Multi-photon microscopic imaging system with ultra-large view field

Abstract

The utility model provides an ultra-large field-of-view multiphoton microscopic imaging system, which adopts a large-field-of-view objective lens based on a large-aperture combined lens, and the objective lens has a high numerical aperture and a large field-of-view number. The method comprises the following steps of: obtaining objective lens parameters, determining sizes and optical parameters of optical elements such as an X-axis scanning mirror, a Y-axis scanning mirror, a scanning lens, a sleeve lens and the like according to the objective lens parameters and an optical invariant principle, and then designing an ultra-large-view-field multi-photon microscopic imaging system by utilizing the objective lens and the optical elements so as to achieve high-resolution large-view-field imaging. In addition, a double-shaft micro-electro-mechanical system rapid scanning mirror is adopted in the laser scanning system, and the double-shaft micro-electro-mechanical system rapid scanning mirror and a double-shaft galvanometer scanning mirror matched with the double-shaft micro-electro-mechanical system rapid scanning mirror are controlled. The imaging speed and the flexibility of switching among different imaging sub-regions are superior to those of a common combination of a galvanometer scanning mirror and a resonance galvanometer, and the system is suitable for carrying out ultra-large field-of-view imaging detection on a rapidly changing physiological process in living biological tissues.

Description

Technical Field

[0001] The utility model relates to the technical field of optical microscopic imaging, in particular to an ultra-large field multi-photon microscopic imaging system. Background Art

[0002] Multiphoton microscopy has the advantages of high resolution, strong three-dimensional tomography, deep imaging depth and weak photobleaching. It is often used to observe the fine structure and function of biological tissues. Therefore, it is widely used in life sciences, especially neuroscience research. The imaging field of view of multiphoton microscopes based on conventional commercial objectives is small (about 1 mm in diameter). The imaging field of view can be increased by stitching two-photon images of small fields of view, but the image stitching process is slow and not suitable for real-time dynamic imaging. In addition, the numerical aperture (NA) of existing large-field objectives is low, which is not conducive to multiphoton excitation, and its resolution needs to be improved compared with imaging based on conventional high-NA objectives. Utility Model Content

[0003] In order to solve the above problems, the utility model provides a large field multi-photon microscopic imaging system, comprising:

[0004] A laser light source, a polarization beam splitter, a first lens, a second lens, a quarter wave plate, a lens group equivalent to an objective lens, a reflector, a dual-axis micro-electromechanical system fast scanning mirror, a third lens, a fourth lens, an X-axis scanning mirror, a fifth lens, a sixth lens, a Y-axis scanning mirror, a seventh lens, an eighth lens, a dichroic mirror, an objective lens, a ninth lens and a photomultiplier tube;

[0005] The X-axis scanning mirror and the Y-axis scanning mirror are used for full-frame scanning imaging at a normal speed or switching between different imaging sub-areas, and the dual-axis micro-electromechanical system fast scanning mirror is used for fast scanning imaging of the sub-areas. When the dual-axis micro-electromechanical system fast scanning mirror scans the sub-areas for imaging, the X / Y scanning mirror is in a stationary state:

[0006] The light beam emitted by the laser light source is reflected by the polarization beam splitter, and then sequentially transmitted through the first lens, the second lens, and the 1 / 4 wave plate, and then transmitted through the lens group equivalent to the objective lens, and then focused on the reflector; the light beam reflected by the reflector is then reversely transmitted through the lens group equivalent to the objective lens, the 1 / 4 wave plate, the second lens, the first lens, and the polarization beam splitter, and then reflected by the dual-axis micro-electromechanical system fast scanning mirror, and the light beam reflected by the dual-axis micro-electromechanical system fast scanning mirror is transmitted through the third lens. The excitation light beam is incident on the X-axis scanning mirror after passing through the fourth lens, and is reflected by the X-axis scanning mirror and sequentially transmitted through the fifth lens and the sixth lens, and then is incident on the Y-axis scanning mirror, and is reflected by the Y-axis scanning mirror and sequentially transmitted through the seventh lens, the eighth lens, the dichroic mirror and the objective lens, and is focused in the biological tissue to be tested; the excitation light beam excites the fluorescent probe in the biological tissue to generate fluorescence; the emitted fluorescence is collected by the objective lens, and is reflected by the dichroic mirror and transmitted through the ninth lens, and is finally detected by the photomultiplier tube;

[0007] The X-axis scanning mirror and the Y-axis scanning mirror are used for ultra-large field of view scanning imaging at conventional speed, or for switching between different imaging sub-areas, and the dual-axis micro-electromechanical system fast scanning mirror performs fast scanning imaging on each sub-area, thereby completing the scanning imaging of all sub-areas in the entire field of view in a cycle in sequence;

[0008] The optical invariants of the first lens, the second lens, the third lens, the fourth lens, the fifth lens, the sixth lens, the seventh lens, the eighth lens, and the ninth lens are close to or equal to the optical invariants of the objective lens.

[0009] Specifically, the ultra-large field multiphoton microscopic imaging system provided by the utility model adopts a large field objective lens based on a large-aperture combined lens, and the objective lens has a high numerical aperture and a large field of view number, for example, the excitation numerical aperture is 0.70, the collection numerical aperture is 1.20, and the imaging field diameter is not less than 3 mm.

[0010] Specifically, the objective lens parameters and the optical invariant principle are used to determine the optical parameters and sizes of the dual-axis micro-electromechanical system fast scanning mirror, the X-axis scanning mirror, the Y-axis scanning mirror, the first lens, the second lens, the third lens, the fourth lens, the fifth lens, the sixth lens, the seventh lens, the eighth lens, the ninth lens, the dichroic mirror and the quarter wave plate.

[0011] The utility model adopts the above technical solution, and its beneficial effects are as follows:

[0012] The ultra-large field-of-view multi-photon microscopic imaging system provided by the utility model adopts a large field-of-view objective lens based on a large-aperture combined lens, and the objective lens has a high numerical aperture and a large field of view number. For example, the excitation numerical aperture is 0.70, the collection numerical aperture is 1.20, and the imaging field diameter is not less than 3 mm. In addition, in order to improve the scanning imaging speed in the ultra-large field of view, the present design adopts a dual-axis micro-electromechanical system fast scanning mirror in the laser scanning system. By controlling the dual-axis micro-electromechanical system fast scanning mirror and the dual-axis galvanometer scanning mirror coordinated therewith, the imaging speed and the flexibility of switching between different imaging sub-areas are better than the combination of the commonly used galvanometer scanning mirror and the resonant oscillator mirror. Therefore, the utility model is very suitable for ultra-large field-of-view imaging detection of rapidly changing physiological processes in living biological tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the technical solutions of the embodiments of the utility model, the drawings required for use in the embodiments of the utility model or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the utility model. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0014] Figure 1 A schematic diagram comparing the sizes and other parameters of a conventional commercial objective lens provided in an embodiment of the utility model and the objective lens used in this embodiment.

[0015] Figure 2 A schematic structural diagram of an ultra-large field-of-view multiphoton microscopic imaging system provided in an embodiment of the utility model. DETAILED DESCRIPTION

[0016] The embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and should not be construed as limiting the present invention.

[0017] In the description of the present invention, it is necessary to understand that the terms "upper", "lower", "horizontal", "inside", "outside", etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present invention.

[0018] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. In the description of the present utility model, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

[0019] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments.

[0020] Example:

[0021] Here, it should be explained that the optical invariant is a constant that is conserved in an ideal aberration-free optical system. It is calculated using the height and angle of the main light and the marginal light. The optical invariant can be calculated on any lateral plane of the optical system. At the same time, in a multiphoton microscopy system, each optical component has a maximum optical invariant. The maximum optical invariant of a multiphoton microscopy system is determined by the optical component with the smallest optical invariant in the system. Therefore, the design of a multiphoton microscopy system with an ultra-large field of view should be considered from two aspects. First, the objective lens plays a vital role in the imaging field of view. The maximum achievable field of view of the multiphoton microscopy system is determined by the maximum optical invariant of the objective lens. Secondly, after designing the objective lens, other optical components are selected or designed and processed according to the parameters of the objective lens and the principle of optical invariants.

[0022] See also Figure 1 , which is a conventional commercial objective lens provided in this embodiment ( Figure 1 The objective lens used in this embodiment ( Figure 1 The scheme for determining the parameters of each optical element in the optical system based on the optical invariant is as follows:

[0023] First, determine the objective lens suitable for the ultra-large field of view multiphoton microscopy imaging system. This design uses a large field of view objective lens based on a large-aperture combined lens. At the same time, the objective lens magnification and field of view number will determine the maximum imaging field of view of the imaging system, and its field of view diameter is:

[0024]

[0025] In the above formula, FN is the field number of the objective lens; M is the magnification of the objective lens; the formula shows that the imaging field of the objective lens is determined by the field number FN and the magnification M, that is, the larger the field number of the objective lens, the smaller the magnification, and the larger the maximum imaging field of view it supports.

[0026] Secondly, in a multiphoton imaging system, an optical quantity can be used to simultaneously measure the diffraction-limited resolution and field of view of each optical element (or system), which is called the optical invariant I, and its expression is:

[0027]

[0028] Among them, NA is the numerical aperture, which determines the diffraction-limited resolution of the optical element; the field number FN and the magnification M determine the imaging field of the optical element (or system). Therefore, based on the parameters of the designed objective lens and the principle of optical invariants, other optical components can be selected or designed and processed.

[0029] Finally, the objective lens parameters and the principle of optical invariants are used to determine the parameters and dimensions of optical components such as the dual-axis micro-electromechanical system fast scanning mirror, X-axis scanning mirror, Y-axis scanning mirror, first lens, second lens, third lens, fourth lens, fifth lens, sixth lens, seventh lens, eighth lens, ninth lens and dichroic mirror, thereby completing the optical design, processing and selection of the ultra-large field of view multi-photon microscopy imaging system.

[0030] In summary, the field of view diameter of conventional commercial objective lenses is generally around 1mm, while the field of view diameter of the objective lens of this design is 3mm, so this system can support a larger imaging field of view. At the same time, the numerical aperture of conventional commercial objective lenses is generally not greater than 1.0, while the photon collection numerical aperture of the objective lens designed by the utility model is 1.20, so compared with the traditional multi-photon imaging system, the fluorescence reception, collection and detection efficiency of this design is greatly improved.

[0031] See also Figure 2 , which is an ultra-large field multiphoton microscopic imaging system provided in this embodiment, specifically includes a laser light source, a polarization beam splitter, a first lens, a second lens, a 1 / 4 wave plate, a lens group equivalent to an objective lens, a reflector, a dual-axis micro-electromechanical system fast scanning mirror, a third lens, a fourth lens, an X-axis scanning mirror, a fifth lens, a sixth lens, a Y-axis scanning mirror, a seventh lens (scanning lens), an eighth lens (tube lens), a dichroic mirror, an objective lens, a ninth lens and a photomultiplier tube;

[0032] The X-axis scanning mirror and the Y-axis scanning mirror are used for full-frame scanning imaging at a normal speed or switching between different imaging sub-areas, and the dual-axis micro-electromechanical system fast scanning mirror is used for fast scanning imaging of the sub-areas. When the dual-axis micro-electromechanical system fast scanning mirror scans the sub-areas for imaging, the X / Y scanning mirror is in a stationary state:

[0033] The light beam emitted by the laser light source is reflected by the polarization beam splitter, and then sequentially transmitted through the first lens, the second lens, and the 1 / 4 wave plate, and then transmitted through the lens group equivalent to the objective lens, and then focused on the reflector; the light beam reflected by the reflector is then reversely transmitted through the lens group equivalent to the objective lens, the 1 / 4 wave plate, the second lens, the first lens, and the polarization beam splitter, and then reflected by the dual-axis micro-electromechanical system fast scanning mirror, and the light beam reflected by the dual-axis micro-electromechanical system fast scanning mirror is transmitted through the third lens. The excitation light beam is incident on the X-axis scanning mirror after passing through the fourth lens, and is reflected by the X-axis scanning mirror and sequentially transmitted through the fifth lens and the sixth lens, and then is incident on the Y-axis scanning mirror, and is reflected by the Y-axis scanning mirror and sequentially transmitted through the seventh lens, the eighth lens, the dichroic mirror and the objective lens, and is focused in the biological tissue to be tested; the excitation light beam excites the fluorescent probe in the biological tissue to generate fluorescence; the emitted fluorescence is collected by the objective lens, and is reflected by the dichroic mirror and transmitted through the ninth lens, and is finally detected by the photomultiplier tube;

[0034] The X-axis scanning mirror and the Y-axis scanning mirror are used for ultra-large field of view scanning imaging at conventional speed, or for switching different imaging sub-areas, and the dual-axis micro-electromechanical system fast scanning mirror performs fast scanning imaging on each sub-area, thereby completing the scanning imaging of all sub-areas in the entire field of view in a cycle in sequence;

[0035] The optical invariants of the first lens, the second lens, the third lens, the fourth lens, the fifth lens, the sixth lens, the seventh lens, the eighth lens and the ninth lens are close to or equal to the optical invariants of the objective lens.

[0036] Specifically, the ultra-large field multiphoton microscopic imaging system provided by the utility model adopts a large field objective lens based on a large-aperture combined lens, and the objective lens has a high numerical aperture and a large field of view number, for example, the excitation numerical aperture is 0.70, the collection numerical aperture is 1.20, and the imaging field diameter is not less than 3 mm.

[0037] Specifically, the objective lens parameters and the optical invariant principle are used to determine the optical parameters and sizes of the dual-axis micro-electromechanical system fast scanning mirror, the X-axis scanning mirror, the Y-axis scanning mirror, the first lens, the second lens, the third lens, the fourth lens, the fifth lens, the sixth lens, the seventh lens, the eighth lens, the ninth lens, the dichroic mirror and the quarter wave plate.

[0038] It should be noted that: the utility model is designed to be a large-field multi-photon imaging system. If only a conventional scanning galvanometer is used, the scanning speed is too slow to meet the time resolution requirements for observing the physiological activities of living biological tissues. The utility model uses a dual-axis micro-electromechanical system fast scanning mirror to greatly improve the scanning speed, and by controlling its cooperation with the conventional galvanometer scanning mirror, the scanning speed and the flexibility of switching between different imaging sub-areas are better than the combination of ordinary galvanometer scanning mirrors and resonant galvanometers. Therefore, it is very suitable for imaging and detecting rapidly changing physiological activities in ultra-large field imaging. In addition, the remote focusing module is added to the system because the large-field objective lens has a large mass, and it is not convenient to use a translation stage to move the objective lens up and down to change the focal plane position. The utility model uses a remote focusing module to achieve the purpose of focusing.

[0039] The utility model adopts a large-viewing-field objective lens based on a large-aperture combined lens, the objective lens has a high numerical aperture and a large field of view number, and determines the parameters and sizes of optical elements such as a dual-axis micro-electromechanical system fast scanning mirror, an X-axis scanning mirror, a Y-axis scanning mirror, a first lens, a second lens, a third lens, a fourth lens, a fifth lens, a sixth lens, a seventh lens, an eighth lens, a ninth lens and a dichroic mirror according to the objective lens parameters and the optical invariant principle, and then, an ultra-large-viewing-field multiphoton microscopic imaging system is designed by using the objective lens and the optical elements to achieve high-resolution ultra-large-viewing-field imaging.

[0040] It can be understood that the technical features of the above-described embodiments can be arbitrarily combined. In order to make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0041] The above are only preferred embodiments of the present invention, and only specifically describe the technical principles of the present invention. These descriptions are only for the purpose of explaining the principles of the present invention, and cannot be interpreted as limiting the scope of protection of the present invention in any way. Based on the explanation here, any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention, and other specific implementation methods of the present invention that can be associated with by technicians in this field without creative labor, should be included in the scope of protection of the present invention.

Claims

1. An ultra-large field multiphoton microscopic imaging system, characterized in that: include: A laser light source, a polarization beam splitter, a first lens, a second lens, a quarter wave plate, a lens group equivalent to an objective lens, a reflector, a dual-axis micro-electromechanical system fast scanning mirror, a third lens, a fourth lens, an X-axis scanning mirror, a fifth lens, a sixth lens, a Y-axis scanning mirror, a seventh lens, an eighth lens, a dichroic mirror, an objective lens, a ninth lens and a photomultiplier tube; The X-axis scanning mirror and the Y-axis scanning mirror are used for full-frame scanning imaging at a normal speed or switching between different imaging sub-areas, and the dual-axis micro-electromechanical system fast scanning mirror is used for fast scanning imaging of the sub-areas. When the dual-axis micro-electromechanical system fast scanning mirror scans the sub-areas for imaging, the X / Y scanning mirror is in a stationary state: The light beam emitted by the laser light source is reflected by the polarization beam splitter and then sequentially transmitted through the first lens, the second lens and the 1 / 4 wave plate, and then transmitted through the lens group equivalent to the objective lens, and then focused on the reflector; the light beam reflected by the reflector is then reversely transmitted through the lens group equivalent to the objective lens, the 1 / 4 wave plate, the second lens, the first lens and the polarization beam splitter, and then reflected by the dual-axis micro-electromechanical system fast scanning mirror, the light beam reflected by the dual-axis micro-electromechanical system fast scanning mirror is transmitted through the third lens and the fourth lens, and then incident on the X-axis scanning mirror, and after being reflected by the X-axis scanning mirror, it is sequentially transmitted through the fifth lens and the sixth lens, and then incident on the Y-axis scanning mirror, and after being reflected by the Y-axis scanning mirror, it is sequentially transmitted through the seventh lens, the eighth lens, the dichroic mirror and the objective lens, and is focused on the biological tissue to be measured; the excitation light beam excites the fluorescent probe in the biological tissue to generate fluorescence; The emitted fluorescence is collected by the objective lens, reflected by the dichroic mirror, transmitted through the ninth lens, and finally detected by the photomultiplier tube; The X-axis scanning mirror and the Y-axis scanning mirror are used for ultra-large field of view scanning imaging at conventional speed, or for switching different imaging sub-areas, and the dual-axis micro-electromechanical system fast scanning mirror performs fast scanning imaging on each sub-area, thereby completing the scanning imaging of all sub-areas in the entire field of view in a cycle in sequence; The optical invariants of the first lens, the second lens, the third lens, the fourth lens, the fifth lens, the sixth lens, the seventh lens, the eighth lens and the ninth lens are close to or equal to the optical invariants of the objective lens.

2. The ultra-large field multiphoton microscopic imaging system according to claim 1, characterized in that: A large-field objective lens based on a large-aperture combined lens is used, and the objective lens has a high numerical aperture and a large field of view number. The excitation numerical aperture of the objective lens is 0.70, the collection numerical aperture of the objective lens is 1.20, and the imaging field diameter of the objective lens is not less than 3 mm.

3. The ultra-large field multiphoton microscopic imaging system according to claim 1, characterized in that: The objective lens parameters and the optical invariant principle are used to determine the optical parameters and sizes of the dual-axis micro-electromechanical system fast scanning mirror, the X-axis scanning mirror, the Y-axis scanning mirror, the first lens, the second lens, the third lens, the fourth lens, the fifth lens, the sixth lens, the seventh lens, the eighth lens, the ninth lens, the dichroic mirror and the 1 / 4 wave plate.

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