Large-view-field multi-photon-body imaging system

Abstract

The invention provides a large-view-field multi-photon-body imaging system, and the system comprises a first scanning galvanometer which is used for the Y-axis scanning of an image; the first optical transmission module comprises a first telecentric flat field scanning lens and a second telecentric flat field scanning lens; the second scanning galvanometer is used for X-axis scanning of the image; the second optical transmission module comprises a third telecentric flat field scanning lens and a barrel lens, and the barrel lens comprises a pair of achromatic doublet lenses which are oppositely placed according to a specific distance; the aberration compensation module is used for correcting optical aberration in the whole light path; and an objective lens driven by a piezoelectric actuator for Z-axis scanning of an image.

Description

Technical Field

[0001] The invention relates to the field of microscopic imaging, and in particular to a large-field multi-photon volume imaging system. Background Art

[0002] In the field of neuroscience research, in vivo deep imaging is a core tool for revealing brain functions, and multiphoton microscopy is currently widely used for imaging brain tissue. However, existing multiphoton imaging technology has significant limitations. For example, in order to improve the efficiency of fluorescence collection, it is usually necessary to use a high-magnification objective lens with a large numerical aperture (such as Olympus's 25X objective lens), which results in a smaller imaging field of view, which is usually limited to 1mm. 2 The following is difficult to meet the needs of large-scale neural network research. If a low-power objective is used to increase the imaging field of view, the fluorescence collection efficiency is only about 4% due to the limited numerical aperture of the low-power objective (usually around 0.28), which is particularly significant when imaging deep tissues. The low fluorescence collection efficiency limits the imaging depth and imaging quality of the system, making the deep tissue imaging signal weak and the signal-to-noise ratio low, making it difficult to provide high-quality data. In addition, existing multi-photon imaging systems have serious optical aberrations at the edge of the field of view, uneven imaging quality, and low resolution and fluorescence intensity in the edge area, which affects the accuracy and consistency of the overall data. Summary of the invention

[0003] In order to solve at least part of the above technical problems, the present application provides a large field of view volume imaging system.

[0004] The present application discloses a large-field-of-view multi-photon volume imaging system, comprising: a first scanning galvanometer, the first scanning galvanometer is used for X-axis scanning; a first optical transfer module, the first optical transfer module comprises a first telecentric flat-field scanning lens and a second telecentric flat-field scanning lens, the first scanning galvanometer is arranged on the front focal plane of the first telecentric flat-field scanning lens, and the back focal plane of the first telecentric flat-field scanning lens coincides with the back focal plane of the second telecentric flat-field scanning lens; a second scanning galvanometer, the second scanning galvanometer is used for Y-axis scanning, and the second scanning galvanometer is arranged on the front focal plane of the second telecentric flat-field scanning lens; a second optical transfer module, the second optical transfer module comprises a third telecentric flat-field scanning lens and a tube lens, the third The front focal plane of the telecentric flat-field scanning lens coincides with the front focal plane of the second telecentric flat-field scanning lens, the tube lens comprises a first achromatic doublet lens and a second achromatic doublet lens, the first achromatic doublet lens and the second achromatic doublet lens are arranged relative to each other at a specific distance, and the front focal plane of the tube lens coincides with the back focal plane of the third telecentric flat-field scanning lens; an aberration compensation module, the aberration compensation module comprises at least two lenses, and 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 an objective lens, the front focal plane of the objective lens coincides with the back focal plane of the tube lens adjusted by the aberration compensation module, and the objective lens is driven by a piezoelectric driver for Z-axis scanning.

[0005] The present 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 arranged opposite to the concave surface of the second lens, the plane of the second lens is arranged opposite to the plane of the third lens, and the concave surface of the third lens is arranged opposite to the convex surface of the fourth lens.

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

[0007] The present application also discloses that the long working distance fluorescent objective lens is a long working distance quadruple fluorescent objective lens, and the long working distance quadruple fluorescent objective lens comprises: a fifth lens, the fifth lens is a meniscus lens, has a negative optical focal length, and the convex surface of the fifth lens is arranged toward the incident direction; a sixth lens, the sixth lens is a meniscus lens, has a negative optical focal length, the sixth lens is arranged closely to the fifth lens, and the concave surface of the sixth lens is arranged toward the concave surface of the fifth lens, and the fifth lens and the sixth lens are used to adjust the back focal length of the objective lens, thereby controlling the working distance of the objective lens ; a seventh lens, the seventh lens is a 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; an eighth lens, the eighth lens is a biconvex lens, has positive optical power; and a ninth lens, the ninth lens is a meniscus lens, has negative optical power, and the convex surface of the ninth lens is arranged toward the eighth lens, the eighth lens and the ninth lens are used to adjust the front focal length of the objective lens, and are also used to correct at least one of the following optical aberrations of the objective lens: spherical aberration, astigmatism, field curvature or chromatic aberration.

[0008] The present application also discloses that the large-field-of-view multi-photon volume imaging system also includes: an optical path time-division multiplexing module arranged 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 the two 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, and each beam is respectively provided with a respective tenth lens, and an eleventh lens is provided on the optical path after combining, and the distance between the respective tenth lens and the eleventh lens is set so that the focus of each beam is staggered by the same distance on the Z axis.

[0009] The present application also discloses that the optical path time-division multiplexing module splits the incident laser pulse light beam into a first optical path, a second optical path, a third optical path and a fourth optical path, and then combines the beams. The optical path time-division multiplexing module includes: a first beam splitter, used to split the laser pulse light beam into the first optical path and a first split optical path; a second beam splitter, used to split the first split optical path into the second optical path and a second split optical path; a third beam splitter, used to split the second split optical path into the third optical path and the fourth optical path; a fourth beam splitter, used to combine the third optical path and the fourth optical path into a first combined optical path; a fifth beam splitter, used to combine the second optical path and the first combined optical path into a second combined optical path; and a sixth beam splitter, used to combine the first optical path and the second combined optical path into an output light beam.

[0010] The present application also discloses that the first beam splitter, the second beam splitter and the third beam splitter are polarization beam splitting prisms, and half-wave plates are respectively arranged on the first beam splitting light path and the second beam splitting light path, so that the first light path after beam splitting is p-polarized light, and the second light path, the third light path and the fourth light path are s-polarized light; the fourth beam splitter and the fifth beam splitter are 1:1 non-polarization beam splitting prisms, the sixth beam splitter is a polarization beam splitting prism, and half-wave plates are respectively arranged on the first light path, the second light path and the third light path, so as to perform beam combining.

[0011] The present application also discloses that the large-field-of-view multi-photon volume imaging system also includes a parabolic lateral fluorescence collection module, which includes: a parabolic lateral reflection module, which has a parabolic reflector, and the imaging sample is arranged at the focus of the parabolic reflector, 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, which is used to focus the light beam emitted in parallel after being reflected by the parabolic lateral reflection module; a filtering module, which is used to filter out interference light other than the fluorescence signal; and a fluorescence detection module, which is used to detect the fluorescence signal.

[0012] The present application also discloses that the first optical transmission module is a single optical transmission module, and the second optical transmission module is a 3.4-fold optical transmission module.

[0013] The present application also discloses that the large-field-of-view multi-photon volume imaging system also includes: a front scanning module, which is arranged upstream of the optical path of the first scanning galvanometer, and is used to determine the target field of view, and the front scanning module includes: a front scanning galvanometer, which is used for scanning the X-axis or Y-axis; a front optical transfer module, which is a single-fold optical transfer 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 relative to each other in opposite directions, and the front optical transfer module is used to conjugately transfer the incident light beam to the first scanning galvanometer; wherein the front scanning galvanometer is arranged on the front focal plane of the first lens pair, and the first scanning galvanometer is arranged on the back focal plane of the second lens pair.

[0014] The large-field-of-view multi-photon volume imaging system disclosed in the present application achieves a key improvement in the performance indicators of the large-field-of-view multi-photon volume imaging system in terms of imaging field of view, imaging resolution, fluorescence collection efficiency, etc. by combining advanced optical design, aberration optimization technology, high-speed galvanometer scanning, piezoelectric displacement scanning, optical path time-division multiplexing technology, long working distance fluorescent objective lens, and parabolic reflection collection technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1The optical path design diagram of an embodiment of a large-field-of-view multi-photon imaging system is shown;

[0016] Figure 2 A schematic diagram of the optical path of another embodiment of a large-field-of-view multiphoton volume imaging system;

[0017] Figure 3 is a schematic structural diagram of an embodiment of an objective lens;

[0018] Figure 4 It is a three-dimensional schematic diagram of an embodiment of a parabolic side fluorescence collection module;

[0019] Figure 5 The figure is a light path schematic diagram of an embodiment of an light path time division multiplexing module. DETAILED DESCRIPTION

[0020] The present application is further described below in conjunction with specific embodiments and drawings. It is to be understood that the illustrative embodiments of the present disclosure are only intended to explain the present application, rather than to limit the present application. In addition, for ease of description, only some, but not all, structures or processes related to the present application are shown in the drawings.

[0021] The following specific embodiments illustrate the implementation of the present application, and those skilled in the art can easily understand other advantages and effects of the present application from the contents disclosed in this specification. Although the description of the present application will be introduced in conjunction with the preferred embodiment, this does not mean that the features of this invention are limited to this implementation. On the contrary, the purpose of introducing the invention in conjunction with the implementation is to cover other options or modifications that may extend based on the claims of the present application. In order to provide an in-depth understanding of the present application, the following description will include many specific details. The present application can also be implemented without using these details. In addition, in order to avoid confusion or blurring the focus of the present application, some specific details will be omitted in the description.

[0022] Unless the context dictates otherwise, the terms "comprising," "having," and "including" are synonymous. 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 the terms "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 only to distinguish one feature from another. For example, without departing from the scope of the exemplary embodiment, a first feature may be referred to as a second feature, and similarly a second feature may be referred to as a first feature. In addition, 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 of each component, these directional terms are only used to indicate the directions in the drawings for the convenience of understanding and cannot be used to limit the scope of protection of this application.

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

[0026] In order to make the objectives, technical solutions and advantages of the present application clearer, the implementation methods of the present application will be further described in detail below in conjunction with the accompanying drawings.

[0027] Figure 1 FIG. 1 is a schematic diagram of the optical path of an embodiment of a large field of view multi-photon volume imaging system involved in the present application. Figure 1 As shown, the large field of view multi-photon volume imaging system involved in the present 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 the present application, the coordinate system of the image includes an X-axis, a Y-axis, and a Z-axis. The X-axis and the Y-axis define a plane (XY plane), and the Z-axis is perpendicular to the XY plane and is used to represent depth information.

[0028] The first scanning galvanometer 5 is used for X-axis scanning. Preferably, the mirror area of ​​the first scanning galvanometer 5 is equivalent to the spot area of ​​the incident light beam. In this embodiment, the first scanning galvanometer 5 is a resonant galvanometer, which has a higher scanning frequency, that is, the X-axis is a fast axis.

[0029] After being scanned by the first scanning galvanometer 5, the incident light beam 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 arranged on the front focal plane of the first telecentric flat field scanning lens 6. The back focal plane of the first telecentric flat field scanning lens 6 coincides with the back 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 arranged 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 in an identical manner. In the present 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 back focal plane of the telecentric flat field scanning lens refers to the focal plane of the telecentric flat field scanning lens in the light exit direction under normal use. That is to say, in the present application, the “front” and “back” of the front focal plane and the back focal plane of the telecentric flat-field scan lens do not refer to the front and back of the optical path itself.

[0030] The second scanning galvanometer 8 is used for Y-axis scanning. Preferably, the mirror area of ​​the second scanning galvanometer 8 is larger than the mirror area of ​​the first scanning galvanometer 5. In this embodiment, the second scanning galvanometer 8 is an ordinary galvanometer, and its scanning frequency is lower than the scanning frequency of the first scanning galvanometer 5, that is, the Y-axis is a slow axis. The second scanning galvanometer 8 is arranged on the front focal plane of the second telecentric flat-field scanning lens 7.

[0031] After the incident light beam is scanned by the first scanning galvanometer, the area irradiated by the second scanning galvanometer will be larger than the spot area. Therefore, it is preferred to select the mirror area of ​​the first scanning galvanometer to be equivalent to the spot area of ​​the incident light beam, and the mirror area of ​​the second scanning galvanometer is larger than the mirror area of ​​the first scanning galvanometer, so that the incident light beam scanned by the first scanning galvanometer on the X-axis can be completely received by the second scanning galvanometer for further scanning on the Y-axis. In addition, since the scanning frequency of the fast axis is relatively high, a resonant galvanometer is required, and the scanning frequency of the resonant galvanometer is related to its own size. Generally speaking, the smaller the size, the smaller the mass and the higher the scanning frequency, and the cost of the resonant galvanometer will also increase with the increase of the size. Therefore, in order to reduce costs and obtain a higher scanning frequency, it is preferred to select the first scanning galvanometer as a resonant galvanometer for fast-axis scanning and the second scanning galvanometer as an ordinary galvanometer for slow-axis scanning. However, those skilled in the art should be able to easily realize that the present application is not limited to this. In other feasible embodiments, an ordinary galvanometer with appropriate parameters can be selected as the first scanning galvanometer to perform slow-axis scanning first, 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 is scanned by the second scanning galvanometer 8 and then incident on the second optical transmission module, which includes a third telecentric flat-field scanning lens 9 and a tube lens. The second scanning galvanometer 8 is arranged on the front focal plane of the third telecentric flat-field scanning lens 9, that is, the front focal plane of the third telecentric flat-field scanning lens 9 coincides with the front focal plane of the second telecentric flat-field scanning lens 7. The tube lens includes a first achromatic doublet lens 10 and a second achromatic doublet lens 10'. The first achromatic doublet lens 10 and the second achromatic doublet lens 10' are arranged relative to each other at a specific distance, and the two together form a lens pair with a specific focal length as the tube lens of the system. The front focal plane of the tube lens coincides with the back focal plane of the third telecentric flat-field scanning lens 9.

[0033] After the incident light beam is transmitted through the second optical transmission module, it is incident on the aberration compensation module 12. The aberration compensation module 12 includes at least two lenses, which are 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. 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 in sequence, 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 arranged opposite to the concave surface of the second lens 122, the plane of the second lens 122 is arranged opposite to the plane of the third lens 123, the concave surface of the third lens 123 is arranged opposite to the convex surface of the fourth lens 124, and the four lenses are arranged at a specific distance to correct the optical aberrations such as spherical aberration, chromatic aberration, astigmatism, field curvature and coma in the entire optical path, so as to improve the imaging quality. Those skilled in the art should be able to easily think that two, three, five or more lenses can be used to form an aberration compensation module for correcting optical aberrations through the selection of lens materials and the design of shapes.

[0034] After the incident light beam passes through the aberration compensation module 12, it is incident on the objective lens 15, and the front focal plane of the objective lens 15 coincides with the back focal plane of the tube lens adjusted by the aberration compensation module 12. The objective lens 15 is driven by a piezoelectric driver (not shown) for Z-axis scanning. It should be pointed out here that the focal length of the aberration compensation module 12 can be designed to be approximately infinite, so that it will hardly affect the back focal plane of the tube lens. In this case, the front focal plane of the objective lens 15 coincides with the back focal plane of the tube lens.

[0035] Figure 2 FIG. 1 is a schematic diagram of the optical path of another embodiment of a large-field-of-view multiphoton volume imaging system. Figure 2 Zhongyu Figure 1 The same reference numerals represent the same components and their description will not be repeated here.

[0036] Figure 2 Also shown is a front scanning module disposed upstream of the optical path of the first scanning galvanometer 5. The scanning speed of the front scanning module is relatively slow, but the imaging is clearer due to its slow scanning speed. Before scanning imaging, it is generally necessary to manually find the target field of view in advance. In order to more clearly identify the area to be imaged in advance, the inventor added the front scanning module to the system. The front scanning module includes: a front scanning galvanometer 1, which is used for scanning the X-axis or Y-axis; a front optical transfer module, which is a single optical transfer 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 the same structure and are arranged relative to each other in opposite directions. The front optical transfer module is used to conjugately transfer the incident light beam to the first scanning galvanometer 5. The front scanning galvanometer 1 is arranged on the front focal plane of the first lens pair 2, and the first scanning galvanometer 5 is arranged on the back focal plane of the second lens pair 4.

[0037] Figure 2 Mark 3 and 11 are elliptical plane reflectors for light path reflection. Mark 14 is a piezoelectric driver, which can drive the objective lens 15 to move in the depth direction at the nanometer level, so as to perform Z-axis scanning. Mark 16 is an imaging surface, i.e., the focal plane of the objective lens. Mark 13 is a dichroic mirror, which is arranged on the front focal plane of the objective lens 15, and is used to reflect the fluorescence received by the objective lens during the imaging process into the photomultiplier tube module 17, so as to perform fluorescence detection. Figure 2 In the embodiment shown, two photomultiplier modules 17 are provided for fluorescence detection of different fluorescence channels.

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

[0039] Figure 3 FIG. 1 is a schematic diagram of a structure of an embodiment of an objective lens. In the present application, the objective lens is preferably a long working distance objective lens. Specifically, Figure 3The objective lens shown is a long working distance quadruple fluorescence objective lens. The objective lens in this embodiment includes a fifth lens 18, a sixth lens 19, a seventh lens 20, an eighth lens 21, and a ninth lens 22 in order from the incident direction to the emission direction, a total of five lenses. The fifth lens 18 is a meniscus lens, with its convex surface facing the incident direction, made of H-K9L material, and having negative optical power; the sixth lens 19 is also a meniscus lens, also made of H-K9L material, and having negative optical power, and the sixth lens 19 is arranged close to the fifth lens 18, and the concave surface of the sixth lens 19 is arranged toward the concave surface of the fifth lens 18; the seventh lens 20 is a triple cemented lens, with positive optical power, and the materials of its three lenses are H-FK61, H-ZF1, and H-ZF62 respectively; the eighth lens 21 is a double convex lens, made of H-FK61 material, and having positive optical power; the ninth lens 22 is a meniscus lens, with its convex surface facing the eighth lens, that is, arranged toward the incident direction, made of H-ZF62 material, and having 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 The figure is a three-dimensional schematic diagram of an embodiment of a parabola side fluorescence collection module. The parabola side fluorescence collection module includes a parabola side reflection module, a focusing module, a filter module, and a fluorescence detection module.

[0041] The reference numeral 23 is a parabolic side reflection module, which has a parabolic reflector and is used to improve the fluorescence stimulation efficiency of the system. When performing scanning imaging, the sample to be tested, such as the brain of an experimental mouse, is set at the focus of the parabolic reflector. The incident light beam (i.e., the excitation light) is converged by the objective lens 15 and then injected through the opening above the parabolic reflection module 23 and focused inside the brain of the experimental mouse. The stimulated fluorescence stimulated at the focus is scattered from the brain of the experimental mouse to all around, and after being reflected by the parabolic side reflection module 23, it is emitted in parallel in a direction perpendicular to the optical axis of the objective lens 15.

[0042] The focusing module is used to focus the light beam emitted in parallel after being reflected by the parabola side reflection module 23, and includes a first focusing lens 24, which is close to the exit of the parabola side reflection module 23 and is used to converge the outgoing light beam emitted in parallel after being reflected by the parabola side reflection module 23 for the first time, thereby reducing the aperture of the outgoing light beam.

[0043] Mark 25 is a filter module, which is arranged after the first focusing lens 24 and is used to filter out interference light such as excitation light and natural light other than the fluorescence signal in the outgoing light beam. The filter module 25 can also be used to separate the fluorescence signals of different channels, for example, the fluorescence signal includes 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 arranged at the entrance of the filter module 25 to filter out interference light such as excitation light and short-wave natural light, and then the outgoing light beam is split into two beams of fluorescence signals by a dichroic mirror arranged inside the filter module 25 and at an angle of 45 degrees to the optical axis of the outgoing light beam. The two band-pass filters are respectively arranged at the two exit ports of the filter module 25 to further filter out interference light.

[0044] The focusing module further includes a second focusing lens 26 and a third focusing lens 27 for further converging the fluorescence signal and projecting it to 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 for heat dissipation of the PMT 29 .

[0046] Figure 5 FIG. 1 is a schematic diagram of an optical path of an embodiment of an optical path time division multiplexing module. Figure 5 As shown, reference numeral 30 is a reflector. Figure 5There are multiple optical path time-division multiplexing modules shown, which are used to change the transmission direction of light, so as to make full use of the system space. Marks 31-33 are polarization beam splitters (PBS), which are used to split the incident light beam from one beam into four beams of the first light path, the second light path, the third light path and the fourth light path, and change the polarization direction of the light beam at the same time, wherein the first light path is p-polarized light, and the second light path, the third light path and the fourth light path are s-polarized light. Marks 34-37 are the tenth lenses respectively arranged on the first light path to the fourth light path, and mark 41 is the eleventh lens arranged on the light path after the first light path to the fourth light path are combined. The tenth lens 34-37 is respectively combined with the eleventh lens 41 to form a four-way focus shifting module to adjust the depth position of the focus of the first light path to the fourth light path, that is, by setting the distance between the tenth lens 34-37 and the eleventh lens 41, the focus of the light beam from the first light path to the fourth light path is staggered by the same distance on the Z axis. Mark 38 is a 1:1 non-polarizing beam splitter (BS), which is used to combine the fourth optical path with the third optical path. The combination here will lose half of the optical power. Mark 39 is also a 1:1 non-polarizing beam splitter (BS), which is used to further combine the fourth optical path and the third optical path with the second optical path after the combination. Here, the combination will also lose half of the optical power. Mark 40 is a polarizing beam splitter (PBS), which is used to further combine the fourth optical path, the third optical path and the second optical path with the first optical path after the combination. Here, due to the use of polarizing beam splitter prism 40, the s-polarized light of the second optical path, the third optical path and the fourth optical path after the combination is combined with the p-polarized light of the first optical path, so there is no optical power loss. Mark 42 is a half-wave plate, there are multiple in the optical path time division multiplexing module, respectively set between two beam splitters, and used to adjust the optical power of the light beams in different optical paths by changing the polarization direction of the light beam. It should be pointed out that in Figure 5 In the figure, due to limited space on paper, it is impossible to fully display the entire optical path of the fourth optical path, so a curve is used to illustrate it.

[0047] The optical path time division multiplexing module is arranged upstream of the optical path of the first scanning galvanometer. Figure 5 In the embodiment shown, the optical path time division multiplexing module splits the incident laser pulse beam into four beams and then combines them, but the present application is not limited to this. The optical path time division multiplexing module is used to split the incident light beam into at least two beams and then combine them. The optical path difference between two adjacent light 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, and each light beam is respectively provided with its own tenth lens, and an eleventh lens is provided on the optical path after the combination, and by setting the distance between the respective tenth lens and the eleventh lens, the focus of each light beam is staggered by the same distance on the Z axis, so that multi-layer scanning of the image can be performed simultaneously.

[0048] According to an embodiment of the large field of view multiphoton volume imaging system of the present application, a 6×6×0.5mm 3 In this embodiment, the two main parameters of the first scanning galvanometer are an effective diameter of 5 mm, a maximum swing angle of plus or minus 13°, and a Figure 3 The long working distance quadruple fluorescent objective lens shown has a focal length of 45mm. If you want to image within a 6mm range, by calculating that the incident light at the rear focal plane of the objective lens requires an incident angle of at least 3.814°, 13 / 3.814≈3.4, so the optical transfer magnification of the entire system should be 3.4 times. The optical transfer magnification of the entire system is determined by the product of the optical transfer magnifications of the first optical transfer module and the second optical transfer module. In this embodiment, the effective diameter of the second scanning galvanometer is selected to be 6mm. In order to make full use of the energy of the incident light beam, the scanning light beam after the first optical transfer module is incident as much as possible into the effective diameter of the second scanning galvanometer. Therefore, the first optical transfer module is selected as a single optical transfer module, and the second optical module is selected as a 3.4-fold optical transfer module.

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

[0050] The entire optical system adopts the Petzval design concept to optimize beam transmission and ensure high-resolution and low-aberration imaging quality. The simulation evaluation is verified based on multiple indicators such as PSF peak, RMS curve, field curvature, etc. Through simulation and optimization, the system utilizes Figure 1 and Figure 2 The aberration compensation module shown effectively corrects the system's spherical aberration, coma, astigmatism, field curvature and other aberration problems, and can ensure the clarity of the entire imaging field of view.

[0051] The system uses a piezoelectric drive to drive the objective lens to move longitudinally at a frequency of 1.1 Hz, thereby achieving volume imaging within a range of 130 μm on the Z axis. The inter-layer distance is set to 18.5 μm, with a total of 7 layers, and 1.1 volume images can be acquired per second. At the same time, this inter-layer 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 in the longitudinal direction, i.e., the Z axis, the system adopts Figure 5 The optical path time-division multiplexing module shown splits the incident laser pulse beam into four beams, with the focus of each beam staggered by the same distance of 130μm on the Z axis and a time delay of 3.125ns. This design, combined with 7-layer longitudinal scanning, achieves a total of 28 layers of 520μm three-dimensional volume imaging, which meets the penetration depth of two-photon calcium imaging.

[0053] The system is inserted between the objective lens and the imaging sample Figure 4 In the parabolic lateral fluorescence collection module shown, most of the fluorescence signals that cannot enter the objective lens will be reflected laterally by the parabolic reflector, so that they are collected and detected by the fluorescence detection module. The existing multi-photon volume imaging system will have a contradiction between the fluorescence collection efficiency and the imaging field of view, which is caused by the size of the objective lens. The system of the present application avoids this theoretical limitation because the fluorescence signal can be collected without passing through the objective lens. The present application combines the parabolic reflector, the independently designed focusing module and the large-area photomultiplier tube to increase the fluorescence collection efficiency from the traditional 4% to more than 40%. The present application combines the limiting conditions such as the focal length of the objective lens, the size of the light source, the aperture size of the commonly used lens and the size of the collection area of ​​the photomultiplier tube, and optimizes the opening size and curve length of the parabolic curve in a targeted manner, so that even at the extreme depth of two-photon calcium imaging (about 600μm), the system can still obtain high-quality imaging data, significantly improving the imaging quality. In addition, the system of the present application is also compatible with the existing fluorescence collection module using the objective lens, which means that the fluorescence returned to the objective lens can be collected at the same time, fully improving the fluorescence collection efficiency.

[0054] Given the system's large 6mm×6mm field of view, the fluorescence collection efficiency at different positions within the target field of view may vary significantly. Through optical simulation, this application predicts the fluorescence collection efficiency at different positions within the target field of view, and further evaluates the fluorescence collection efficiency at different positions within the target field of view based on the relationship between the filter spectrum and the incident light angle, ensuring balanced imaging quality throughout the field of view. Specifically, the present application comprehensively considers the fluorescence collection efficiency and the incident light angle at different positions in the target field of view. First, the parabolic curve of the parabolic reflector is optimized, and the size of the parabola opening is increased while the length of the curve is reduced, so that the photons at different points on the imaging surface can pass through the same transmission distance as much as possible before being reflected by the parabola, so that the fluorescence emitted from different points on the imaging surface can be emitted from the side of the parabola 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 light beam reflected from the parabolic reflector can be converged to the collection surface of the photomultiplier tube while its incident light angle is not too large, and finally achieve the goal of significantly improving the total fluorescence collection efficiency of the imaging surface while having no obvious difference in the collection efficiency of each point.

[0055] To match the parabolic side fluorescence collection module, the system uses Figure 3 The long working distance quadruple fluorescence objective shown in the figure has a working distance of 30.5-31 mm. The longer working distance allows more design space for the parabola side collection module and can achieve higher efficiency. Through optical simulation optimization, while ensuring the long working distance of the objective, the aberration compensation and imaging performance are balanced, so that the objective can meet the needs of the parabola side fluorescence collection system while having near-ideal imaging quality.

[0056] The present invention proposes a breakthrough large-field multiphoton volume imaging system, which has significant advantages in biomedical engineering and neuroscience research. By combining advanced optical design, aberration optimization technology, high-speed galvanometer scanning, piezoelectric displacement scanning, optical path time-division multiplexing technology, long working distance fluorescent objective lens, and parabolic reflection collection technology, a large-field multiphoton volume imaging system is achieved in the imaging field of view, imaging resolution, fluorescence collection efficiency and other performance indicators. The following are the main beneficial effects of this application compared with the prior art.

[0057] Ultra-large field of view and high resolution improvement: The imaging field of this application is increased from the traditional 1mm 2 The following expansion to 6×6×0.5mm 3 , the resolution reaches 1×1×19μm 3This significant improvement allows researchers to capture the activity of millions of neurons in a single image, which not only expands the scope of observation but also improves the ability to analyze the coordinated activities of neural networks. Compared with existing multiphoton microscopy systems, this invention can perform high-quality three-dimensional imaging in a larger volume, greatly improving data throughput and the breadth of research.

[0058] Significant enhancement of fluorescence collection efficiency: In order to improve the imaging field of view, existing multiphoton microscopy systems usually use a standard four-fold objective lens, and its fluorescence collection efficiency is limited by the numerical aperture of the objective lens (taking the four-fold objective lens as an example), which is usually only about 4%. However, this application uses an innovative parabolic lateral fluorescence collection module to place the fluorescence collection module between the objective lens and the sample. Fluorescence collection is no longer limited by the objective lens, and the fluorescence collection efficiency is increased to more than 40%. Combined with the independently designed long working distance fluorescent objective lens, this design solves the contradiction between the objective lens size limitation and the fluorescence collection efficiency, greatly improving the signal-to-noise ratio and signal quality of deep imaging, ensuring that high-quality image data can still be obtained in two-photon calcium imaging at a depth of 600μm.

[0059] The above are only specific embodiments of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be imagined by any technician familiar with the technical field within the technical scope disclosed in the present application should be included in the protection scope of the present application; in the absence of conflict, the embodiments of the present application and the features in the embodiments can be combined with each other. Therefore, the protection scope of the present application shall be based on the protection scope of the claims.

Claims

1. A large-field-of-view multiphoton volume imaging system, characterized in that: include: A first scanning galvanometer, wherein the first scanning galvanometer is used for X-axis scanning; A first optical transmission module, wherein the first optical transmission module comprises a first telecentric flat field scanning lens and a second telecentric flat field scanning lens, wherein the first scanning galvanometer is arranged on a front focal plane of the first telecentric flat field scanning lens, and a back focal plane of the first telecentric flat field scanning lens coincides with a back focal plane of the second telecentric flat field scanning lens; A second scanning galvanometer, the second scanning galvanometer is used for Y-axis scanning, and the second scanning galvanometer is arranged on the front focal plane of the second telecentric flat-field scanning lens; a second optical transmission module, wherein the second optical transmission module comprises a third telecentric flat-field scanning lens and a tube lens, wherein a front focal plane of the third telecentric flat-field scanning lens coincides with a front focal plane of the second telecentric flat-field scanning lens, wherein the tube lens comprises a first achromatic doublet lens and a second achromatic doublet lens, wherein the first achromatic doublet lens and the second achromatic doublet lens are arranged relative to each other at a specific distance, and the front focal plane of the tube lens coincides with a back focal plane of the third telecentric flat-field scanning lens; an aberration compensation module, the aberration compensation module comprising 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; and An objective lens, wherein the front focal plane of the objective lens coincides with the rear focal plane of the tube lens adjusted by the aberration compensation module, and the objective lens is driven by a piezoelectric driver for Z-axis scanning.

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

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

4. The large-field-of-view multiphoton volume imaging system according to claim 3, characterized in that: The long working distance fluorescent objective lens is a long working distance quadruple fluorescent objective lens, and the long working distance quadruple fluorescent objective lens comprises: a fifth lens, wherein the fifth lens is a meniscus lens having a negative optical power, and a convex surface of the fifth lens is arranged toward an incident direction; a sixth lens, wherein the sixth lens is a meniscus lens having a negative optical power, the sixth lens is arranged closely to the fifth lens, the concave surface of the sixth lens is arranged toward the concave surface of the fifth lens, and the fifth lens and the sixth lens are used to adjust the back focal length of the objective lens, thereby controlling the working distance of the objective lens; a seventh lens, the seventh lens being a triplet lens, having positive optical power, and being used to correct at least one of the following optical aberrations of the objective lens: astigmatism, field curvature or chromatic aberration; an eighth lens, wherein the eighth lens is a biconvex lens and has positive optical power; and A ninth lens, wherein the ninth lens is a meniscus lens having negative optical power, wherein the convex surface of the ninth lens is arranged toward the eighth lens, and the eighth lens and the ninth lens are used to adjust the front focal length of the objective lens, and are also used 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 volume imaging system according to claim 1, characterized in that: Also includes: An optical path time division multiplexing module is arranged upstream of the optical path of the first scanning galvanometer, and the optical path time division multiplexing module splits the incident laser pulse light beam into at least two light beams and then combines them, and the optical path difference between the two adjacent light 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, and each light beam is respectively provided with a respective tenth lens, and an eleventh lens is provided on the optical path after combining, and the distance between the respective tenth lens and the eleventh lens is set so that the focus of each light beam is staggered by the same distance on the Z axis.

6. The large-field-of-view multiphoton volume 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 the beams. The optical path time division multiplexing module includes: A first beam splitter, used for splitting the laser pulse beam into the first optical path and a first split optical path; A second beam splitter, used for splitting the first split light path into the second light path and a second split light path; A third beam splitter, used for splitting the second split light path into the third light path and the fourth light path; a fourth beam splitter, used for combining the third light path and the fourth light path into a first combined light path; a fifth beam splitter, used for combining the second light path and the first combined light path into a second combined light path; The sixth beam splitter is used to combine the first light path and the second combined light path into an output light beam.

7. The large-field-of-view multiphoton volume imaging system according to claim 6, characterized in that: The first beam splitter, the second beam splitter and the third beam splitter are polarization beam splitting prisms, and half-wave plates are respectively arranged on the first beam splitting light path and the second beam splitting light path, so that the first light path after beam splitting is p-polarized light, and the second light path, the third light path and the fourth light path are s-polarized light; the fourth beam splitter and the fifth beam splitter are 1:1 non-polarization beam splitting prisms, and the sixth beam splitter is a polarization beam splitting prism, and half-wave plates are respectively arranged on the first light path, the second light path and the third light path, so as to perform beam combining.

8. The large-field-of-view multiphoton volume imaging system according to claim 3, characterized in that: It also includes a parabola side fluorescence collection module, the parabola side fluorescence collection module includes: A parabolic side reflection module, which has a parabolic reflector, the imaging sample is arranged at the focus of the parabolic reflector, and the parabolic side reflection module has an opening facing the objective lens for allowing the light beam to be incident on the imaging sample; A focusing module, the focusing module is used to focus the light beams emitted in parallel after being reflected by the parabola side reflection module; A filter module, wherein the filter module is used to filter out interference light other than the fluorescent signal; A fluorescence detection module is used to detect fluorescence signals.

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

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

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