Large-view-field ultrafast multi-photon microscopic imaging system based on polyhedral rotating scanning mirror

Abstract

The invention discloses a large-view-field ultrafast multi-photon microscopic imaging system based on a polyhedral rotating scanning mirror. According to the large-field-of-view ultrafast multi-photon microscopic imaging system provided by the invention, the polyhedral rotary scanning mirror is adopted to replace a traditional resonance galvanometer, so that the scanning imaging speed is greatly improved; the system comprises a laser light source, a laser control module, a reflector, a polyhedral rotary scanning mirror (X-axis), a first lens, a second lens, a galvanometer scanning mirror (Y-axis), a third lens (scanning lens), a fourth lens (sleeve lens), a dichroscope, an objective lens, a fifth lens and a photomultiplier. The polyhedral rotary scanning mirror does not have repeated acceleration and deceleration processes, and the rotary scanning speed is ultra-high, so that the line scanning speed can be increased by several times compared with a resonance galvanometer or a galvanometer; in addition, the polyhedral rotary scanning mirror has a larger optical aperture and a larger scanning angle range. Therefore, multi-photon microscopic imaging based on the polyhedral rotating scanning mirror has the advantages of large field of view and ultra-high scanning speed at the same time.

Description

Technical Field

[0001] This application relates to the field of optical microscopy imaging technology, and in particular to a large field-of-view, ultrafast multiphoton microscopy imaging system based on a polyhedral rotating scanning mirror. Background Technology

[0002] Multiphoton microscopy is a nonlinear optical microscopy technique that, with its advantages of deep imaging, low optical damage, and high resolution, has become a powerful tool in life science research, particularly suitable for high spatiotemporal resolution imaging of live animals. Currently, conventional multiphoton microscopy systems mostly use resonant mirrors or galvanometer mirrors as their laser scanning devices. Electromagnetic induction deflects the scanning mirrors at a specific frequency or amplitude, thereby achieving rapid scanning of the excitation beam.

[0003] The resonant galvanometer moves in simple harmonic motion at a natural resonant frequency, and its bidirectional linear scanning speed can reach 12 kHz, which is suitable for high-speed imaging. However, its scanning angle range is small and cannot meet the requirements of large field of view imaging. On the other hand, the scanning speed of the galvanometer galvanometer is insufficient and cannot meet the requirements of rapid imaging. Summary of the Invention

[0004] This application provides a large field-of-view, ultrafast multiphoton microscopy imaging system based on a polyhedral rotating scanning module, including a laser source and laser control module, a reflector, a polyhedral rotating scanning mirror, a first lens, a second lens, a galvanometer scanning mirror, a third lens, a fourth lens, a dichroic mirror, an objective lens, a fifth lens, and a photomultiplier tube; wherein:

[0005] The excitation beam emitted from the laser source and laser control module is incident on the reflecting mirror, then reflected by the reflecting mirror and incident on the polyhedral rotating scanning mirror. The polyhedral rotating scanning mirror reflects the incident excitation beam and transmits it through the first lens and the second lens, and then focuses it on the galvanometer scanning mirror. The beam reflected by the galvanometer scanning mirror is transmitted sequentially through the third lens and the fourth lens to complete beam expansion. The expanded beam is transmitted through the dichroic mirror and incident on the objective lens, and then focused by the objective lens on the biological tissue to be tested. The emitted light (such as fluorescence, second harmonic, and third harmonic) generated by laser excitation is transmitted through the objective lens and incident on the dichroic mirror, then reflected by the dichroic mirror and focused by the fifth lens, and finally detected by the photomultiplier tube. During the scanning process, by controlling the deflection of the X-axis polyhedral rotating scanning mirror and the galvanometer scanning mirror, a two-dimensional image scan within the same focal plane is completed.

[0006] In some embodiments, the polyhedral rotating scanning mirror and the galvanometer scanning mirror deflect the beam in two orthogonal directions.

[0007] In some embodiments, the first lens and the second lens have identical optical structures and are combined into a cemented doublet lens group, which performs optical relay between the polyhedral rotating scanning mirror and the galvanometer scanning mirror.

[0008] The technical solution provided in this application uses a polyhedral rotating scanning mirror to replace the traditional galvanometer or resonant mirror to achieve ultra-high-speed scanning in a large field of view for multiphoton microscopy. Because the polyhedral rotating scanning mirror does not involve repeated acceleration and deceleration processes and has an extremely fast rotational scanning speed, its line scanning speed can be several times higher than that of a resonant or galvanometer mirror. Furthermore, the polyhedral rotating scanning mirror has a larger optical aperture and scanning angle range. Therefore, multiphoton microscopy based on a polyhedral rotating scanning mirror simultaneously possesses the advantages of a large field of view and ultra-fast scanning speed. Attached Figure Description

[0009] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0010] Figure 1 This is a schematic diagram of the imaging system structure provided in an embodiment of this application; Detailed Implementation

[0011] To help those skilled in the art better understand the technical solutions of this application, the technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the description of the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0012] The terms "first," "second," etc., in the specification, claims, and accompanying drawings of this application are used to distinguish different objects, not to describe a specific order. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, software, product, or apparatus that includes a series of steps or units is not limited to the listed steps or units, but also includes steps or units not listed, or other steps or units inherent to such processes, methods, products, or apparatus.

[0013] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0014] Please see Figure 1 This embodiment provides a large field-of-view, ultrafast multiphoton microscopy imaging system based on a polyhedral rotating scanning mirror, including a laser source and laser control module, a reflector, a polyhedral rotating scanning mirror (X-axis), a first lens, a second lens, a galvanometer scanning mirror (Y-axis), a third lens, a fourth lens, a dichroic mirror, an objective lens, a fifth lens, and a photomultiplier tube. The specific implementation method is described in detail below.

[0015] The excitation beam emitted from the laser source and laser control module is incident on the reflecting mirror, then reflected by the reflecting mirror and incident on the polyhedral rotating scanning mirror. The polyhedral rotating scanning mirror reflects the incident excitation beam and transmits it through the first lens and the second lens, and then focuses it on the galvanometer scanning mirror. The beam reflected by the galvanometer scanning mirror is transmitted sequentially through the third lens and the fourth lens to complete beam expansion. The expanded beam is transmitted through the dichroic mirror and incident on the objective lens, and then focused by the objective lens on the biological tissue to be tested. The emitted light (such as fluorescence, second harmonic, and third harmonic) generated by laser excitation is transmitted through the objective lens and incident on the dichroic mirror, then reflected by the dichroic mirror and focused by the fifth lens, and finally detected by the photomultiplier tube. During the scanning process, by controlling the deflection of the X-axis polyhedral rotating scanning mirror and the galvanometer scanning mirror, a two-dimensional image scan within the same focal plane is completed.

[0016] Example

[0017] This application uses imaging a mouse brain as an example for illustration. In this embodiment, the center wavelength of the laser source is 920 nm. The 920 nm excitation beam is incident on the reflecting mirror, then reflected by the reflecting mirror and incident on the polyhedral rotating scanning mirror. The polyhedral rotating scanning mirror reflects the incident excitation beam and transmits it through the first lens and the second lens, and then focuses it on the galvanometer scanning mirror. The beam reflected by the galvanometer scanning mirror is transmitted through the third lens and the fourth lens in sequence to complete beam expansion. The expanded beam is transmitted through the dichroic mirror and incident on the objective lens, and then focused by the objective lens onto the mouse brain to be tested. The fluorescence, second harmonic, and third harmonic generated by laser excitation are transmitted through the objective lens and incident on the dichroic mirror, then reflected by the dichroic mirror and focused by the fifth lens, and finally detected by the photomultiplier tube. During the scanning process, the scanning imaging of mouse brain neurons in the imaging field of view is completed by controlling the deflection of the polyhedral rotating scanning mirror and the galvanometer scanning mirror.

[0018] The large field-of-view, ultrafast multiphoton microscopy imaging system provided in this application uses a polyhedral rotating scanning mirror instead of a traditional resonant galvanometer to significantly improve the scanning speed during imaging. The improved imaging system includes a laser source and laser control module, a reflector, a polyhedral rotating scanning mirror, a first lens, a second lens, a galvanometer scanning mirror, a third lens, a fourth lens, an objective lens, a fifth lens, and a photomultiplier tube. At the same scanning angle, because the polyhedral rotating scanning mirror does not have repeated acceleration and deceleration processes and has an ultrafast rotational scanning speed, its line scanning speed can be increased several times compared to the resonant galvanometer. Furthermore, the polyhedral rotating scanning mirror has a larger optical aperture and a wider scanning angle range. Therefore, the multiphoton microscopy imaging system based on the polyhedral rotating scanning mirror has the advantages of both a larger imaging field of view and a faster scanning speed.

[0019] It should be understood that "at least one" in the embodiments of this application refers to one or more, and "more than one" refers to two or more. "And / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A alone, A and B simultaneously, or B alone, where A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects are in an "or" relationship. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or plural items. For example, at least one of a, b, or c can represent: a, b, c, ab, ac, bc, or abc, where a, b, and c can be single or multiple.

[0020] Furthermore, unless otherwise stated, the ordinal numbers such as "first" and "second" mentioned in the embodiments of this application are used to distinguish multiple objects and are not used to limit the order, sequence, priority or importance of multiple objects.

[0021] It should be noted that, for the sake of simplicity, the foregoing embodiments are all described as a series of actions. However, those skilled in the art should understand that this application is not limited to the described order of actions, as some steps may be performed in other orders or simultaneously according to this application. Furthermore, those skilled in the art should also understand that the embodiments described in the specification are preferred embodiments, and the actions and modules involved are not necessarily essential to this application.

[0022] In the embodiments provided in this application, it should be understood that the disclosed apparatus can be implemented in other ways. For example, the apparatus embodiments described above are merely illustrative; for instance, the division of the units described above is only a logical functional division, and in actual implementation, there may be other division methods. For example, multiple units or components may be combined or integrated into another system, or some features may be ignored or not executed. Furthermore, the coupling or direct coupling or communication connection shown or discussed may be through some interfaces; the indirect coupling or communication connection between devices or units may be electrical or other forms.

[0023] The embodiments of this application have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of this application. The description of the above embodiments is only for the purpose of helping to understand the method and core ideas of this application. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the ideas of this application. Therefore, the content of this specification should not be construed as a limitation of this application.

Claims

1. A large field of view, ultrafast multi-photon microscopy imaging system based on a polyhedral rotating scanning mirror, characterized in that, The laser light source and the laser control module, a mirror, a polyhedral rotating scanning mirror, a first lens, a second lens, a galvanometer scanning mirror, a third lens, a fourth lens, a dichroic mirror, an objective lens, a fifth lens, and a photomultiplier tube are included. The excitation light beam emitted by the laser light source and the laser control module is incident to the mirror, and then is reflected by the mirror to the polyhedral rotating scanning mirror. The polyhedral rotating scanning mirror reflects and then transmits the incident excitation light beam through the first lens and the second lens, and then focuses the excitation light beam on the galvanometer scanning mirror. The light beam reflected by the galvanometer scanning mirror is transmitted through the third lens and the fourth lens in sequence, and then is expanded. The expanded light beam is transmitted through the dichroic mirror and is incident to the objective lens, and then is focused on the biological tissue to be measured by the objective lens. The emission light generated by laser excitation is transmitted through the objective lens and is incident to the dichroic mirror, and then is reflected by the dichroic mirror and is focused by the fifth lens, and finally is detected by the photomultiplier tube. In the scanning process, the deflection of the X-axis polyhedral rotating scanning mirror and the galvanometer scanning mirror is controlled to complete the two-dimensional image scanning in the same focal plane.

2. The large field-of-view, ultrafast multi-photon microscopic imaging system based on a polyhedral rotating scanning mirror according to claim 1, wherein, The polyhedral rotating scanning mirror and the galvanometer scanning mirror deflect the light beam in two orthogonal directions.

3. The large field-of-view, ultrafast multi-photon microscopic imaging system based on a polyhedral rotating scanning mirror according to claim 1, wherein, The first lens and the second lens have the same optical structure and are combined into a double-cemented lens. The double-cemented lens completes optical relay between the polyhedral rotating scanning mirror and the galvanometer scanning mirror.

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