Wide field-of-view multiphoton imaging system

Abstract

This application provides a large field-of-view multiphoton imaging system, comprising: a first scanning galvanometer for Y-axis scanning of an image; a first optical transmission module including a first telecentric plan scanning lens and a second telecentric plan scanning lens; a second scanning galvanometer for X-axis scanning of an image; a second optical transmission module including a third telecentric plan scanning lens and a telescope, the telescope including a pair of achromatic cemented doublet lenses placed at a specific distance; an aberration compensation module for correcting optical aberrations in the entire optical path; and an objective lens driven by a piezoelectric actuator for Z-axis scanning of the image.

Description

Technical Field

[0001] This invention relates to the field of microscopic imaging, and in particular to a large field-of-view multiphoton imaging system. Background Technology

[0002] In the field of neuroscience research, in vivo depth imaging is a core tool for revealing brain function, and multiphoton microscopy is currently widely used for imaging brain tissue. However, existing multiphoton imaging techniques have significant limitations. For example, to improve fluorescence collection efficiency, high-power objectives with large numerical apertures (such as the Olympus 25X objective) are usually required, which results in a small imaging field of view, typically limited to 1 mm. 2 The following limitations make it difficult to meet the needs of large-scale neural network research. If low-magnification objectives are used to increase the imaging field of view, the limited numerical aperture (typically around 0.28) results in a fluorescence collection efficiency of only about 4%, which is particularly significant in deep tissue imaging. This low fluorescence collection efficiency limits the imaging depth and quality, leading to weak signals and low signal-to-noise ratios in deep tissue imaging, making it difficult to provide high-quality data. Furthermore, existing multiphoton imaging systems suffer from severe optical aberrations at the edges of the field of view, resulting in uneven image quality, lower resolution and fluorescence intensity in edge regions, and affecting the overall accuracy and consistency of the data. Summary of the Invention

[0003] To address at least some of the technical problems, this application provides a large field-of-view volume imaging system.

[0004] This application discloses a large field-of-view multiphoton imaging system, comprising: a first scanning galvanometer for X-axis scanning; a first optical transmission module including a first telecentric plan scanning lens and a second telecentric plan scanning lens, wherein the first scanning galvanometer is disposed on the front focal plane of the first telecentric plan scanning lens, and the rear focal plane of the first telecentric plan scanning lens coincides with the rear focal plane of the second telecentric plan scanning lens; a second scanning galvanometer for Y-axis scanning, wherein the second scanning galvanometer is disposed on the front focal plane of the second telecentric plan scanning lens; and a second optical transmission module including a third telecentric plan scanning lens and a tube lens, wherein the third... The front focal plane of the telecentric plan scanning lens coincides with the front focal plane of the second telecentric plan scanning lens. The tube lens includes a first achromatic cemented doublet and a second achromatic cemented doublet, which are arranged opposite each other at a specific distance. The front focal plane of the tube lens coincides with the rear focal plane of the third telecentric plan scanning lens. An aberration compensation module includes at least two lenses for correcting at least one of the following optical aberrations in the entire optical path: spherical aberration, chromatic aberration, astigmatism, field curvature, or coma. An objective lens has its front focal plane coincide with the rear focal plane of the tube lens, which is adjusted by the aberration compensation module. The objective lens is driven by a piezoelectric actuator for Z-axis scanning.

[0005] This application also discloses that the aberration compensation module includes: a first lens, which is a plano-convex lens; a second lens, which is a plano-concave lens; a third lens, which is a plano-concave lens; and a fourth lens, which is a plano-convex lens; wherein the convex surface of the first lens is disposed opposite to the concave surface of the second lens, the plane of the second lens is disposed opposite to the plane of the third lens, and the concave surface of the third lens is disposed opposite to the convex surface of the fourth lens.

[0006] This application also discloses that the objective lens is a long working distance fluorescence objective lens.

[0007] This application also discloses that the long working distance fluorescence objective is a long working distance four-fold fluorescence objective, which includes: a fifth lens, which is a meniscus lens with negative optical power, and the convex surface of the fifth lens is oriented towards the incident direction; and a sixth lens, which is also a meniscus lens with negative optical power, and is positioned close to the fifth lens, with the concave surface of the sixth lens facing the concave surface of the fifth lens. The fifth and sixth lenses are used to adjust the back focal length of the objective, thereby controlling the working distance of the objective. The seventh lens, a cemented triplet lens, has positive optical power and is used to correct at least one of the following optical aberrations of the objective lens: astigmatism, field curvature, or chromatic aberration; the eighth lens, a biconvex lens, has positive optical power; and the ninth lens, a meniscus lens, has negative optical power, with its convex surface facing the eighth lens. The eighth and ninth lenses are used to adjust the front focal length of the objective lens and also to correct at least one of the following optical aberrations of the objective lens: spherical aberration, astigmatism, field curvature, or chromatic aberration.

[0008] This application also discloses that the large field-of-view multiphoton imaging system further includes: an optical path time-division multiplexing module disposed upstream of the optical path of the first scanning galvanometer, wherein the optical path time-division multiplexing module splits the incident laser pulse beam into at least two beams and then combines them, wherein the optical path difference between adjacent beams is the same, thereby making each incident laser pulse separated into at least two laser pulses with the same time interval after passing through the optical path time-division multiplexing module, and each beam is provided with its own tenth lens, and an eleventh lens is provided on the optical path after beam combining, wherein the distance between the respective tenth lens and the eleventh lens is set so that the focal points of each beam are offset by the same distance on the Z-axis.

[0009] This application also discloses that the optical path time-division multiplexing module splits an incident laser pulse beam into a first optical path, a second optical path, a third optical path, and a fourth optical path, and then combines them. The optical path time-division multiplexing module includes: a first beam splitter for splitting the laser pulse beam into the first optical path and the first split optical path; a second beam splitter for splitting the first split optical path into the second optical path and the second split optical path; a third beam splitter for splitting the second split optical path into the third optical path and the fourth optical path; a fourth beam splitter for combining the third optical path and the fourth optical path into a first combined optical path; a fifth beam splitter for combining the second optical path and the first combined optical path into a second combined optical path; and a sixth beam splitter for combining the first optical path and the second combined optical path into an output beam.

[0010] This application also discloses that the first, second, and third beam splitters are polarizing beam splitting prisms, and half-wave plates are respectively provided on the first and second beam splitting optical paths, so that the first optical path after beam splitting is p-polarized light, and the second, third, and fourth optical paths are s-polarized light; the fourth and fifth beam splitters are 1:1 unpolarized beam splitting prisms, the sixth beam splitter is a polarizing beam splitting prism, and half-wave plates are respectively provided on the first, second, and third optical paths to perform beam combining.

[0011] This application also discloses that the large field-of-view multiphoton imaging system further includes a parabolic lateral fluorescence collection module, which comprises: a parabolic lateral reflection module having a parabolic mirror, wherein the imaging sample is positioned at the focal point of the parabolic mirror, and the parabolic lateral reflection module has an opening facing the objective lens for allowing the light beam to be incident on the imaging sample; a focusing module for focusing the light beam that is reflected parallel to the objective lens; a filtering module for filtering out interfering light other than the fluorescence signal; and a fluorescence detection module for detecting the fluorescence signal.

[0012] This application also discloses that the first optical transmission module is a single-time optical transmission module and the second optical transmission module is a 3.4-time optical transmission module.

[0013] This application also discloses that the large field-of-view multiphoton imaging system further includes: a front scanning module, which is disposed upstream of the optical path of the first scanning galvanometer and is used to determine the target field of view. The front scanning module includes: a front scanning galvanometer for scanning along the X-axis or Y-axis; and a front optical transmission module, which is a single-lens optical transmission module, including a first lens pair and a second lens pair. The first lens pair and the second lens pair have the same structure and are arranged opposite to each other in opposite directions. The front optical transmission module is used to conjugate the incident beam to the first scanning galvanometer. The front scanning galvanometer is disposed on the front focal plane of the first lens pair, and the first scanning galvanometer is disposed on the rear focal plane of the second lens pair.

[0014] The large field-of-view multiphoton imaging system disclosed in this application achieves key improvements in performance indicators such as imaging field of view, imaging resolution, and fluorescence collection efficiency by combining advanced optical design, aberration optimization technology, high-speed galvanometer scanning, piezoelectric displacement scanning, optical path time-division multiplexing technology, long working distance fluorescence objective lens, and parabolic reflection collection technology. Attached Figure Description

[0015] Figure 1An optical path design diagram for an embodiment of a large field-of-view multi-subject imaging system;

[0016] Figure 2 This is a schematic diagram of the optical path for another embodiment of a large field-of-view multiphoton imaging system.

[0017] Figure 3 This is a schematic diagram of the structure of one embodiment of the objective lens;

[0018] Figure 4 A three-dimensional schematic diagram of one embodiment of a parabolic lateral fluorescence collection module;

[0019] Figure 5 This is a schematic diagram of the optical path of an embodiment of an optical path time division multiplexing module. Detailed Implementation

[0020] The present application will be further described below with reference to specific embodiments and accompanying drawings. It is to be understood that the illustrative embodiments of this disclosure are merely for explaining the present application and not for limiting it. Furthermore, for ease of description, the accompanying drawings show only the parts relevant to the present application, and not all of the structures or processes.

[0021] The following specific embodiments illustrate the implementation of this application. Those skilled in the art can easily understand other advantages and effects of this application from the content disclosed in this specification. Although the description of this application is presented in conjunction with preferred embodiments, this does not mean that the features of this invention are limited to these embodiments. On the contrary, the purpose of describing the invention in conjunction with embodiments is to cover other options or modifications that may arise based on the claims of this application. To provide a thorough understanding of this application, many specific details will be included in the following description. This application may also be implemented without using these details. Furthermore, to avoid confusion or obscuring the focus of this application, some specific details will be omitted in the description.

[0022] Unless the context otherwise specifies, the terms “contains,” “has,” and “includes” are synonyms. The phrase “A / B” means “A or B.” The phrase “A and / or B” means “(A and B) or (A or B).”

[0023] It should be understood that although terms such as "first," "second," "one," "another," etc., may be used herein to describe various components, units, data, or things, these components, units, data, or things should not be limited by these terms. These terms are used merely to distinguish one feature from another. For example, without departing from the scope of the exemplary embodiments, a first feature may be referred to as a second feature, and similarly, a second feature may be referred to as a first feature; additionally, one may be referred to as another, and similarly, another may be referred to as one.

[0024] It should be understood that although directional terms such as "up," "down," "left," and "right" may be used here to describe the positional relationship between the various components, these directional terms are only for the convenience of understanding and are not intended to limit the scope of protection of this application.

[0025] It should be noted that in this specification, similar reference numerals and letters in the accompanying drawings represent the same or similar items. Therefore, once an item is defined in one drawing, it does not need to be further defined and explained in subsequent drawings.

[0026] To make the objectives, technical solutions, and advantages of this application clearer, the embodiments of this application will be described in further detail below with reference to the accompanying drawings.

[0027] Figure 1 This is a schematic diagram of the optical path of one embodiment of the large field-of-view multiphoton imaging system involved in this application. Figure 1 As shown, the large field-of-view multiphoton imaging system of this application includes: a first scanning galvanometer 5, a first optical transmission module, a second scanning galvanometer 8, a second optical transmission module, an aberration compensation module, and an objective lens 15. In this application, the coordinate system of the image includes an X-axis, a Y-axis, and a Z-axis. The X-axis and Y-axis define a plane (XY plane), and the Z-axis, perpendicular to the XY plane, is used to represent depth information.

[0028] The first scanning mirror 5 is used for X-axis scanning. Preferably, the mirror area of ​​the first scanning mirror 5 is comparable to the spot area of ​​the incident light beam. In this embodiment, the first scanning mirror 5 is a resonant mirror with a high scanning frequency, i.e., the X-axis is a fast axis.

[0029] The incident light beam, after being scanned by the first scanning galvanometer 5, is incident on the first optical transmission module. The first optical transmission module includes a first telecentric flat-field scanning lens 6 and a second telecentric flat-field scanning lens 7. The first scanning galvanometer 5 is disposed on the front focal plane of the first telecentric flat-field scanning lens 6, and the rear focal plane of the first telecentric flat-field scanning lens 6 coincides with the rear focal plane of the second telecentric flat-field scanning lens 7. Preferably, the first telecentric flat-field scanning lens 6 and the second telecentric flat-field scanning lens 7 are two identical telecentric flat-field scanning lenses, and the second telecentric flat-field scanning lens 7 is disposed in the opposite direction to the first telecentric flat-field scanning lens 6. The first optical transmission module can transmit the incident light beam from the front focal plane of the first telecentric flat-field scanning lens 6 to the front focal plane of the second telecentric flat-field scanning lens 7 completely. In this application, the front focal plane of the telecentric flat-field scanning lens refers to the focal plane of the telecentric flat-field scanning lens in the light incident direction under normal use. In contrast, the rear focal plane of the telecentric flat-field scanning lens refers to the focal plane of the telecentric flat-field scanning lens in the light emitting direction under normal use. In other words, in this application, the "front" and "back" of the front and rear focal planes of the telecentric flat scanning lens do not refer to the front and back of the optical path itself.

[0030] The second scanning mirror 8 is used for Y-axis scanning. Preferably, the mirror area of ​​the second scanning mirror 8 is larger than that of the first scanning mirror 5. In this embodiment, the second scanning mirror 8 is a conventional mirror with a scanning frequency lower than that of the first scanning mirror 5, i.e., the Y-axis is a slow axis. The second scanning mirror 8 is disposed on the front focal plane of the second telecentric plan scanning lens 7.

[0031] After the incident beam is scanned by the first scanning mirror, the area illuminating the second scanning mirror is larger than the spot area. Therefore, it is preferable to select a first scanning mirror with a mirror area comparable to the spot area of ​​the incident beam, and a second scanning mirror with a larger mirror area than the first. This ensures that the entire incident beam scanned along the X-axis by the first scanning mirror can be received by the second scanning mirror for further scanning along the Y-axis. Furthermore, since the fast axis scanning frequency is relatively high, a resonant mirror is required. The scanning frequency of a resonant mirror is related to its size; generally, smaller size results in lower mass and higher scanning frequency. The cost of a resonant mirror also increases with its size. Therefore, to reduce costs and obtain a higher scanning frequency, it is preferable to use a resonant mirror for the first scanning mirror for fast axis scanning and a conventional mirror for the second scanning mirror for slow axis scanning. However, those skilled in the art should readily realize that this application is not limited thereto. In other feasible embodiments, a conventional galvanometer with appropriate parameters can be selected as the first scanning galvanometer to perform slow-axis scanning, and then a resonant galvanometer with appropriate parameters can be selected as the second scanning galvanometer to perform fast-axis scanning.

[0032] The incident light beam, after being scanned by the second scanning galvanometer 8, is incident on the second optical transmission module, which includes a third telecentric plan scanning lens 9 and a telescope. The second scanning galvanometer 8 is positioned on the front focal plane of the third telecentric plan scanning lens 9, meaning that the front focal plane of the third telecentric plan scanning lens 9 coincides with the front focal plane of the second telecentric plan scanning lens 7. The telescope includes a first achromatic cemented doublet 10 and a second achromatic cemented doublet 10'. The first achromatic cemented doublet 10 and the second achromatic cemented doublet 10' are positioned opposite each other at a specific distance, together forming a lens pair with a specific focal length as the telescope of the system. The front focal plane of the telescope coincides with the rear focal plane of the third telecentric plan scanning lens 9.

[0033] After being transmitted through the second optical transmission module, the incident light beam is incident on the aberration compensation module 12. The aberration compensation module 12 includes at least two lenses for correcting at least one of the following optical aberrations in the entire optical path: spherical aberration, chromatic aberration, astigmatism, field curvature, or coma. Specifically, in this embodiment, the aberration compensation module 12 includes a first lens 121, a second lens 122, a third lens 123, and a fourth lens 124 arranged sequentially. The first lens 121 is a plano-convex lens, the second lens 122 is a plano-concave lens, the third lens 123 is a plano-concave lens, and the fourth lens 124 is a plano-convex lens. The plane of the first lens 121 faces the second optical transmission module. The convex surface of the first lens 121 is positioned opposite the concave surface of the second lens 122. The plane of the second lens 122 is positioned opposite the plane of the third lens 123. The concave surface of the third lens 123 is positioned opposite the convex surface of the fourth lens 124. The four lenses are arranged at specific distances to correct optical aberrations such as spherical aberration, chromatic aberration, astigmatism, field curvature, and coma in the entire optical path, thereby improving image quality. Those skilled in the art should readily recognize that by selecting lens materials and designing shapes, two, three, five, or more lenses can be used to form an aberration compensation module for correcting optical aberrations.

[0034] The incident light beam passes through the aberration compensation module 12 and then onto the objective lens 15. The front focal plane of the objective lens 15 coincides with the rear focal plane of the tube lens, which has been adjusted by the aberration compensation module 12. The objective lens 15 is driven by a piezoelectric actuator (not shown) for Z-axis scanning. It should be noted that the focal length of the aberration compensation module 12 can be designed to be approximately infinite, so that it hardly affects the rear focal plane of the tube lens. In this case, the front focal plane of the objective lens 15 coincides with the rear focal plane of the tube lens.

[0035] Figure 2 This is a schematic diagram of the optical path for another embodiment of a large field-of-view multiphoton imaging system. Figure 2 Zhongyu Figure 1 The same markings represent the same parts, which will not be elaborated further here.

[0036] Figure 2 The diagram also shows a front-scanning module positioned upstream of the optical path of the first scanning galvanometer 5. While the front-scanning module has a slower scanning speed, this slower speed results in clearer imaging. Before scanning and imaging, it is generally necessary to manually locate the target field of view. To more clearly identify the area to be imaged in advance, the inventors added this front-scanning module to the system. The front-scanning module includes: a front-scanning galvanometer 1 for scanning along the X or Y axis; and a front-scanning optical transmission module, which is a single-lens optical transmission module, including a first lens pair 2 and a second lens pair 4. The first lens pair 2 and the second lens pair 4 have identical structures and are arranged opposite each other. The front-scanning optical transmission module is used to conjugate the incident beam to the first scanning galvanometer 5. The front-scanning galvanometer 1 is positioned on the front focal plane of the first lens pair 2, and the first scanning galvanometer 5 is positioned on the rear focal plane of the second lens pair 4.

[0037] Figure 2 Marks 3 and 11 indicate elliptical plane mirrors used for light reflection. Mark 14 indicates a piezoelectric actuator that drives objective lens 15 to move in the depth direction at the nanometer scale, thereby performing Z-axis scanning. Mark 16 indicates the imaging plane, i.e., the focal plane of the objective lens. Mark 13 indicates a dichroic mirror, which is located on the front focal plane of objective lens 15 and is used to reflect the fluorescence received by the objective lens during imaging to the photomultiplier tube module 17 for fluorescence detection. Figure 2 The illustrated embodiment includes two photomultiplication modules 17 for fluorescence detection in different fluorescence channels.

[0038] Before scanning imaging, the front scanning galvanometer 1 is driven to scan, while the first scanning galvanometer 5 and the second scanning galvanometer 8 are kept stationary. Fluorescence detection is performed by the photomultiplier module 17, and the area to be imaged is determined by observation. Then, the front scanning galvanometer 1 is stopped to keep it stationary, and the first scanning galvanometer 5, the second scanning galvanometer 8, and the piezoelectric actuator 14 are driven to perform scanning imaging.

[0039] Figure 3 This is a schematic diagram of one embodiment of the objective lens. In this application, the objective lens is preferably a long working distance objective lens. Specifically, Figure 3The objective lens shown is a long working distance 4x fluorescence objective lens. In this embodiment, the objective lens includes, from the incident direction to the exit direction, a fifth lens 18, a sixth lens 19, a seventh lens 20, an eighth lens 21, and a ninth lens 22, for a total of five lenses. The fifth lens 18 is a meniscus lens with its convex surface facing the incident direction. It is made of H-K9L material and has negative optical power. The sixth lens 19 is also a meniscus lens, also made of H-K9L material, and has negative optical power. The sixth lens 19 is positioned close to the fifth lens 18, with its concave surface facing the concave surface of the fifth lens 18. The seventh lens 20 is a cemented triode lens with positive optical power. The materials of its three lenses are H-FK61, H-ZF1, and H-ZF62, respectively. The eighth lens 21 is a biconvex lens, made of H-FK61 material, and has positive optical power. The ninth lens 22 is a meniscus lens with its convex surface facing the eighth lens, i.e., facing the incident direction. It is made of H-ZF62 material and has negative optical power. The fifth lens 18 and the sixth lens 19 are used to adjust the back focal length of the objective lens and control the working distance of the objective lens; the seventh lens 20 is used to correct aberrations such as astigmatism, field curvature, and chromatic aberration of the objective lens; the eighth lens 21 and the ninth lens 22 are used to adjust the front focal length of the objective lens, and are also used to optimize aberrations such as spherical aberration, astigmatism, chromatic aberration, and field curvature of the objective lens.

[0040] Figure 4 This is a three-dimensional schematic diagram of one embodiment of a parabolic lateral fluorescence collection module. The parabolic lateral fluorescence collection module includes a parabolic lateral reflection module, a focusing module, a filtering module, and a fluorescence detection module.

[0041] Mark 23 is a parabolic lateral reflection module, which has a parabolic mirror to improve the fluorescence stimulation efficiency of the system. During scanning imaging, the sample to be tested, such as the brain of a laboratory mouse, is placed at the focal point of the parabolic mirror. The incident light beam (i.e., the excitation light) is converged by the objective lens 15 and enters through the opening above the parabolic reflection module 23 and is focused inside the brain of the laboratory mouse. The stimulated fluorescence excited at the focal point is scattered from inside the brain of the laboratory mouse to the surroundings, and after being reflected by the parabolic lateral reflection module 23, it is emitted parallel to the optical axis of the objective lens 15.

[0042] The focusing module is used to focus the parallel beam emitted after being reflected by the parabolic lateral reflection module 23. It includes a first focusing lens 24, which is located near the exit of the parabolic lateral reflection module 23. It is used to converge the emitted beam after being reflected by the parabolic lateral reflection module 23 for the first time, thereby reducing the aperture of the emitted beam.

[0043] Designated as a filter module 25, it is positioned after the first focusing lens 24 and is used to filter out interference light such as excitation light and natural light, in addition to the fluorescence signal in the emitted beam. The filter module 25 can also be used to separate fluorescence signals from different channels, for example, fluorescence signals including red fluorescence and green fluorescence. The filter module 25 includes a long-pass filter, a dichroic mirror, and two band-pass filters. The long-pass filter is positioned at the entrance of the filter module 25 to filter out interference light such as excitation light and short-wavelength natural light. Then, the dichroic mirror, positioned inside the filter module 25 and forming a 45-degree angle with the optical axis of the emitted beam, splits the emitted beam into two fluorescence signals. The two band-pass filters are positioned at the two exit points of the filter module 25 to further filter out interference light.

[0044] The focusing module also includes a second focusing lens 26 and a third focusing lens 27, which are used to further converge the fluorescence signal and project it onto the fluorescence detection module.

[0045] The fluorescence detection module includes a photomultiplier tube (PMT) 29 and a water-cooling module 28. The PMT 29 is used for fluorescence detection, and the water-cooling module 28 is used to dissipate heat from the PMT 29.

[0046] Figure 5 This is a schematic diagram of the optical path of one embodiment of an optical time-division multiplexing module. Figure 5 As shown, mark 30 is a reflector. Figure 5The time-division multiplexing module shown contains multiple lenses to change the transmission direction of light, thereby making full use of system space. Labels 31-33 are polarizing beam splitters (PBS), used to split the incident beam into four beams: a first beam, a second beam, a third beam, and a fourth beam, while simultaneously changing the polarization direction of the beams. The first beam is p-polarized light, while the second, third, and fourth beams are s-polarized light. Labels 34-37 are the tenth lenses respectively placed on the first to fourth beam paths, and label 41 is the eleventh lens placed on the beam path after the first to fourth beam paths are combined. Tenth lenses 34-37 and eleventh lens 41 combine to form a four-way focusing module to adjust the depth position of the focal points of the first to fourth beam paths. In other words, by setting the distance between tenth lenses 34-37 and eleventh lens 41, the focal points of the beams in the first to fourth beam paths are offset by the same distance on the Z-axis. Label 38 is a 1:1 unpolarized beam splitter (BS), used to combine the fourth and third optical paths. This beam combining results in a loss of half the optical power. Label 39 is also a 1:1 unpolarized beam splitter (BS), used to further combine the combined fourth and third optical paths with the second optical path. Here, beam combining also results in a loss of half the optical power. Label 40 is a polarized beam splitter (PBS), used to further combine the combined fourth, third, and second optical paths with the first optical path. Because polarized beam splitter 40 is used, it combines the s-polarized light from the combined second, third, and fourth optical paths with the p-polarized light from the first optical path, thus eliminating optical power loss. Label 42 is a half-wave plate, which exists in multiple times in the time-division multiplexing module, positioned between two beam splitters. It is used to adjust the optical power of the beams in different optical paths by changing the polarization direction of the beam. It should be noted that... Figure 5 In this paper, due to the limited space, the entire optical path of the fourth optical path cannot be fully displayed, so a curve is used for illustration.

[0047] The optical path time-division multiplexing module is positioned upstream of the optical path of the first scanning galvanometer. Figure 5 In the illustrated embodiment, the optical path time-division multiplexing module splits the incident laser pulse beam into four beams and then combines them. However, this application is not limited to this. The optical path time-division multiplexing module is used to split the incident beam into at least two beams and then combine them. The optical path difference between adjacent beams is the same, so that each incident laser pulse is separated into at least two laser pulses with the same time interval after passing through the optical path time-division multiplexing module. Each beam is provided with its own tenth lens, and an eleventh lens is provided on the optical path after beam combining. By setting the distance between the tenth and eleventh lenses, the focal points of each beam are offset by the same distance on the Z-axis, thereby enabling simultaneous multi-layer scanning of images.

[0048] According to one embodiment of the large field-of-view multiphoton imaging system of this application, a 6×6×0.5mm... 3 The imaging range. In this embodiment, the two main parameters of the selected first scanning galvanometer are an effective diameter of 5 mm and a maximum swing angle of ±13°, and it uses... Figure 3 The illustrated long working distance 4x fluorescence objective has a focal length of 45mm. To achieve imaging within a 6mm range, calculations show that the incident light at the rear focal plane of the objective requires an incident angle of at least 3.814°. Since 13 / 3.814 ≈ 3.4, the optical transmission magnification of the entire system should be 3.4x. The overall optical transmission magnification is determined by the product of the optical transmission magnifications of the first and second optical transmission modules. In this embodiment, the effective diameter of the second scanning mirror is selected as 6mm. To fully utilize the energy of the incident beam and ensure that the scanning beam after the first optical transmission module is incident within the effective diameter of the second scanning mirror as much as possible, a 1x optical transmission module is selected for the first optical transmission module, and a 3.4x optical transmission module is selected for the second optical module.

[0049] The laser source was an 80MHz two-photon laser, and the second scanning mirror was an 8kHz resonant mirror. The system sampled at 2048×2048 pixels / frame, with a frame rate of 7.7 frames / second. Each pixel was 3×3μm in size. 2 Furthermore, each pixel can only be allocated 1-2 laser pulses, which means that the pixel sampling speed limit has been reached at a laser pulse repetition frequency of 80MHz. In this design, the resolution of large field-of-view high-speed imaging is determined by the pixel size, because the pixel size (3×3μm)... 2 It has exceeded the lateral optical resolution (1×1μm) 2 This means that if the field of view is increased further, pixel resolution will be sacrificed.

[0050] The entire optical system adopts the Petzval design concept to optimize beam propagation and ensure high-resolution and low-aberration imaging quality. Simulation evaluation is performed based on multiple indicators such as PSF peak value, RMS curve, and field curvature. Through simulation and optimization, the system utilizes... Figure 1 and Figure 2 The aberration compensation module shown effectively corrects aberrations such as spherical aberration, coma, astigmatism, and field curvature in the system, ensuring the clarity of the entire imaging field of view.

[0051] The system uses a piezoelectric actuator to drive the objective lens longitudinally at a frequency of 1.1 Hz, thereby achieving volumetric imaging within a 130 μm range along the Z-axis. The interlayer distance is set at 18.5 μm, with a total of 7 layers, and 1.1 volumetric images can be acquired per second. Simultaneously, this interlayer distance is very close to the system's axial resolution of 19 μm, ensuring the integrity and continuity of data acquisition.

[0052] To further expand the imaging range along the longitudinal direction, i.e., the Z-axis, the system employs... Figure 5 The optical path time-division multiplexing module shown splits the incident laser pulse beam into four beams, with the focal points of each beam offset by the same distance of 130 μm on the Z-axis and a time delay of 3.125 ns. This design, combined with a longitudinal 7-layer scan, achieves a total of 28 layers and a 520 μm three-dimensional volumetric imaging, which meets the penetration depth requirements of two-photon calcium imaging.

[0053] The system adds between the objective lens and the imaging sample. Figure 4 The parabolic lateral fluorescence collection module shown in this paper reflects most of the fluorescence signals that cannot enter the objective lens laterally by the parabolic mirror, allowing them to be collected and detected by the fluorescence detection module. Existing multiphoton imaging systems suffer from a trade-off between fluorescence collection efficiency and imaging field of view, caused by the objective lens size. This application's system avoids this theoretical limitation because the fluorescence signal no longer needs to pass through the objective lens for collection. By combining a parabolic mirror, a self-designed focusing module, and a large-area photomultiplier tube, this application increases the fluorescence collection efficiency from the traditional 4% to over 40%. This application comprehensively considers limitations such as objective lens focal length, light source size, commonly used lens aperture size, and photomultiplier tube collection area size, specifically optimizing the opening size and curve length of the parabolic curve. This allows the system to obtain high-quality imaging data even at the extreme depths of two-photon calcium imaging (approximately 600 μm), significantly improving imaging quality. Furthermore, this application's system is also compatible with existing fluorescence collection modules utilizing the objective lens, meaning that fluorescence returning to the objective lens can be collected simultaneously, further enhancing fluorescence collection efficiency.

[0054] Given the system's large 6mm × 6mm field of view, the fluorescence collection efficiency at different locations within the target field of view may vary significantly. Through optical simulation, this application predicts the fluorescence collection efficiency at different locations within the target field of view and further evaluates the fluorescence collection efficiency at different locations based on the relationship between the filter spectral lines and the incident light angle, ensuring consistent imaging quality across the entire field of view. Specifically, this application comprehensively considers the fluorescence collection efficiency and incident light angle at different positions within the target field of view. First, the parabolic curve of the parabolic reflector was optimized by increasing the size of the parabolic opening while reducing its curve length. This ensures that photons from different points on the imaging surface travel the same transmission distance before being reflected by the parabolic surface. In this way, the fluorescence emitted from different points on the imaging surface can be emitted from the side of the parabolic surface as parallel as possible. Then, we adjusted the focal length and spacing of the three focusing lenses in the focusing module to ensure that the 2-inch aperture beam reflected from the parabolic reflector can be converged to the photomultiplier tube collecting surface while its incident light angle is not too large. Ultimately, this achieves a significant improvement in the overall fluorescence collection efficiency of the imaging surface while maintaining no significant difference in collection efficiency at each point.

[0055] To match the parabolic lateral fluorescence collection module, the system used Figure 3 The long working distance 4x fluorescence objective shown has a working distance of 30.5–31 mm. The longer working distance allows for more design flexibility in the parabolic lateral fluorescence collection module, enabling higher efficiency. Through optical simulation optimization, aberration compensation and imaging performance are balanced while ensuring the long working distance of the objective, allowing the objective to meet the requirements of the parabolic lateral fluorescence collection system while achieving near-ideal imaging quality.

[0056] This invention proposes a groundbreaking large field-of-view multiphoton imaging system with significant advantages in biomedical engineering and neuroscience research. By combining advanced optical design, aberration optimization techniques, high-speed galvanometer scanning, piezoelectric displacement scanning, time-division multiplexing of the optical path, long working distance fluorescence objectives, and parabolic reflection collection technology, key improvements in performance indicators such as imaging field of view, imaging resolution, and fluorescence collection efficiency are achieved for the large field-of-view multiphoton imaging system. The following are the main beneficial effects of this application compared with existing technologies.

[0057] Larger field of view and improved resolution: The imaging field of view of this application is increased from the traditional 1mm. 2 The following is extended to 6×6×0.5mm 3 The resolution reached 1×1×19μm 3This significant improvement allows researchers to capture the activity of millions of neurons in a single image, expanding the scope of observation and enhancing the resolution of coordinated neural network activities. Compared to existing multiphoton microscopy systems, this invention enables high-quality three-dimensional imaging within a larger volume, greatly increasing data throughput and the breadth of research.

[0058] Significantly enhanced fluorescence collection efficiency: Existing multiphoton microscopy systems typically use standard 4x objectives to improve the imaging field of view. Their fluorescence collection efficiency is limited by the numerical aperture of the objective (taking a 4x objective as an example), usually only around 4%. This application, however, utilizes an innovative parabolic lateral fluorescence collection module, placing the module between the objective and the sample. Fluorescence collection is no longer limited by the objective, increasing the efficiency to over 40%. Combined with a self-designed long working distance fluorescence objective, this design resolves the contradiction between objective size limitations and fluorescence collection efficiency, significantly improving the signal-to-noise ratio and signal quality of deep imaging, ensuring high-quality image data can still be acquired in two-photon calcium imaging at a depth of 600 μm.

[0059] The above are merely specific embodiments of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Where there is no conflict, the embodiments and features described in the embodiments of this application can be combined with each other. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A large field-of-view multiphoton imaging system, characterized in that, include: The first scanning galvanometer is used for X-axis scanning. The first optical transmission module includes a first telecentric flat-field scanning lens and a second telecentric flat-field scanning lens. The first scanning galvanometer is disposed on the front focal plane of the first telecentric flat-field scanning lens, and the rear focal plane of the first telecentric flat-field scanning lens coincides with the rear focal plane of the second telecentric flat-field scanning lens. The second scanning galvanometer is used for Y-axis scanning and is disposed on the front focal plane of the second telecentric flat scanning lens. The second optical transmission module includes a third telecentric plan scanning lens and a tube lens. The front focal plane of the third telecentric plan scanning lens coincides with the front focal plane of the second telecentric plan scanning lens. The tube lens includes a first achromatic cemented doublet lens and a second achromatic cemented doublet lens. The first achromatic cemented doublet lens and the second achromatic cemented doublet lens are arranged opposite each other at a specific distance. The front focal plane of the tube lens coincides with the rear focal plane of the third telecentric plan scanning lens. An aberration compensation module, comprising at least two lenses, is used to correct at least one of the following optical aberrations in the entire optical path: spherical aberration, chromatic aberration, astigmatism, field curvature, or coma; and The objective lens has its front focal plane coincide with the rear focal plane of the tube lens, which is adjusted by the aberration compensation module. The objective lens is driven by a piezoelectric actuator for Z-axis scanning.

2. The large field-of-view multiphoton imaging system according to claim 1, characterized in that, The aberration compensation module includes: The first lens is a plano-convex lens; The second lens is a plano-concave lens; A third lens, wherein the third lens is a plano-concave lens; and The fourth lens is a plano-convex lens; wherein, The convex surface of the first lens is disposed opposite to the concave surface of the second lens, the plane of the second lens is disposed opposite to the plane of the third lens, and the concave surface of the third lens is disposed opposite to the convex surface of the fourth lens.

3. The large field-of-view multiphoton imaging system according to claim 1, characterized in that, The objective lens is a long working distance fluorescence objective lens.

4. The large field-of-view multiphoton imaging system according to claim 3, characterized in that, The long working distance fluorescence objective is a long working distance four-fold fluorescence objective, which includes: The fifth lens is a meniscus lens with negative optical power, and the convex surface of the fifth lens is oriented towards the incident direction. The sixth lens is a meniscus lens with negative optical power. The sixth lens is closely attached to the fifth lens, with the concave surface of the sixth lens facing the concave surface of the fifth lens. The fifth and sixth lenses are used to adjust the back focal length of the objective lens, thereby controlling the working distance of the objective lens. The seventh lens, which is a cemented triplicate lens, has positive optical power and is used to correct at least one of the following optical aberrations of the objective lens: astigmatism, field curvature, or chromatic aberration; The eighth lens, which is a biconvex lens, has positive optical power; and The ninth lens is a meniscus lens with negative optical power. The convex surface of the ninth lens is positioned facing the eighth lens. The eighth lens and the ninth lens are used to adjust the front focal length of the objective lens and also to correct at least one of the following optical aberrations of the objective lens: spherical aberration, astigmatism, field curvature, or chromatic aberration.

5. The large field-of-view multiphoton imaging system according to claim 1, characterized in that, Also includes: An optical path time-division multiplexing module is disposed upstream of the optical path of the first scanning galvanometer. The optical path time-division multiplexing module splits the incident laser pulse beam into at least two beams and then combines them. The optical path difference between adjacent beams is the same, so that each incident laser pulse is separated into at least two laser pulses with the same time interval after passing through the optical path time-division multiplexing module. Each beam is provided with its own tenth lens, and an eleventh lens is provided in the optical path after beam combining. The distance between the respective tenth and eleventh lenses is set so that the focal points of each beam are offset by the same distance on the Z-axis.

6. The large field-of-view multiphoton imaging system according to claim 5, characterized in that, The optical path time-division multiplexing module splits the incident laser pulse beam into a first optical path, a second optical path, a third optical path, and a fourth optical path, and then combines them. The optical path time-division multiplexing module includes: A first beam splitter is used to split the laser pulse beam into the first optical path and the first beam-splitting optical path; The second beam splitter is used to split the first beam splitting optical path into the second optical path and the second beam splitting optical path. The third beam splitter is used to split the second beam-splitting optical path into the third optical path and the fourth optical path; The fourth beam splitter is used to combine the third optical path and the fourth optical path into a first combined optical path; The fifth beam splitter is used to combine the second optical path and the first beam combiner into a second beam combiner. The sixth beam splitter is used to combine the first optical path and the second beam combining optical path into an output beam.

7. The large field-of-view multiphoton imaging system according to claim 6, characterized in that, The first, second, and third beam splitters are polarizing beam splitting prisms, and half-wave plates are respectively provided on the first and second beam splitting optical paths, so that the first optical path after beam splitting is p-polarized light, and the second, third, and fourth optical paths are s-polarized light; the fourth and fifth beam splitters are 1:1 unpolarized beam splitting prisms, and the sixth beam splitter is a polarizing beam splitting prism, and half-wave plates are respectively provided on the first, second, and third optical paths, thereby performing beam combining.

8. The large field-of-view multiphoton imaging system according to claim 3, characterized in that, It also includes a parabolic lateral fluorescence collection module, which comprises: A parabolic lateral reflection module has a parabolic mirror, and an imaging sample is placed at the focal point of the parabolic mirror. The parabolic lateral reflection module has an opening on the side facing the objective lens for allowing a light beam to be incident on the imaging sample. A focusing module is used to focus the light beam that is reflected in parallel by the parabolic lateral reflection module; A filter module, which is used to filter out interfering light other than the fluorescence signal; A fluorescence detection module, which is used to detect fluorescence signals.

9. The large field-of-view multiphoton imaging system according to claim 1, characterized in that, The first optical transmission module is a single-time optical transmission module, and the second optical transmission module is a 3.4-time optical transmission module.

10. The large field-of-view multiphoton imaging system according to claim 1, characterized in that, Also includes: A front-scanning module, disposed upstream of the optical path of the first scanning galvanometer, is used to determine the target field of view. The front-scanning module includes: Front scanning galvanometer, used for scanning the X-axis or Y-axis; A front-mounted optical transmission module, which is a single-magnification optical transmission module, includes a first lens pair and a second lens pair. The first lens pair and the second lens pair have identical structures and are arranged opposite to each other in opposite directions. The front-mounted optical transmission module is used to conjugately transmit the incident light beam to the first scanning galvanometer. The front scanning galvanometer is disposed on the front focal plane of the first lens pair, and the first scanning galvanometer is disposed on the rear focal plane of the second lens pair.

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