Microscope imaging system

Abstract

The invention provides a microscope imaging system, and relates to the technical field of microscope imaging. The microscope imaging system comprises a total internal reflection light source, a field diaphragm, a focusing lens, an aperture diaphragm, a secondary imaging lens, an objective lens and an imaging receiving module, the total internal reflection light source, the field diaphragm, the focusing lens, the aperture diaphragm and the secondary imaging lens are sequentially arranged and form a first light path, and the secondary imaging lens is used for imaging to a rear focal plane of the objective lens; the objective lens is used for focusing the exciting light of the total internal reflection light source on a sample and collecting fluorescent light emitted by the sample; the microscope imaging system can be adapted to a total internal reflection light source, and the illumination effect is more uniform.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microscope imaging technology, in particular, to a microscope imaging system. BACKGROUND

[0002] Fluorescence microscope is an important tool widely used in biomedical research, its basic principle is to use a specific wavelength of excitation light to excite fluorescent markers, and obtain sample information by detecting the emitted fluorescence signal. Kohler illumination as a standard illumination method of microscope, through the condenser system to image the light source in the back focal plane of the objective, so as to realize uniform sample illumination.

[0003] The Kohler illumination component of the traditional wide-field fluorescence microscope is mainly designed for halogen lamp or extended light source, which is not suitable for total internal reflection light source, and uneven illumination phenomenon will occur, thereby affecting the imaging quality. SUMMARY

[0004] The purpose of the present application includes, for example, providing a microscope imaging system which can adapt to total internal reflection light source, and the illumination effect is more uniform.

[0005] Embodiments of the present application can be implemented as follows: The embodiment of the present application provides a microscope imaging system, which comprises a total internal reflection light source, a field diaphragm, a focusing lens, an aperture diaphragm, a secondary imaging lens, an objective and an imaging receiving module; the total internal reflection light source, the field diaphragm, the focusing lens, the aperture diaphragm and the secondary imaging lens are arranged in sequence and form a first light path, and the secondary imaging lens is used for imaging to the back focal plane of the objective; the objective is used for focusing the excitation light of the total internal reflection light source on the sample and collecting the fluorescence emitted by the sample; and the imaging receiving module is used for receiving and forming a fluorescence image.

[0006] Optionally, the imaging receiving module comprises a tube lens and an imaging sensor, a second light path is formed between the objective, the tube lens and the imaging sensor, the tube lens is used for magnifying the image, and the imaging sensor is used for forming the fluorescence image.

[0007] Optionally, the focal length of the tube lens is 150-180mm.

[0008] Optionally, a dichroic mirror is arranged on the side of the secondary imaging lens away from the aperture diaphragm, and the dichroic mirror is simultaneously in the first light path and the second light path.

[0009] Optionally, the focusing lens and the secondary imaging lens both satisfy: Wherein, z is the vector height, c is the curvature, k is the conic coefficient, r is the radial coordinate under the lens unit, and a1-a8 are high-order aspherical coefficients.

[0010] Optionally, the secondary imaging lens is provided with a filter on the side away from the aperture stop.

[0011] Optionally, the effective focal length of the objective lens is 4mm-4.25mm.

[0012] Optionally, the numerical aperture N A of the objective lens is 0.6.

[0013] The microscope imaging system provided by the embodiments of the present application has the beneficial effects, for example: through the ordered arrangement and functional cooperation of the total internal reflection light source, the field stop, the focusing lens, the aperture stop, the secondary imaging lens, the objective lens, and the imaging receiving module, the excitation light of the total internal reflection light source is uniformly distributed on the sample surface, the system is fully adapted to the total internal reflection light source, the consistency of fluorescence excitation is effectively improved, the fluorescence signal is finally received by the imaging receiving module and forms a fluorescence image, and then the imaging quality is improved. BRIEF DESCRIPTION OF DRAWINGS

[0014] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the present application, and therefore should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can also be obtained without creative labor.

[0015] Figure 1 The figure is a schematic diagram of the microscope imaging system in the embodiments of the present application.

[0016] Figure: 1-total internal reflection light source; 2-field stop; 3-focusing lens; 4-aperture stop; 5-secondary imaging lens; 6-objective lens; 7-imaging receiving module; 71-tube lens; 72-imaging sensor; 8-dichroic mirror; 9-filter. DETAILED DESCRIPTION

[0017] In order to make the purpose, technical solutions and advantages of the embodiments of the present application more clear, the following will combine the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are some of the embodiments of the present application, not all. The components of the embodiments of the present application described and shown in the drawings here can be arranged and designed in various different configurations.

[0018] Therefore, the following detailed description of the embodiments of this application provided in the accompanying drawings is not intended to limit the scope of the claimed application, but merely to illustrate selected embodiments of the application. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

[0019] It should be noted that similar labels and letters in the following figures indicate similar items. Therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0020] In the description of this application, it should be noted that if terms such as "upper," "lower," "inner," or "outer" are used to indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, or the orientation or positional relationship in which the product of the invention is usually placed during use, they are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of this application.

[0021] Furthermore, the terms "first" and "second" are used only to distinguish descriptions and should not be interpreted as indicating or implying relative importance.

[0022] It should be noted that, where there is no conflict, the features in the embodiments of this application can be combined with each other.

[0023] Traditional Kohler lighting systems are mostly designed for halogen lamps or extended light sources, while total internal reflection (TIR) ​​light sources have the characteristics of strong collimation and concentrated energy. Directly applying the original light path will lead to uneven lighting.

[0024] Please refer to Figure 1 The embodiments of this application provide a microscope imaging system, including a total internal reflection light source 1, a field stop 2, a focusing lens 3, an aperture stop 4, a secondary imaging lens 5, an objective lens 6, and an imaging receiving module 7; the total internal reflection light source 1, the field stop 2, the focusing lens 3, the aperture stop 4, and the secondary imaging lens 5 are arranged sequentially and form a first optical path, the secondary imaging lens 5 is used to image onto the back focal plane of the objective lens 6; the objective lens 6 is used to focus the excitation light of the total internal reflection light source 1 onto the sample and collect the fluorescence emitted by the sample; the imaging receiving module 7 is used to receive and form a fluorescence image.

[0025] The collimated or nearly collimated excitation light emitted by the total internal reflection light source 1 first passes through the field stop 2, which is used to define the size of the illumination area and thus control the effective brightness range of the sample surface. Then the light enters the focusing lens 3, forming a primary image of the light source near its rear focal plane. An aperture stop 4 is introduced at this position as a pupil plane control element to adjust the aperture angle of the illumination beam and match the numerical aperture of the objective lens 6.

[0026] The secondary imaging lens 5 is positioned after the aperture stop 4. Its core function is to re-image the primary image of the light source on the plane of the aperture stop 4 onto the back focal plane of the objective lens 6, thereby satisfying the key condition of Kohler illumination—the image of the light source is conjugate to the back focal plane of the objective lens 6. In other words, this embodiment, through the orderly arrangement and functional synergy of the above optical elements, enables the excitation light to achieve a spatially uniform distribution on the sample surface, effectively improving the consistency of fluorescence excitation. At the same time, the objective lens 6 is not only responsible for focusing the excitation light onto the sample interface to achieve total internal reflection excitation, but also has the function of collecting the fluorescence emitted by the sample. These fluorescence signals are ultimately received by the imaging receiving module 7 and form a fluorescence image.

[0027] This embodiment can be understood as follows: by redesigning the microscope imaging system, the total internal reflection light source 1 is combined with the Kohler illumination system. The entire system is fully adapted to the total internal reflection light source 1, achieving high uniformity excitation illumination of the sample, thereby ensuring imaging quality.

[0028] In this embodiment, the imaging receiving module 7 includes a tube lens 71 and an imaging sensor 72. A second optical path is formed between the objective lens 6, the tube lens 71 and the imaging sensor 72. The tube lens 71 is used to magnify the imaging, and the imaging sensor 72 is used to form a fluorescence image.

[0029] Objective lens 6, while focusing the excitation light, also collects the fluorescence signal emitted by the sample. This fluorescence exits objective lens 6 and enters tube lens 71, which, together with objective lens 6, forms part of the infinity-corrected optical system. Its function is to magnify the intermediate image formed by objective lens 6 and transmit it to the image plane of imaging sensor 72. In this process, objective lens 6, tube lens 71, and imaging sensor 72 are arranged sequentially to form a second optical path. The focal length of tube lens 71 is designed to match the overall magnification requirement of the system, allowing the fluorescence signal to achieve appropriate spatial resolution magnification. Subsequently, the magnified optical image is projected onto the surface of imaging sensor 72, which converts the received light signal into an electrical signal and further processes it to form a digital fluorescence image.

[0030] In this embodiment, the focal length of the tube lens 71 is 150~180mm.

[0031] The tube lens 71 is positioned after the objective lens 6 to further magnify the intermediate image formed by the objective lens 6. When the focal length of the tube lens 71 is set in the range of 150~180mm, it can be used with the objective lens 6, which has an effective focal length of about 4.125mm, thereby achieving a total magnification of about 40 times. Preferably, the focal length of the tube lens 71 is set to 165mm, which optimizes the optical parameters with those of the objective lens 6, thus improving the resolution and field of view uniformity of the image.

[0032] This embodiment can be understood as achieving a reasonable match between the tube lens 71 and the objective lens 6 by limiting the focal length range of the tube lens 71 to obtain stable magnification imaging performance.

[0033] In this embodiment, a dichroic mirror 8 is provided on the side of the secondary imaging lens 5 away from the aperture stop 4, and the dichroic mirror 8 is simultaneously in the first optical path and the second optical path.

[0034] A dichroic mirror 8 is disposed on the side of the secondary imaging lens 5 away from the aperture stop 4, and is positioned at the intersection of the first and second optical paths. In practical applications, the excitation light from the total internal reflection source 1 propagates along the first optical path, is controlled by the secondary imaging lens 5, and then enters the dichroic mirror 8. Due to its wavelength characteristics, the excitation light is reflected by the dichroic mirror 8 and guided to the objective lens 6, ultimately focusing on the sample plane. Simultaneously, the fluorescence signal emitted by the sample after excitation returns to the objective lens 6 in the opposite direction and passes through the dichroic mirror 8 again. Because the fluorescence wavelength is different from the excitation light, it can pass through the dichroic mirror 8 into the second optical path, where it is amplified by the tube lens 71 and received by the imaging sensor 72 to form an image. In this process, the dichroic mirror 8, as a wavelength-selective optical element, achieves efficient separation and path multiplexing of the excitation and emission light.

[0035] In this embodiment, both the focusing lens 3 and the secondary imaging lens 5 satisfy the following: , where z is the vector height, c is the curvature, k is the conic coefficient, r is the radial coordinate in lens units, and α1~α8 are all higher-order aspherical coefficients.

[0036] In the first optical path, the collimated or nearly collimated beam emitted by the total internal reflection source 1 passes through the field stop 2, is converged by the focusing lens 3 to form a primary image, and then the beam angle is controlled by the aperture stop 4. Finally, the secondary imaging lens 5 accurately projects the image of the source onto the back focal plane of the objective lens 6. During this process, because the total internal reflection source 1 has strong collimation and high energy concentration, it is prone to introducing aberrations such as spherical aberration and coma, which affect the uniformity of illumination. By designing both the focusing lens 3 and the secondary imaging lens 5 as high-order aspherical structures, these aberrations can be effectively corrected, making the image of the source clearer and more regular on the back focal plane of the objective lens 6, thereby improving the uniformity and stability of the sample surface illumination.

[0037] It can be understood that this embodiment effectively improves the imaging quality by using a high-order aspherical focusing lens 3 and a secondary imaging lens 5.

[0038] In this embodiment, a filter 9 is provided on the side of the secondary imaging lens 5 away from the aperture stop 4.

[0039] A filter 9 is disposed on the side of the secondary imaging lens 5 away from the aperture stop 4. This filter 9 is located in the first optical path and in front of the dichroic mirror 8. It is used to allow excitation light of a specific wavelength band to pass through while blocking other stray light or unwanted wavelength components. In this process, the light beam from the total internal reflection source 1 is controlled by the focusing lens 3 and the aperture stop 4, and then passes through the filter 9 for wavelength selection. This ensures that the light entering the subsequent optical elements is pure excitation light, which helps to improve the spectral purity of the illumination, reduce background noise, and improve fluorescence excitation efficiency.

[0040] In this embodiment, the effective focal length of objective lens 6 is 4mm~4.25mm.

[0041] The effective focal length of objective lens 6 is set in the range of 4mm to 4.25mm, which can be matched with tube lens 71 with a focal length of 150 to 180mm, thus forming an infinity-corrected optical system and achieving a total magnification of approximately 40x. Preferably, the effective focal length of objective lens 6 is set to 4.125mm, which provides better matching with the optical parameters of tube lens 71 and is beneficial for improving imaging resolution and field-of-view uniformity. In this process, objective lens 6 is not only responsible for focusing the excitation light onto the sample plane to achieve total internal reflection illumination, but also for efficiently collecting the fluorescence signal emitted by the sample.

[0042] This embodiment can be understood as achieving stable magnification performance by limiting the effective focal length range of the objective lens 6 and coordinating it with the tube lens 71.

[0043] In this embodiment, the numerical aperture N of objective lens 6 A It is 0.6.

[0044] The numerical aperture of objective lens 6 determines its light-gathering ability and illumination-converging characteristics. When the numerical aperture N... A When the aperture is 0.6, the use of a high refractive index medium can ensure that the incident angle of the excitation light is greater than the critical angle, thereby generating a total internal reflection effect on the sample plane. In this process, the numerical aperture also ensures a high collection capability for the fluorescence emitted by the sample, improving the signal intensity and signal-to-noise ratio.

[0045] This can be understood as follows: In this embodiment, the numerical aperture N of the objective lens 6 is set... A The value is 0.6 to achieve effective total internal reflection of the excitation light at the sample interface while also taking into account fluorescence collection efficiency.

[0046] In summary, the embodiments of this application provide a microscope imaging system. Through the orderly arrangement and functional coordination of the total internal reflection light source 1, the field aperture 2, the focusing lens 3, the aperture diaphragm 4, the secondary imaging lens 5, the objective lens 6, and the imaging receiving module 7, the excitation light of the total internal reflection light source 1 is uniformly distributed on the sample surface. The system is fully compatible with the total internal reflection light source 1, effectively improving the consistency of fluorescence excitation. The fluorescence signal is finally received by the imaging receiving module 7 and forms a fluorescence image, thereby improving the imaging quality.

[0047] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A microscope imaging system, characterized in that, It includes a total internal reflection light source, a field stop, a focusing lens, an aperture stop, a secondary imaging lens, an objective lens, and an imaging receiving module; the total internal reflection light source, the field stop, the focusing lens, the aperture stop, and the secondary imaging lens are arranged in sequence and form a first optical path, and the secondary imaging lens is used to image onto the back focal plane of the objective lens; The objective lens is used to focus the excitation light from the total internal reflection light source onto the sample and collect the fluorescence emitted by the sample; the imaging receiving module is used to receive and form a fluorescence image.

2. The microscope imaging system according to claim 1, characterized in that, The imaging receiving module includes a tube lens and an imaging sensor. A second optical path is formed between the objective lens, the tube lens, and the imaging sensor. The tube lens is used to magnify the imaging, and the imaging sensor is used to form a fluorescence image.

3. The microscope imaging system according to claim 2, characterized in that, The focal length of the tube lens is 150~180mm.

4. The microscope imaging system according to claim 2, characterized in that, A dichroic mirror is provided on the side of the secondary imaging lens away from the aperture stop, and the dichroic mirror is simultaneously located in the first optical path and the second optical path.

5. The microscope imaging system according to claim 1, characterized in that, Both the focusing lens and the secondary imaging lens satisfy the following: , where z is the vector height, c is the curvature, k is the conic coefficient, r is the radial coordinate in lens units, and α1~α8 are all higher-order aspherical coefficients.

6. The microscope imaging system according to claim 1, characterized in that, A filter is provided on the side of the secondary imaging lens away from the aperture stop.

7. The microscope imaging system according to claim 1, characterized in that, The effective focal length of the objective lens is 4mm to 4.25mm.

8. The microscope imaging system according to claim 1, characterized in that, The numerical aperture N of the objective lens A It is 0.6.

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