanded by the beam expander and then adjusted in direction by the first plane mirror and the second plane mirror to form a desired initial light beam.

[0013] Preferably, the Bessel beam modulation device includes a prism, a first lens, a one-dimensional scanning galvanometer and a second lens; in step S2, the initial light beam passes through the prism to generate a one-dimensional Bessel beam, and then passes through the first lens to form two parallel light beams and passes through the one-dimensional scanning galvanometer to scan the light beam, and finally passes through the second lens to converge on a focusing plane to form a one-dimensional Bessel beam that can be scanned.

[0014] Preferably, the selective illumination device comprises a transmissive spatial light modulator, a third lens, a first objective lens and a sample; the transmissive spatial light modulator is placed on a primary focusing plane to modulate the intensity of a one-dimensional Bessel light beam, which is focused on the sample surface after propagating through a relay optical path composed of the third lens and the first objective lens.

[0015] Preferably, the multi-plane imaging device comprises an objective lens, a dichroic mirror, a fourth lens, a beam splitter prism, a fifth lens, a tube lens, a filter and an image sensor; the light generated by the sample is collected by the objective lens and then focused on an image plane after being reflected by the dichroic mirror and converged by the fourth lens; the beam splitter prism maps images at different axial positions to different positions on the same plane; the image is then transmitted to the image sensor after passing through the fifth lens, the tube lens and the filter; the image sensor cooperates with the movement direction and speed of the scanning galvanometer to achieve simultaneous imaging of multiple planes.

[0016] Preferably, the beam splitter prism is composed of a plurality of reflectors and a beam splitter, and by setting different optical paths between the beam splitter prism entrance and each exit in advance, different axial positions of the object side are mapped to different lateral areas at the same axial position on the image plane.

[0017] Preferably, it is used for generating, collimating and amplifying a light beam; a Bessel beam modulator is used to generate a one-dimensional Bessel beam and scan it; a selective lighting device is used to generate an excitation pattern and select a sample excitation area; a multi-plane imaging device is used to map object planes at different axial positions to different areas of the image sensor; a synchronous control device is used to synchronize the electronic control elements in the device and feedback the image analysis results; the light beam generating device, the Bessel beam modulator and the selective lighting device constitute an illumination optical path; the multi-plane imaging device constitutes an imaging optical path.

[0018] Beneficial effects of the present invention: The present invention discloses a high-speed wide-field multi-plane tomography method and device based on Bessel beams. The surface scanning Bessel beams and multi-plane imaging technology are used to realize tomography while mapping objects in different axial positions to different areas of the image sensor, thereby realizing high-speed three-dimensional imaging and improving the volume imaging acquisition rate. At the same time, the lighting technology is selected, and the lighting area can be adjusted in real time according to different sample structures and work, further reducing background light and light damage to the sample, and realizing high-speed wide-field multi-plane tomography.

[0019] The features and advantages of the present invention will be described in detail through embodiments in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a flow chart of a high-speed wide-field multi-plane tomography method based on Bessel beam of the present invention; Figure 2 It is a structural schematic diagram of a high-speed wide-field multi-plane tomography device based on Bessel beams of the present invention; Figure 3 It is a schematic diagram of the illumination optical path of a high-speed wide-field multi-plane tomography device based on Bessel beams of the present invention; Figure 4 is a schematic diagram of a point spread function simulation of an axial large-range lighting device according to an embodiment of the present invention; Figure 5 It is a schematic structural diagram of an imaging optical path of a high-speed wide-field multi-plane tomography imaging device based on a Bessel beam according to the present invention; Figure 6 It is a beam splitter prism structure and light propagation diagram in a multi-plane imaging device according to an embodiment of the present invention; Figure 7 Schematic diagram of a high-speed wide-field multi-plane tomography device based on Bessel beam according to an embodiment of the present invention. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below through the accompanying drawings and embodiments. However, it should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the scope of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention.

[0022] The present invention provides a high-speed wide-field multi-plane tomography method based on Bessel beam, the method comprising the following steps: S1, excitation beam adjustment step: generating, collimating and amplifying the excitation beam through a beam generating device to obtain an initial beam; S2, Bessel beam modulation step: phase modulating the initial beam obtained in step S1 by a Bessel beam modulation device to obtain a one-dimensional surface Bessel beam; and scanning the one-dimensional Bessel beam by a scanning device to extend the axial illumination range while maintaining the lateral resolution of the one-dimensional surface Bessel beam; S3, a selective illumination step: generating a selective illumination pattern by selecting an illumination device, performing binary modulation on the one-dimensional Bessel light beam, and then transmitting the modulated one-dimensional Bessel light beam to the sample position through a relay optical path, thereby achieving selective illumination of the biological sample; The illumination pattern may be analyzed based on the image acquired in step S4 when the light beam passes completely through.

[0023] S4, multi-plane imaging step: through the multi-plane imaging device, the four specific axial positions of the biological sample are mapped to different areas of the image sensor, and the rolling shutter exposure position of the image sensor is ensured to be consistent with the position of the sample illuminated area mapped on the image sensor through the synchronous control circuit, so as to realize multi-plane tomography. After the data is analyzed, the illumination pattern is selected and input into step S3; According to the high-speed wide-field multi-plane tomography method based on Bessel beams in an embodiment of the present invention, through axial large-range excitation and selective illumination, intensity and phase modulation of the illumination beam are implemented to achieve selective axial large-range scanning illumination; then, through multi-plane imaging, images at different axial positions are mapped to specific areas of the image sensor to achieve multi-plane imaging. Furthermore, the image sensor used in the present invention is a camera with a two-way rolling shutter, and by synchronizing the rolling shutter with the scanning position and speed, tomography similar to confocal technology is achieved. The illumination efficiency of the imaging spatial resolution is guaranteed, and the rate and tomography capability of volume imaging are improved.

[0024] In addition, another aspect of the present invention proposes a high-speed wide-field multi-plane tomography device based on Bessel beams, including: a beam generating device for generating, collimating and amplifying beams; a Bessel beam modulating device for generating and scanning one-dimensional Bessel beams; a selective illumination device for generating an excitation pattern and selecting a sample excitation area; a multi-plane imaging device for mapping object planes at different axial positions to different areas of the image sensor; and a synchronous control device for synchronously controlling the electronic control elements in the device and feeding back image analysis results. Figure 2 , which is a schematic diagram of a high-speed wide-field multi-plane tomography device based on Bessel beams of the present invention.

[0025] The light beam generating device, the Bessel light beam modulating device and the selective lighting device together constitute the lighting optical path of the high-speed wide-field multi-plane tomography device based on the Bessel light beam, such as Figure 3 As shown, each device is described in detail below; In some examples, the light beam generating device includes: a light source 301, a first plane mirror 303, a second plane mirror 304 and a beam expander 302. The light beam is emitted by the light source 301, expanded by the beam expander 302, and then adjusted in direction by the first plane mirror 303 and the second plane mirror 304 to form a desired initial light beam; In some examples, the Bessel beam modulation device includes: a prism 305, a first lens 306, a one-dimensional scanning galvanometer 307, and a second lens 308. The light beam generated by the light beam generating device passes through the prism 305 to generate a one-dimensional Bessel beam, and then passes through the first lens 306 to form two parallel light beams and passes through the one-dimensional scanning galvanometer 307 to scan the light beam, and finally passes through the second lens 308 to converge on the primary focusing plane of the system to form a one-dimensional Bessel beam that can be scanned.

[0026] In some examples, the selective illumination device includes: a transmissive spatial light modulator 309, a third lens 310, an objective lens 311, and a sample 312. The transmissive spatial light modulator 309 is placed on a primary focusing plane to modulate the intensity of the one-dimensional Bessel beam, and after propagating through a relay optical path composed of the third lens 310 and the objective lens 311, the beam is focused on the surface of the sample 312; the point spread function of the one-dimensional Bessel beam on the sample surface is as follows: Figure 4 shown.

[0027] Among them, the multi-plane imaging device constitutes the imaging optical path of the high-speed wide-field multi-plane tomography imaging device; Figure 5As shown, in some examples, the multi-plane imaging device includes: an objective lens 311, a dichroic mirror 503, a fourth lens 504, a beam splitter prism 505, a fifth lens 506, a tube lens 507, a filter 508 and an image sensor 509. The light generated by the sample 312 is collected by the objective lens 311 and then focused on the primary image plane after being reflected by the dichroic mirror 503 and converged by the fourth lens 504; the beam splitter prism 505 is installed near the primary image plane to map images at different axial positions to different positions on the same plane. The image is then transmitted to the image sensor 509 through a relay system composed of the fifth lens 506 and the tube lens 507, wherein the filter 508 can eliminate the illumination light and retain the fluorescence. The image sensor 509 cooperates with the movement direction and speed of the one-dimensional scanning galvanometer 307 to achieve multi-plane simultaneous imaging. The beam splitter prism 505 is composed of a plurality of reflectors and a beam splitter. By setting different optical paths from the beam splitter entrance to each exit in advance, different axial positions of the object are mapped to different transverse areas of the same axial position on the image plane. Figure 6 As shown. The image sensor used in the device is a camera with a two-way rolling shutter function. In order to cooperate with scanning to achieve tomographic imaging, the position and direction corresponding to the image on each sub-field of view can be completely corresponding after modulation by the beam splitter prism 505. At the same time, the camera based on the rolling shutter can also eliminate the side lobe light of the illuminated Bessel beam, thereby improving the background light elimination capability of the device.

[0028] As a specific example, Figure 7 FIG. 1 is a schematic diagram of a high-speed wide-field multi-plane tomography imaging device based on a Bessel beam according to an embodiment of the present invention, which is a high-speed wide-field multi-plane tomography imaging device based on a Bessel beam composed of a beam generating device 201, a Bessel beam modulating device 202, a selective illumination device 203, a multi-plane imaging device 204 and a synchronization control device 205 described in the above embodiments. In a specific example, the imaging process is as follows: Combination Figure 7The light beam is emitted by the light source 301, and after the beam is expanded by the beam expander 302 and the collimating plane mirror group composed of the first plane mirror 303 and the second plane mirror 304 adjusts the direction of the light beam, it enters the prism 305 to form a surface Bessel beam. The surface Bessel beam is scanned and reflected by the first lens 306, the one-dimensional scanning galvanometer 307, and focused by the second lens 308, and then converges on the transmission type spatial light modulator 309. After the light beam is modulated by the transmission type spatial light modulator 309, it passes through the third lens 310, the dichroic mirror 503 and the objective lens 311 to illuminate the sample 312. The signal light generated by the sample is collected by the objective lens 311, reflected by the dichroic mirror 503 and converged by the fourth lens 504, and then enters the beam splitter prism 505 to realize multi-plane imaging, and finally passes through the fifth lens 506, the tube lens 507 and the filter 508 to form an image on the image sensor 509. The controller synchronously controls the one-dimensional scanning galvanometer 307, the transmissive spatial light modulator 309 and the image sensor 509 to complete high-speed wide-field multi-plane tomography. The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A high-speed wide-field multi-plane tomography method based on Bessel beam, characterized in that: The steps include: S1, excitation beam adjustment step: generating, collimating and amplifying the excitation beam through a beam generating device to obtain an initial beam; S2, Bessel beam modulation step: phase modulating the initial beam obtained in step S1 by a Bessel beam modulation device to obtain a one-dimensional surface Bessel beam; and scanning the one-dimensional Bessel beam by a scanning device to extend the axial illumination range while maintaining the lateral resolution of the one-dimensional surface Bessel beam; S3, a selective illumination step: generating a selective illumination pattern by selecting an illumination device, performing binary modulation on the one-dimensional Bessel light beam, and then transmitting the modulated one-dimensional Bessel light beam to the sample position through a relay optical path, thereby achieving selective illumination of the biological sample; S4, multi-plane imaging step: through the multi-plane imaging device, the four specific axial positions of the biological sample are mapped to different areas of the image sensor, and the rolling shutter exposure position of the image sensor is ensured to be consistent with the position of the sample illuminated area mapped on the image sensor through the synchronization control circuit, so as to realize multi-plane tomographic imaging.

2. A high-speed wide-field multi-plane tomography method based on Bessel beams as claimed in claim 1, characterized in that: The light beam generating device comprises a light source, a first plane mirror, a second plane mirror and a beam expander. In step S1, the light source emits a light beam, which is expanded by the beam expander and then adjusted in direction by the first plane mirror and the second plane mirror to form a desired initial light beam.

3. The high-speed wide-field multi-plane tomography method based on Bessel beams according to claim 1, characterized in that: The Bessel beam modulation device includes a prism, a first lens, a one-dimensional scanning galvanometer and a second lens; in step S2, the initial light beam passes through the prism to generate a one-dimensional Bessel beam, and then passes through the first lens to form two parallel light beams and passes through the one-dimensional scanning galvanometer to scan the light beam, and finally passes through the second lens to converge on a primary focusing plane to form a one-dimensional Bessel beam that can be scanned.

4. A high-speed wide-field multi-plane tomography method based on Bessel beams as claimed in claim 3, characterized in that: The selective illumination device comprises a transmissive spatial light modulator, a third lens, a first objective lens and a sample; the transmissive spatial light modulator is placed on a primary focusing plane to modulate the intensity of a one-dimensional Bessel light beam, which is focused on the sample surface after propagating through a relay optical path composed of the third lens and the first objective lens.

5. A high-speed wide-field multi-plane tomography method based on Bessel beams as claimed in claim 4, characterized in that: The multi-plane imaging device comprises an objective lens, a dichroic mirror, a fourth lens, a beam splitter prism, a fifth lens, a tube lens, a filter and an image sensor; the light generated by the sample is collected by the objective lens and then focused on a primary image plane after being reflected by the dichroic mirror and converged by the fourth lens; the beam splitter prism maps images at different axial positions to different positions on the same plane; the image is then transmitted to the image sensor after passing through the fifth lens, the tube lens and the filter; the image sensor cooperates with the movement direction and speed of the one-dimensional scanning galvanometer to realize simultaneous imaging of multiple planes.

6. A high-speed wide-field multi-plane tomography method based on Bessel beams as claimed in claim 5, characterized in that: The beam splitter prism is composed of a plurality of reflectors and a beam splitter. By setting different optical paths between the beam splitter entrance and each exit in advance, different axial positions of the object side are mapped to different lateral areas of the same axial position on the image plane.

7. A high-speed wide-field multi-plane tomography device based on Bessel beam, characterized in that: It includes a beam generating device for generating, collimating and amplifying the beam; A Bessel beam modulation device is used to generate a one-dimensional Bessel beam and scan it; a selective illumination device is used to generate an excitation pattern and select an excitation area of ​​the sample; A multi-plane imaging device for mapping object planes at different axial positions to different areas of the image sensor; The synchronous control device is used for the electronically controlled elements in the synchronous control device and the feedback image analysis results; the light beam generating device, the Bessel light beam modulating device and the selective lighting device constitute the lighting light path; the multi-plane imaging device constitutes the imaging light path.

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