A large-field fluorescence microscopy imaging system

Abstract

The present invention relates to the field of optical neuroimaging technology, and specifically provides a large-field fluorescence microscopic imaging system, which is mainly used as a miniature head-mounted fluorescence microscope for small animals, and includes: an objective lens, a liquid lens, a tube lens, and a detector. The light emitted by a point on the object passes through the three lenses in the objective lens in sequence and becomes a parallel light beam. The parallel light beam enters the liquid lens, and the radius change of the two curved surfaces of the liquid lens is controlled by voltage to achieve focusing; the light beam then passes through the two lenses in the tube lens to image the light beam on the image plane of the detector. The present invention has a small number of components, light weight, and the lenses are all spherical lenses, which have the advantages of simple structure, light weight, and low cost. The overall field of view can reach 8mm, which reduces the complexity and cost of the system on the basis of improving the imaging performance of the system.

Description

Technical Field

[0001] The present invention relates to the technical field of optical nerve imaging, and specifically provides a large-field fluorescence microscopy imaging system. Background Art

[0002] In the past few decades, neuroscience has made great progress, and its research methods have also undergone a great change, entering a new era of brain science research. With the emergence and development of genetically encoded calcium indicators (GECIs), optical neuroimaging technology combines optical microscopy, fluorescent dyes, and fluorescent proteins to directly image the calcium ion activity of brain neural signals, providing unprecedented insights into the function of the brain and the impact and damage of various neurological diseases on brain function.

[0003] In recent years, with the development of micro-optical components and electronic devices, optical neuroimaging equipment has gradually shifted from a "desktop" platform to a "head-mounted" platform. The micro head-mounted fluorescence microscope is fixed on the skull of the animal and cannot affect its free movement. Taking mice as an example, its head can only bear a weight no greater than 1 / 4 of its body weight. The strict weight constraint greatly limits the imaging performance of the microscope. At present, the field of view of a micro head-mounted fluorescence microscope is generally less than 1mm, and the resolution is about 1μm. Its optical system generally consists of two parts, the objective lens and the tube lens. The objective lens is generally a gradient refractive index GRIN lens, and the tube lens is generally a single aspheric lens or a double cemented lens. The diameter of the GRIN lens is generally 1mm-2.2mm, and the field of view is very small. It can only image the light near the optical axis well. When imaging off-axis object points, the aberration is large and difficult to correct. Due to the small field of view, the head-mounted fluorescence microscope can generally only image one brain area.

[0004] For example, in 2020, Bagramyan et al. designed a micro-microscope weighing only 1.3g, which is the lightest system officially published so far. The objective lens consists of a GRIN lens with a diameter of only 0.5mm and a relay GRIN lens; the tube lens is a plano-convex lens. The advantages of this microscope are its light weight and the use of a small-diameter GRIN lens, which causes less damage to tissues when implanted in the animal brain. The disadvantage is that the imaging field of view is very small, only 105μm.

[0005] In 2021, Rynes and others designed a micro microscope mScope with an ultra-large field of view, weighing only 3.8g. The optical system of mScope is extremely simple, using only a biconvex lens for imaging. mScope achieves an ultra-large field of view with a very light weight, but sacrifices the resolution of the system, and the final system resolution is only tens of microns.

[0006] In 2023, Guo et al. designed and open-sourced a miniature fluorescence microscope MiniLFOV, which weighs 13.9g. The optical system consists of multiple spherical and aspherical lenses, has good imaging performance, and the highest resolution of the central field of view reaches 2.5µm. In addition, an electrowetting lens (EWL) is added in front of the image sensor, which can electrically adjust the working distance within the range of 100µm. It can monitor the neural activity of rats in a free-moving state, but due to its heavy weight, it cannot be worn on smaller animals such as mice without affecting their free movement.

[0007] As brain science research deepens, scientists have begun to study the connections between nerve cells in different brain regions, hoping to image multiple brain regions simultaneously, or even the entire brain at once. To meet this demand, it is necessary to increase the field of view of the microscope optical system while maintaining a high numerical aperture. As the field of view angle increases, the vertical axis aberration nonlinearity of the optical system increases rapidly. In order to balance the aberrations, the number and complexity of components in the optical system increase dramatically, and the size and weight of the equipment increase significantly. In order to meet the needs of head-mounted equipment, it is a scientific problem that needs to be solved to achieve high resolution and large field of view of miniature fluorescence microscopes under strict weight constraints. Summary of the invention

[0008] In order to solve the above problems, the present invention provides a large-field-of-view fluorescence microscopy imaging system, which is mainly used in animal head-mounted fluorescence microscopy equipment. Its optical field of view reaches 8mm, and it has the advantages of single-cell resolution, small size and light weight. It also reduces the complexity and cost of the system on the basis of improving the imaging performance of the system.

[0009] The invention provides a large-field fluorescence microscopic imaging system, comprising: an objective lens, a liquid lens and a tube lens arranged in sequence along an optical axis;

[0010] The objective lens includes a first lens, a second lens and a third lens made of different optical materials in sequence along the optical axis, the first lens is a negative lens, the front surface of which is a convex spherical surface, and the rear surface of which is a concave spherical surface; the second lens is a negative lens, the front surface of which is a convex spherical surface, and the rear surface of which is a concave spherical surface; the third lens is a positive lens, the front and rear surfaces of which are both convex spherical surfaces;

[0011] The curved surface radius of the liquid lens is controlled by voltage to achieve focusing effect;

[0012] The tube lens includes a fourth lens and a fifth lens made of different optical materials in sequence along the optical axis. The fourth lens is a positive lens, and its front and rear surfaces are both convex spherical surfaces; the fifth lens is a negative lens, and its front surface is a concave spherical surface, and its rear surface is a convex spherical surface.

[0013] Preferably, the first lens is made of N-LAK12 material; the second lens is made of N-SF66 material; and the third lens is made of N-PSK53A material.

[0014] Preferably, the fourth lens is made of N-LAK33B material; and the fifth lens is made of N-SF66 material.

[0015] Preferably, an aperture stop is provided at the rear end of the objective lens.

[0016] Preferably, behind the tube lens, along the optical axis, there are provided in sequence: a filter, a detector protection window and an image plane.

[0017] Preferably, the optical radius of the front surface of the first lens is (7.13±0.05) mm, and the clear aperture thereof is 4.4 mm; the optical radius of the rear surface of the first lens is (3.11±0.01) mm, and the clear aperture thereof is 3.64 mm;

[0018] The optical radius of the front surface of the second lens is (5.61±0.01) mm, and the clear aperture thereof is 2.74 mm; the optical radius of the rear surface of the second lens is (3.43±0.01) mm, and the clear aperture thereof is 2.48 mm;

[0019] The front surface optical radius of the third lens is (5.16±0.01) mm, and the clear aperture thereof is 2.50 mm; the rear surface optical radius of the first lens is (7.22±0.02) mm, and the clear aperture thereof is 2.40 mm;

[0020] The front surface optical radius of the fourth lens is (12.01±0.02) mm, and the clear aperture thereof is 3.30 mm; the rear surface optical radius of the fourth lens is (6.37±0.01) mm, and the clear aperture thereof is 3.36 mm;

[0021] The front surface optical radius of the fifth lens is (3.4±0.01) mm, and the clear aperture thereof is 2.64 mm; the rear surface optical radius of the fifth lens is (11.51±0.02) mm, and the clear aperture thereof is 2.96 mm.

[0022] Preferably, the optical field of view is 8 mm.

[0023] Compared with the prior art, the present invention can achieve the following beneficial effects:

[0024] The present invention is mainly used as a head-mounted fluorescence microscope for small animals. The curvature radius of the liquid lens is controlled by voltage to achieve optical focusing. The lens elements are only 5 pieces, and the materials are specially designed. The maximum diameter of the lens elements is 5.1 mm, and the overall weight is only 0.35 g. It has the advantages of simple structure, light weight and low cost. The lens elements in the present invention all use spherical lenses, which greatly reduces the difficulty of preparing the lens elements, and effectively eliminates chromatic aberration by combining lenses of different materials. The present invention greatly improves the object field of view by specially designing the optical radius and the clear aperture of each lens. The field of view can reach 8 mm, which is much larger than the field of view of the existing miniature single-photon fluorescence microscope. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is an optical schematic diagram of a large-field fluorescence microscopy imaging system provided according to an embodiment of the present invention;

[0026] Figure 2 is a modulation transfer function MTF curve diagram of different fields of view provided according to an embodiment of the present invention;

[0027] Figure 3 is a spot diagram of different fields of view provided according to an embodiment of the present invention;

[0028] Figure 4 is a field curvature and distortion curve diagram provided according to an embodiment of the present invention;

[0029] Figure 5 is a light aberration curve diagram of different fields of view provided according to an embodiment of the present invention.

[0030] Reference numerals include:

[0031] Object plane 1, objective lens 2, aperture stop 3, liquid lens 4, tube lens 5, filter 6, detector protection window 7, image plane 8, first lens 21, second lens 22, third lens 23, fourth lens 51, fifth lens 52. DETAILED DESCRIPTION

[0032] Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. In the following description, the same modules are represented by the same reference numerals. In the case of the same reference numerals, their names and functions are also the same. Therefore, the detailed description thereof will not be repeated.

[0033] In order to make the purpose, technical solution 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 specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and do not constitute a limitation of the present invention.

[0034] like Figure 1As shown, an embodiment of the present invention provides a fluorescence microscopic imaging system for a mouse head-mounted imaging device with an optical field of view of 8 mm, which mainly includes: an objective lens 2, an aperture diaphragm 3, a liquid lens 4, a tube lens 5, a filter 6 and a detector. In the application process, the light beam directly emitted or reflected on the object plane 1 first enters the objective lens 2, and the objective lens 2 is composed of a first lens 21, a second lens 22 and a third lens 23 coaxially arranged in sequence, and an aperture diaphragm 3 is arranged behind the objective lens 2. The aperture diaphragm 3 can be directly mounted on the rear surface of the third lens 23, or the aperture diaphragm 3 can be fixed in other ways. The incident light beam passes through the first lens 21, the second lens 22, the third lens 23 and the aperture diaphragm 3 in sequence, and the objective lens 2 and the aperture diaphragm 3 process the incident light beam into a parallel light beam.

[0035] The first lens 21, the second lens 22 and the third lens 23 are all spherical lenses, that is, the front and rear surfaces are all spherical, and the materials used to make them are different, so as to eliminate chromatic aberration. The first lens 21 is a negative lens, made of N-LAK12 glass material, and its front surface is a convex spherical surface, the optical radius of the front surface is (7.13±0.05) mm, the thickness is (1.0±0.02) mm, and the aperture is 4.4 mm; the rear surface of the first lens 21 is a concave spherical surface, the optical radius of the rear surface is (3.11±0.01) mm, the thickness is (6.79±0.02) mm, and the aperture is 3.64 mm. The second lens 22 is a negative lens, made of N-SF66 glass material, with a convex spherical front surface, an optical radius of the front surface of (5.61±0.01) mm, a thickness of (0.8±0.02) mm, and a light-clearing aperture of 2.74 mm; the rear surface of the second lens 22 is a concave spherical surface, with an optical radius of the rear surface of (3.43±0.01) mm, a thickness of (0.3±0.01) mm, and a light-clearing aperture of 2.48 mm. The third lens 23 is a positive lens, made of N-PSK53A glass material, with a convex spherical front surface, an optical radius of the front surface of (5.16±0.01) mm, a thickness of (1.2±0.02) mm, and a light aperture of 2.50 mm; the rear surface of the third lens 23 is a convex spherical rear surface, with an optical radius of (7.22±0.02) mm, a thickness of (0.3±0.01) mm, and a light aperture of 2.40 mm. The thickness of the aperture 3 is (0.4±0.02) mm, and the light aperture is 2.20 mm.

[0036] After being emitted by the objective lens 2 and the aperture diaphragm 3, the parallel light beam enters the liquid lens 4. The front surface thickness of the liquid lens 4 is 1.65 mm, and the adjustable thickness of the rear surface is (1.38 ± 0.02) mm. The refractive ability of the liquid in the liquid lens 4 is changed by changing the shape of the liquid, thereby affecting the focal point of the light. In the embodiment of the present invention, the radius of the curved surface of the liquid lens 4 can be controlled by voltage, and the shape of the liquid surface is adjusted by changing the voltage of the electrode, thereby realizing the focusing function. The liquid lens 4 can quickly adjust the focal length by changing the voltage, without the need for additional mechanical components. While having a faster response speed, it also greatly compresses the volume and weight of the entire optical system.

[0037] After the parallel light beam is focused by the liquid lens 4, it is incident on the tube lens 5. The tube lens 5 is composed of a fourth lens 51 and a fifth lens 52 which are coaxially arranged in sequence. The fourth lens 51 and the fifth lens 52 are both spherical lenses, that is, the front and rear surfaces are both spherical, and the fourth lens 51 and the fifth lens 52 are made of different materials to eliminate chromatic aberration. The fourth lens 51 is a positive lens made of N-LAK33B glass material, and its front surface is a convex spherical surface, the optical radius of the front surface is (12.01±0.02) mm, the thickness is (1.0±0.02) mm, and the light aperture is 3.30 mm; the rear surface of the fourth lens 51 is a convex spherical surface, the optical radius of the rear surface is (6.37±0.01) mm, the thickness is (2.6±0.01) mm, and the light aperture is 3.36 mm. The fifth lens 52 is a negative lens made of N-SF66 glass material, with a front surface that is a concave spherical surface, an optical radius of the front surface that is (3.4±0.01) mm, a thickness of (1.0±0.02) mm, and a light-clearing aperture of 2.64 mm; the rear surface of the fifth lens 52 is a convex spherical surface, with an optical radius of the rear surface that is (11.51±0.02) mm, a thickness of (0.6±0.02) mm, and a light-clearing aperture of 2.96 mm.

[0038] After the light beam is emitted from the tube lens 5, it continues to transmit through the filter 6 to eliminate stray light. By changing the voltage applied to the liquid lens 4, the focal length is adjusted, and the light beam emitted through the liquid lens 4 is imaged on the image plane 8 of the detector through the tube lens 5. A detector protection window 7 is also provided between the filter 6 and the image plane 8.

[0039] In order to verify the effect of the large-field fluorescence microscopic imaging system of the present invention, simulation and testing are performed according to the above structure and optical parameters, as follows:

[0040] First, set the working band to 500-550nm, the object field of view to 8mm, the object numerical aperture to 0.0833, and the pixel size of the detector to 2.2μm×2.2μm. The optical data of the large-field fluorescence microscopy system constructed in the optical simulation software are as follows:

[0041] Table 1 Optical data of large field fluorescence microscopy system

[0042]

[0043] It should be noted that Table 1 is the output optical data of the optical simulation software. In the optical software, the lens settings are determined one by one in units of surfaces, and the radius of curvature of each surface is determined relative to a standard surface. Therefore, the "-" before the optical radius value does not mean that the mirror body is a concave spherical surface, but it indicates the surface shape of the surface relative to the standard surface. For example, although the rear surface of the second lens 22 is a concave spherical surface relative to the mirror body itself, it is a convex spherical surface relative to the standard surface. Therefore, its radius value is preceded by "+" (omitted in the table). Although the surface of the third lens 23 is a convex spherical surface relative to the mirror body itself, it is a concave spherical surface relative to the standard surface. Therefore, its radius value is preceded by "-".

[0044] According to the above table, a large field of view fluorescence microscopy system is constructed, and the following simulation results are obtained: Figure 2 The MTF curves of the modulation transfer function in different fields of view show that the MTF approaches the diffraction limit within the Nyquist sampling frequency range of 227lp / mm of the detector. Figure 3 Spot diagram of the imaging system at different fields of view, from Figure 3 It can be seen that the system's Airy disk radius is 2.757μm, and the spot diagram radius of different fields of view is within the Airy disk range. The simulation results are as follows Figure 4 The field curvature and distortion curves shown are from Figure 4 It can be seen from the figure that under the condition of maximum field of view, the deformation of the edge field of view is small and no obvious bending phenomenon occurs. Figure 5 The light aberration curves of different fields of view are shown. Figure 5 It can be seen that the smoothness and consistency of the curve indicate that the objective has a better focusing effect on light under different fields of view, with smaller aberrations, and can produce clear and sharp images.

[0045] Although the embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and cannot be understood as limiting the present invention. Those skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

[0046] The above specific implementations of the present invention do not constitute a limitation on the protection scope of the present invention. Any other corresponding changes and modifications made based on the technical concept of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A large field of view fluorescence microscopy system, characterized in that: Applied to head-mounted fluorescence microscope, the optical field of view is 8mm, the object numerical aperture is 0.0833, and along the optical axis are arranged in sequence: objective lens, liquid lens and tube lens; The objective lens is composed of a first lens, a second lens and a third lens made of different optical materials arranged in sequence along the optical axis. The first lens is a negative lens, whose front surface is a convex spherical surface and whose rear surface is a concave spherical surface; the second lens is a negative lens, whose front surface is a convex spherical surface and whose rear surface is a concave spherical surface; the third lens is a positive lens, whose front and rear surfaces are both convex spherical surfaces; the optical radius of the front surface of the first lens is (7.13±0.05) mm, and its clear aperture is 4.4 mm; the optical radius of the rear surface of the first lens is (3.11±0.01) mm, and its clear aperture is 3.64 mm; The optical radius of the front surface of the second lens is (5.61±0.01) mm, and the clear aperture thereof is 2.74 mm; the optical radius of the rear surface of the second lens is (3.43±0.01) mm, and the clear aperture thereof is 2.48 mm; The front surface optical radius of the third lens is (5.16±0.01) mm, and the clear aperture thereof is 2.50 mm; the rear surface optical radius of the first lens is (7.22±0.02) mm, and the clear aperture thereof is 2.40 mm; The curved surface radius of the liquid lens is controlled by voltage to achieve focusing effect; The tube lens is composed of a fourth lens and a fifth lens made of different optical materials arranged in sequence along the optical axis, wherein the fourth lens is a positive lens, and both the front and rear surfaces thereof are convex spherical surfaces; the fifth lens is a negative lens, and the front surface thereof is a concave spherical surface, and the rear surface thereof is a convex spherical surface; The front surface optical radius of the fourth lens is (12.01±0.02) mm, and the clear aperture thereof is 3.30 mm; the rear surface optical radius of the fourth lens is (6.37±0.01) mm, and the clear aperture thereof is 3.36 mm; The front surface optical radius of the fifth lens is (3.4±0.01) mm, and the clear aperture thereof is 2.64 mm; the rear surface optical radius of the fifth lens is (11.51±0.02) mm, and the clear aperture thereof is 2.96 mm; An aperture stop is arranged at the rear end of the objective lens.

2. The large-field fluorescence microscopy imaging system according to claim 1, characterized in that: The first lens is made of N-LAK12 material; the second lens is made of N-SF66 material; and the third lens is made of N-PSK53A material.

3. The large-field fluorescence microscopy imaging system according to claim 2, characterized in that: The fourth lens is made of N-LAK33B material; the fifth lens is made of N-SF66 material.

4. The large-field fluorescence microscopy imaging system according to claim 1, characterized in that: Behind the tube lens, along the optical axis, a filter, a detector protection window and an image plane are arranged in sequence.

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