An excitation light system for super-resolution fluorescence imaging

Abstract

An excitation light system for super-resolution fluorescence imaging relates to the technical field of super-resolution fluorescence imaging microscope, and solves the problems of uneven excitation light energy distribution, small field of view angle and poor imaging effect of the prior art. The system comprises a multi-wavelength excitation light device, a first reflector, an aspherical beam homogenization shaping device and a focusing lens. The multi-wavelength excitation light device emits a combined beam, the light field energy distribution of which is Gaussian type. After being reflected by the first reflector, the combined beam enters the aspherical beam homogenization shaping device to homogenize the combined beam. The light beam emitted from the aspherical beam homogenization shaping device is a flat-top type light field energy, which is coupled into the microscope system through the focusing lens. The distance from the outlet of the aspherical beam homogenization shaping device to the focusing lens is 360mm-400mm.

Description

Technical Field

[0001] This invention relates to the field of super-resolution fluorescence imaging microscopy, and more specifically to an excitation light system for super-resolution fluorescence imaging. Background Technology

[0002] like Figure 1-3 As shown, since the excitation light uses a laser source output from a single-mode fiber, its emitted light energy distribution is Gaussian. When the beam directly shines on the sample, due to the uneven energy distribution, the excited fluorescence also exhibits a Gaussian distribution. The energy at the center of the field of view is too strong, which will accelerate the quenching of the fluorescence. In addition, due to the weak light field intensity at the edge of the field of view, there are very few effectively clustered points, resulting in a very small imaging field of view.

[0003] Existing Gaussian beam homogenization techniques are typically used in the field of laser processing, where Gaussian beams are shaped using microlens arrays or diffractive optical elements. However, this approach can only achieve homogenized light field energy distribution near the focal point and cannot achieve homogenization over long distances. Furthermore, diffractive optical elements are very sensitive to light wavelengths, making them unsuitable for the field of super-resolution fluorescence imaging microscopy.

[0004] In the field of super-resolution fluorescence imaging microscopy, in order to solve the above-mentioned technical problems, existing technologies have also adopted the method of using multimode fiber instead of single-mode fiber. Since multimode fiber contains multiple laser modes, the effect of multimode combination can avoid the problem of uneven energy distribution of a single Gaussian mode to a certain extent. However, the energy distribution of excitation light is not ideal, and because different modes of light will interfere inside the multimode fiber, it will also cause speckle in the outgoing light, which seriously affects the imaging effect. Summary of the Invention

[0005] This invention solves the problems of uneven excitation light energy distribution, small field of view, and poor imaging effect in existing methods.

[0006] The present invention discloses an excitation light system for super-resolution fluorescence imaging, the system comprising a multi-wavelength exciter, a first reflecting mirror, an aspherical beam homogenization and shaping device, and a focusing lens; A multi-wavelength exciter emits a combined beam with a Gaussian energy distribution. After being reflected by the first mirror, the beam enters an aspherical beam homogenization and shaping device to homogenize it. The beam emitted from the aspherical beam homogenization and shaping device has a flat-top light field energy and is coupled into the microscope system through a focusing lens. The distance from the exit of the aspherical beam homogenization and shaping equipment to the focusing lens is 360mm-400mm.

[0007] Furthermore, in one embodiment of the present invention, the multi-wavelength exciter includes multiple wavelength light source emitting ends; Multiple wavelength light source emitters are collimated by lenses. One wavelength light source emitter is reflected by a second mirror and then combined with multiple light source emitters that are respectively passed through dichroic mirrors to form a single beam.

[0008] Furthermore, in one embodiment of the present invention, the plurality of wavelength light source emitting ends include light sources in the 405nm, 488nm, 532nm and 638nm wavelength bands.

[0009] Furthermore, in one embodiment of the present invention, the system further includes a third reflecting mirror; The beam emitted from the aspherical beam homogenization and shaping device is reflected by the third reflecting mirror and then coupled into the microscope system through the focusing lens.

[0010] Furthermore, in one embodiment of the present invention, the lens is a 40mm lens made by bonding a first lens and a second lens together, wherein the radius of curvature of the front surface of the first lens is 61.313, the radius of curvature of the rear surface is 18.679, the thickness is 1.8, the refractive index is 1.67, and the Abbe number is 32.2. The second lens has a front surface radius of curvature of 18.679, a rear surface radius of curvature of -22.647, a thickness of 6.7, a refractive index of 1.52, and an Abbe number of 64.2. The aspherical beam homogenization and shaping device includes a third lens and a fourth lens. The third lens has an infinite radius of curvature on its front surface, a radius of curvature of 87.111 on its rear surface, a thickness of 4, a refractive index of 1.52, and an Abbe number of 64.2. The fourth lens has a radius of curvature of 87.111 on the front surface, a radius of curvature of 130.667 on the rear surface, a thickness of 81.523, a refractive index of 1.52, and an Abbe number of 64.2. The focusing lens is a 400mm lens cemented together with a fifth lens and a sixth lens. The fifth lens has a radius of curvature of -220.133 on the front surface, a radius of curvature of 193.143 on the rear surface, a thickness of -4, a refractive index of 1.52, and an Abbe number of 64.2. The sixth lens has a radius of curvature of 193.143 on its front surface, a radius of curvature of 698.743 on its rear surface, a thickness of -2.5, a refractive index of 1.67, and an Abbe number of 32.2.

[0011] Furthermore, in one embodiment of the present invention, the system further includes a beam-expanding lens group; After being reflected by the third reflecting mirror, the beam passes through the beam expander lens group to increase the field of view of the image, and then is coupled into the microscope system through the focusing lens.

[0012] Furthermore, in one embodiment of the present invention, the multi-wavelength exciter includes optical fibers coupled to the emitters of multiple wavelength light sources; The light beam emitted from the optical fiber is collimated by a collimating lens and then outputs a combined beam.

[0013] This invention solves the problems of uneven excitation light energy distribution, small field of view, and poor imaging effect in existing methods. Specific beneficial effects include: The present invention discloses an excitation light system for super-resolution fluorescence imaging. In order to solve the technical problems that have not been solved in the prior art, the excitation light system designed in the field of super-resolution fluorescence imaging microscopy is designed with a multi-wavelength exciter, a first reflecting mirror, an aspherical beam homogenization and shaping device, and a focusing lens. Experimental verification shows that the uniformity of the fluorescence signal of the system is significantly improved, while the imaging field of view is increased, providing a larger effective illumination area, without affecting the imaging effect. Attached Figure Description

[0014] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments taken in conjunction with the accompanying drawings, wherein: Figure 1 The image shows a measured effect of the laser source described in the background technology being output through a single-mode optical fiber. Figure 2 This is the light field distribution diagram of the laser source described in the background technology, output through a single-mode fiber; Figure 3 This is a one-dimensional energy distribution diagram of the laser source described in the background technology, output through a single-mode optical fiber; Figure 4 This is a schematic diagram of the excitation light system without a third reflector as described in Embodiment 2; Figure 5 This is a schematic diagram of the excitation light system of the third reflecting mirror described in Embodiment 2; Figure 6 This is the actual optical path diagram produced according to Implementation Method 2; Figure 7 This is a schematic diagram of the excitation light system without a third reflector as described in Embodiment 2; Figure 8 This is a schematic diagram of the non-spherical beam homogenization and shaping device described in Embodiment 2; Figure 9 This is a structural diagram of the excitation light system for super-resolution fluorescence imaging as described in Embodiment 3; Figure 10 These are the parameter diagrams of each lens described in Embodiment 4; Figure 11 This is the reflection spectrum curve of the mirror described in Embodiment Six at 400-750nm; Figure 12This is a structural diagram of the excitation light system for super-resolution fluorescence imaging as described in Embodiment Six; Figure 13 This is a comparison diagram of the functions of the beam-expanding lens group described in Implementation Method 5; Figure 14 This is a comparison diagram of the effects of the beam-expanding lens group described in Embodiment 5; Figure 15 This is a comparison chart of the homogenization effect described in Example 1; Figure 16 This is a comparison chart of the homogenization effect described in Example 1; Figure 17 This is a comparison chart of the homogenization effect described in Example 2; Figure 18 This is the measured effect diagram after homogenization as described in Implementation Method 4; Figure 19 It is the homogenized light field distribution diagram described in Implementation Method 4; Figure 20 It is the homogenized one-dimensional energy distribution map described in Implementation Method 4; Figure 21 This is a schematic diagram of the microlens array described in Embodiment 1; Figure 22 This is a schematic diagram of the aspherical beam homogenization and shaping device described in Embodiment 1. Figure 23 This is an illustration of the effect of the aspherical beam homogenization and shaping device described in Embodiment 1. Detailed Implementation

[0015] Various embodiments of the present invention will now be clearly and completely described with reference to the accompanying drawings. The embodiments described with reference to the drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0016] Implementation Method 1: In the field of laser processing, regarding Gaussian beam homogenization technology, such as... Figure 21 As shown, microlens arrays or diffractive optical elements have also been used because if a Gaussian intensity distribution is used in laser material processing (such as laser cutting), the quality of the cut edge of the workpiece will be reduced. This is because of the shape of the Gaussian profile, which has high intensity at the center and low intensity at the edges. Due to the inhomogeneity of the laser beam, the melting of the workpiece is uneven. A simple and effective solution to increase the uniformity of the laser beam is to use a flat-top beam to optimize the quality of the cut edge, thereby inputting heat evenly to the surface to be processed.

[0017] However, the working distance of homogenization equipment in the field of laser processing is very short, and the light energy distribution is uniform only at the conjugate position. The homogenization effect deteriorates rapidly after deviating from the conjugate position, which actually increases the difficulty of optical system assembly and reliability in the field of super-resolution fluorescence imaging microscopy.

[0018] Although the aspherical cylindrical lens groups disclosed in Chinese patent CN119270520A ("A Beam Homogenization and Shaping Method Based on Aspherical Cylindrical Lens Group") and Chinese patent CN117031766A ("A Beam Homogenization and Collimation Method Based on Aspherical Cylindrical Lens Group") are not suitable for the field of super-resolution fluorescence imaging microscopy, this is because aspherical cylindrical lenses can only modulate the beam in one dimension, shaping a circular Gaussian light field into a long, uniform light field. They are mainly used in non-illumination applications, such as laser processing. However, in the field of super-resolution fluorescence imaging microscopy, the laser source primarily provides illumination to the microscopic system, requiring a uniformly distributed circular illumination area. Therefore, aspherical cylindrical lens groups are not suitable for this field.

[0019] To address the persistent technical challenge of achieving uniform excitation light energy distribution in super-resolution fluorescence imaging microscopy, this embodiment proposes an excitation light system for super-resolution fluorescence imaging. This system redistributes the Gaussian energy distribution of the laser to form a flat-topped energy distribution, thus resolving the aforementioned problems of uneven energy distribution and small field of view, and resulting in superior imaging performance. Specifically: The system includes a multi-wavelength exciter, a first reflecting mirror, an aspherical beam homogenization and shaping device, and a focusing lens; A multi-wavelength exciter emits a combined beam with a Gaussian energy distribution. After being reflected by the first mirror, the beam enters an aspherical beam homogenization and shaping device to homogenize it. The beam emitted from the aspherical beam homogenization and shaping device has a flat-top light field energy and is coupled into the microscope system through a focusing lens. The distance from the exit of the aspherical beam homogenization and shaping equipment to the focusing lens is 360mm-400mm.

[0020] Aspherical beam homogenization and shaping equipment changes the light refraction ability of a local area by controlling the surface shape of a lens. In other words, it changes the refraction of light. Because the surface shape of each local area of ​​an aspherical lens is different, the refraction effect of light is also different. However, the combination of the refracted light from each local area can redistribute the energy distribution of light, which means that the Gaussian energy distribution can be transformed into a flat-top distribution.

[0021] For example, Figure 22The left image shows that after parallel light passes through a conventional spherical lens, the light energy can converge to a single focal point. If you want to change the distribution of light energy at the focal point, you can use an aspherical surface (by altering the local shape of the spherical surface) to control the direction of local light refraction, deflecting the local light energy away from the original focal point. This disperses the energy, effectively reshaping the light energy distribution. Figure 22 As shown in the right figure.

[0022] The advantages of aspherical beam homogenization and shaping equipment are mainly: 1) It is not sensitive to light wavelength and can simultaneously shape multiple wavelengths such as 405nm, 488nm, 532nm, and 638nm. Optical diffraction elements (DOEs) have also been used in beam shaping equipment in other fields, but diffraction elements are very sensitive to light wavelength, and a single device can only shape light for one wavelength. 2) The working distance is relatively long. Aspherical homogenization and shaping equipment can achieve good shaping results over a relatively long working distance (approximately 300mm). Even when deviating from the conjugate position, the homogenization effect will not suddenly become very poor. Figure 23 As shown, this can reduce the difficulty of assembling and adjusting the optical system (such as the offset of the conjugate surface caused by factors such as device processing tolerances and vibration).

[0023] Implementation Method Two: This implementation method further defines the excitation light system for super-resolution fluorescence imaging described in Implementation Method One, specifically as follows: like Figure 4 As shown, because the conjugate relationship must be satisfied, the working distance of the aspherical beam homogenization and shaping device described in Embodiment 1 is required. The farther the aspherical beam homogenization and shaping device is from the device, the more demanding it becomes. Figure 7 As shown, the homogenization effect will deteriorate to some extent, and the structure will be difficult to lay out.

[0024] To achieve better homogenization, such as Figure 8 As shown, therefore, in this embodiment, a distance of 360mm-400mm (designed as 360mm in this embodiment) is required between the aspherical beam homogenization and shaping device and the focusing lens. A third reflecting mirror is designed, as follows: Figure 5 and Figure 6 As shown, adding a third reflecting mirror allows the entire optical path system to be designed as a box. This third reflecting mirror reflects the light beam emitted from the aspherical beam homogenizing and shaping device, and then couples it into the microscope system through a focusing lens, conjugating it onto the sample to achieve optimal homogenization of the light illuminating the sample. Conversely, without the third reflecting mirror, the box design would be very difficult to implement because the subsequent optical path would be too long.

[0025] Implementation Method 3: This implementation method further defines the excitation light system for super-resolution fluorescence imaging described in Implementation Method 2, specifically as follows: The designed optical system, such as Figure 9 As shown, coupling and homogenization of multi-wavelength excitation light at 405nm, 488nm, 532nm, and 638nm are achieved. For the 405nm, 488nm, 532nm, and 638nm bands, 4μm single-mode fiber is used as the light source emitter. The emitted light is collimated by a lens with a focal length of 40mm, and then combined by a dichroic mirror. The combined beam has a size of 10mm and a Gaussian energy distribution. After reflection by the first mirror, the beam enters an aspherical beam homogenization and shaping device for homogenization. The beam exiting the aspherical beam homogenization and shaping device has a size of 15mm and a flat-top energy distribution. To satisfy the conjugate relationship between the microscope object surface and the homogenization surface, the distance from the outlet of the aspherical beam homogenization and shaping device to the focusing lens (focal length 400mm) is designed to be 360mm. After reflection by the third mirror, the beam is coupled into the microscope system through the focusing lens.

[0026] A 40mm lens is universal for the 400-700nm wavelength range. Although there are chromatic differences in different wavelengths, the same collimation effect can be achieved for different wavelengths by controlling and adjusting the distance between the 40mm lens and the light source.

[0027] Implementation Method Four: This implementation method further defines the excitation light system for super-resolution fluorescence imaging described in Implementation Method Three, such as... Figure 10 As shown, the 405 band shares the same lens as other optical paths in the system, and the lens parameters are the same, specifically: The first and second lenses, combined to form an achromatic cemented lens with a focal length of 40mm, are used to collimate the light emitted from a point source. The third and fourth lenses form an aspherical beam homogenizing and shaping device, used to shape and expand the Gaussian spot. The fifth and sixth lenses, combined to form an achromatic cemented lens with a focal length of 400mm, are used to focus the excitation light onto the back focal plane of the microscope, so that the light, after passing through the objective lens, illuminates the sample surface in a parallel manner. The combination of parameters for the lens radius of curvature, thickness, refractive index, and Abbe number in Table 1 is designed to meet the lens's optical power (its ability to deflect light, i.e., to ensure the lens's focal length) while correcting for chromatic aberration in the 400-700nm wavelength range (the deviation of different wavelengths after passing through the lens).

[0028] Table 1 Parameters of Each Lens

[0029] like Figures 18-20As shown, after homogenization, the uniformity of the fluorescence signal is significantly improved, and the imaging field of view is increased by 35%, resulting in a larger effective illumination area.

[0030] Implementation Method 5: This implementation method further defines the excitation light system for super-resolution fluorescence imaging described in Implementation Method 4. An additional beam expander lens can be added to further increase the imaging field of view, such as... Figure 13 As shown, when a large field-of-view fluorescence imaging is required, a pair of beam-expanding lenses can be introduced; when high-intensity fluorescence imaging is required, the beam-expanding lenses can be removed. The two operating modes can be switched, and the imaging effects corresponding to the two optical paths are as follows: Figure 14 As shown, adding a beam expander increases the field of view of the illumination, but reduces the light density.

[0031] Implementation Method Six: In order to minimize light loss over long distances, this implementation method further improves the excitation light system for super-resolution fluorescence imaging described in Implementation Method One, specifically as follows: In practical application, this implementation method has found that, as Figure 11 As shown, although the reflectors used in Embodiments 1-5 can achieve a reflectivity of over 99% through coating, resulting in relatively low light loss during light transmission, the laser beams experience significant losses primarily at the dichroic plates.

[0032] To solve the above technical problems, such as Figure 12 As shown, this embodiment uses fiber optic coupling to couple multiple laser beams to a single fiber output. This eliminates the need for dichroic beam splitting and combining; a single mirror can be used to combine multiple beams before they enter the aspherical beam homogenization and shaping device.

[0033] The specific parameters of each lens in this embodiment are as described in the embodiment.

[0034] To better illustrate the technical effects of the systems described in Embodiments 1-6, the following experimental verifications are provided in detail: Example 1 like Figure 15 and Figure 16 As shown, the left and right images are comparison images of the fluorescent target taken with a microscope before and after adding the system described in the above embodiments. The improvement in uniformity and field of view can be clearly seen.

[0035] Example 2 like Figure 17The image shows a comparison of the effects of microscopic fluorescence imaging on cells before and after adding the system described in Embodiments 1-6. Without homogenization, the uniformity of the intracellular fluorescence signal distribution is poor. Due to the excessively high light field intensity in the central field of view, fluorescence quenching is accelerated. In addition, the edge field of view has very weak light field intensity, resulting in very few effectively clustered points and a small imaging field of view. After beam homogenization, the uniformity of the fluorescence signal is significantly improved, and the number of effectively clustered points is also significantly increased in the reconstructed image. At the same time, the imaging field of view after homogenization increases by 35%, providing a larger effective illumination area.

[0036] The above provides a detailed description of the excitation light system for super-resolution fluorescence imaging proposed in this invention. Specific examples have been used to illustrate the principles and implementation methods of this invention. The descriptions of the above embodiments are only for the purpose of helping to understand the method and core ideas of this invention. 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 invention. Therefore, the content of this specification should not be construed as a limitation of this invention.

Claims

1. An excitation light system for super-resolution fluorescence imaging, characterized in that, The system includes a multi-wavelength exciter, a first reflecting mirror, an aspherical beam homogenization and shaping device, and a focusing lens; A multi-wavelength exciter emits a combined beam with a Gaussian energy distribution. After being reflected by the first mirror, the beam enters an aspherical beam homogenization and shaping device to homogenize it. The beam emitted from the aspherical beam homogenization and shaping device has a flat-top light field energy and is coupled into the microscope system through a focusing lens. The distance from the exit of the aspherical beam homogenization and shaping equipment to the focusing lens is 360mm-400mm.

2. The excitation light system for super-resolution fluorescence imaging according to claim 1, characterized in that, The multi-wavelength exciter includes multiple wavelength light source emitting ends; Multiple wavelength light source emitters are collimated by lenses. One wavelength light source emitter is reflected by a second mirror and then combined with multiple light source emitters that are respectively passed through dichroic mirrors to form a single beam.

3. The excitation light system for super-resolution fluorescence imaging according to claim 2, characterized in that, The multiple wavelength light source emitters include light sources in the 405nm, 488nm, 532nm, and 638nm wavelength bands.

4. The excitation light system for super-resolution fluorescence imaging according to claim 2, characterized in that, The system also includes a third reflecting mirror; The beam emitted from the aspherical beam homogenization and shaping device is reflected by the third reflecting mirror and then coupled into the microscope system through the focusing lens.

5. The excitation light system for super-resolution fluorescence imaging according to claim 4, characterized in that, The lens is a 40mm lens made by bonding a first lens and a second lens together. The first lens has a radius of curvature of 61.313 on the front surface, a radius of curvature of 18.679 on the rear surface, a thickness of 1.8, a refractive index of 1.67, and an Abbe number of 32.

2. The second lens has a front surface radius of curvature of 18.679, a rear surface radius of curvature of -22.647, a thickness of 6.7, a refractive index of 1.52, and an Abbe number of 64.

2. The aspherical beam homogenization and shaping device includes a third lens and a fourth lens. The third lens has an infinite radius of curvature on its front surface, a radius of curvature of 87.111 on its rear surface, a thickness of 4, a refractive index of 1.52, and an Abbe number of 64.

2. The fourth lens has a radius of curvature of 87.111 on the front surface, a radius of curvature of 130.667 on the rear surface, a thickness of 81.523, a refractive index of 1.52, and an Abbe number of 64.

2. The focusing lens is a 400mm lens cemented together with a fifth lens and a sixth lens. The fifth lens has a radius of curvature of -220.133 on the front surface, a radius of curvature of 193.143 on the rear surface, a thickness of -4, a refractive index of 1.52, and an Abbe number of 64.

2. The sixth lens has a radius of curvature of 193.143 on its front surface, a radius of curvature of 698.743 on its rear surface, a thickness of -2.5, a refractive index of 1.67, and an Abbe number of 32.

2.

6. The excitation light system for super-resolution fluorescence imaging according to claim 5, characterized in that, The system also includes a beam expander lens group; After being reflected by the third reflecting mirror, the beam passes through the beam expander lens group to increase the field of view of the image, and then is coupled into the microscope system through the focusing lens.

7. The excitation light system for super-resolution fluorescence imaging according to claim 1, characterized in that, The multi-wavelength exciter includes optical fibers coupled to the emitters of multiple wavelength light sources. The light beam emitted from the optical fiber is collimated by a collimating lens and then outputs a combined beam.

Images