Microscopic illumination system and method based on mask projection layer-cut imaging

Abstract

The invention provides a microscopic illumination system and method based on mask plate projection layer tangent imaging, and the system comprises a light source, a first lens, a field diaphragm, a second lens, an aperture diaphragm, a third lens, a mask plate switching device, an illumination cylinder lens and a microscopic objective lens which are sequentially arranged along an illumination light path. The field diaphragm is located on the rear focal plane of the first lens and the front focal plane of the second lens, the aperture diaphragm is located on the rear focal plane of the second lens and the front focal plane of the third lens, the mask plate switching device is located on the rear focal plane of the third lens and the front focal plane of the lighting tube lens, the mask plate switching device comprises a base and a rotating wheel, and the rotating wheel is erected on the base and can rotate. A plurality of mask plates with different shapes are embedded in the rotating wheel, the mask plates are distributed in a circumferential array, and the central point of any one mask plate is overlapped with the illumination light path in an initial state. The invention has the beneficial effects that structured light in multiple directions can be provided in the same focal plane, and the mask plates can be conveniently switched.

Description

Technical Field

[0001] The present invention relates to the technical field of microscopic illumination systems, and in particular to a microscopic illumination system and method based on mask projection layer-cut imaging. Background Art

[0002] Structured light illumination is a technology that uses a projector to project specific light information onto the surface of an object, and then uses a camera to collect changes in light signals to calculate information such as the object's position and depth. This technology consists of a system structure consisting of a projector and a camera, and is often used for the restoration of three-dimensional space.

[0003] In structured light illumination fluorescence microscopy (SIM), structured light illumination breaks through the optical diffraction limit through a specific illumination method to achieve super-resolution imaging. SIM technology inserts a structured light generator (such as a grating, spatial light modulator, or digital micromirror array DMD, etc.) into the illumination light path to form a pattern with regularly changing brightness. The illumination pattern is then moved and rotated to cover various areas of the sample, and the multiple images taken are combined and reconstructed using software to ultimately obtain a super-resolution image.

[0004] Structured light illumination is one of the main technologies used for three-dimensional microscopy, and there are many ways to generate structured light. Among them, the structured light generation method based on mask projection is relatively simple in structure, economical, and easy to operate. Mask projection produces structured light. In principle, a special mask with a certain structure (such as a stripe structure) is projected onto the focal plane of the microscope through optical imaging. The precise adjustment of the conjugate position relationship between the mask and the object plane is very important.

[0005] Conventional optical path construction methods are not suitable for the fine positioning required for the mask plate. It is necessary to design a mask plate positioning method that is convenient and adjustable in real time. The grating structure projection produces structured light, and then multiple stripe distribution directions are designed to obtain the required structured light stripes in various directions, which increases the complexity of the program and the image acquisition time; the microscope system needs to switch the illumination light source to collect wide-field images and structured light images. Therefore, it is urgent to design a microscope illumination system and method that can provide structured light in multiple directions within the same focal plane and can conveniently switch the mask plate. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide structured light in multiple directions within the same focal plane and to be able to conveniently switch the mask plate. In order to overcome the defects of the above-mentioned prior art (or related technology), the present invention provides a microscopic illumination system and method based on mask plate projection layer section imaging.

[0007] The present invention provides a microscopic illumination system based on mask projection layer cutting imaging, comprising a light source, a first lens, a field stop, a second lens, an aperture stop, a third lens, a mask switching device, an illumination tube lens and a microscope objective lens, which are sequentially arranged along an illumination light path, wherein the light source is located at a front focal plane of the first lens, the field stop is located at a back focal plane of the first lens and a front focal plane of the second lens, the aperture stop is located at a back focal plane of the second lens and a front focal plane of the third lens, the mask switching device is located at a back focal plane of the third lens and a front focal plane of the illumination tube lens, and the mask switching device comprises a base and a rotating wheel, wherein the rotating wheel is mounted on the base and can rotate, wherein a plurality of masks of different shapes are embedded in the rotating wheel, and each of the mask plates is distributed in a circular array with the axis of the rotating wheel as the center line, and in an initial state, the center point of any one of the mask plates coincides with the illumination light path.

[0008] Compared with the prior art, the present invention provides a microscopic illumination system based on mask projection layer section imaging, which has the following advantages: In the present application, a Kohler illumination part is composed of a light source, a first lens, a field stop, a second lens, an aperture stop and a third lens; a mask projection part is composed of a mask switching device, an illumination tube lens and a microscope objective lens; the mask switching device is designed between the third lens and the illumination tube lens; a rotating wheel is arranged in the mask switching device, and masks of different shapes are arranged on the rotating wheel, so that after determining the conjugate position, structured light in multiple directions can be provided in the same focal plane by switching masks of different shapes, and masks of different shapes can be embedded in the rotating wheel to facilitate switching of the masks.

[0009] In a possible implementation manner, the focal lengths of the first lens and the second lens are the same and are 60 mm, and the focal length of the third lens is 30 mm.

[0010] In a possible implementation manner, the diameters of the first lens and the second lens are the same and are 50.8 mm, and the diameter of the third lens is 25.4 mm.

[0011] In a possible implementation manner, each of the mask plates is a soda lithography mask plate and the surface is plated with chrome.

[0012] In a possible implementation manner, the shape of each of the mask plates includes a cross shape, a grid shape, and a regular hexagonal shape.

[0013] In a possible implementation manner, a diameter of each of the mask plates is 20-100 μm.

[0014] In a possible implementation, the base is mounted on a slide rail disposed along the illumination light path and the base is slidably connected to the slide rail, and the slide rail is located between the third lens and the illumination tube lens.

[0015] The present invention also provides a microscopic illumination method based on mask projection layer section imaging, which is applied to the above-mentioned microscopic illumination system and includes the following steps: Step S1, assembling the first lens, the field stop, the second lens, the aperture stop, the third lens, the mask switching device, the illumination tube lens and the microscope objective lens in sequence along a set illumination light path; Step S2, placing the specimen sample, focusing the microscope objective lens until it is clear under the bright field of the microscope, and then locking the focus of the microscope objective lens; Step S3, removing the specimen sample, adding a light source to input a parallel light beam in the forward direction along the illumination light path, placing a light baffle at the rear end of the microscope objective lens, observing that the light spot on the light baffle is elliptical in distribution, and then adjusting the position of the illumination tube lens on the illumination light path until the long axis direction of the light spot is about to change and stopping the position adjustment; Step S4, repositioning the specimen sample, switching the cross-shaped mask on the rotating wheel into the illumination light path, and adjusting the position of the mask switching device on the illumination light path to a clear stop position of the cross image observed through the microscope objective lens; Step S5, switching the mask plates of different shapes to capture structured light images.

[0016] Compared with the prior art, the present invention provides a microscopic illumination method based on mask projection layer section imaging, which has the following advantages: In the present application, the overall microscope illumination structure is assembled through step S1, the microscope objective is focused through step S2, the position of the illumination tube lens is adjusted through step S3, the position of the mask plate switching device is adjusted through step S4, and different structured light images are collected through step S5. After the conjugate position is determined in step S4, structured light in multiple directions can be provided in the same focal plane by switching mask plates of different shapes, and mask plates of different shapes can be embedded in the rotating wheel to facilitate switching of the mask plates. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a structural schematic diagram of the present invention; Figure 2 It is a structural schematic diagram of the mask plate switching device of the present invention; Figure 3 A schematic structural diagram of a grid-shaped mask plate of the present invention; Figure 4It is a structural schematic diagram of a regular hexagonal mask plate of the present invention; Figure 5 It is a schematic diagram of position adjustment of the lighting tube mirror of the present invention; Figure 6 It is a schematic diagram of position adjustment of the mask plate switching device of the present invention; Figure 7 is a flow chart of the method steps of the present invention; Explanation of the reference numerals: 1. light source; 2. first lens; 3. field diaphragm; 4. second lens; 5. aperture diaphragm; 6. third lens; 7. mask switching device; 71. base; 72. rotating wheel; 73. mask; 8. illumination tube lens; 9. microscope objective lens. DETAILED DESCRIPTION

[0018] First, those skilled in the art should understand that these implementations are only used to explain the technical principles of the embodiments of the present application, and are not intended to limit the protection scope of the embodiments of the present application. Those skilled in the art can make adjustments to them as needed to adapt to specific application scenarios.

[0019] The present application is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0020] See also Figure 1 The embodiment of the present application discloses a microscope illumination system based on mask projection layer-cut imaging. The entire optical path system includes a Kohler illumination part and a mask projection part, wherein the Kohler illumination part is composed of three lenses and two apertures, and the mask projection part is composed of a mask switching device (mask), an illumination tube lens (tube) and a microscope objective lens. A specially-structured mask and a light field switching method are designed for the light field switching required for microscopic illumination, and a method for accurately positioning the position of the mask switching device is proposed, which has the advantages of simple structure, low cost, and easy assembly and adjustment.

[0021] Continue to see Figure 1In a specific implementation, the microscopic illumination system includes, from front to back along the illumination light path, a light source 1, a first lens 2, a field stop 3, a second lens 4, an aperture stop 5, a third lens 6, a mask switching device 7, an illumination tube lens 8, and a microscope objective lens 9. The light source 1, the first lens 2, the field stop 3, the second lens 4, the aperture stop 5, and the third lens 6 constitute the Kohler illumination part of the microscopic illumination system; the focal length f1 and diameter d1 of the first lens 2, the focal length f2 and diameter d2 of the second lens 4, and the focal length f3 and diameter d3 of the third lens 6 satisfy: f1=f2=60mm, f3=30mm, d1=d2=50.8mm, d3=25.4mm; for In the Kohler illumination part, the light source 1 is located at the front focal plane of the first lens 2, and the field stop 3 is placed between the first lens 2 and the second lens 4. For the mask projection part, the light source 1 is located at the front focal plane of the second lens 2, and the aperture stop 5 is placed at the back focal plane of the second lens 4, which is also the front focal plane of the third lens 6. The mask switching device 7 is placed at the back focal plane of the third lens 6 and the front focal plane of the illumination tube lens 8. The light beam emitted by the light source 1 passes through the first lens 2 and is incident on the second lens 4 as parallel light. The back focal plane of the second lens 4 coincides with the front focal plane of the third lens 6. The light emitted by the light source 1 passes through the third lens 6 and is parallel to the surface of the mask 73 (located at the back focal plane of the third lens 6), which is the Kohler illumination method.

[0022] See also Figure 2 In the present application, a manual or electric rotating wheel 72 is provided, which is mounted on a base 71 that can move back and forth in the light path. The position of the rotating wheel 72 in the lighting light path is adjusted by moving the base 71 back and forth.

[0023] Continue to see Figure 2 The rotating wheel 72 in the present application can be used to install multiple mask discs. By rotating and switching the mask 73 placed in the illumination light path, the light beam illuminates the mask 73 and projects its structure onto the focal plane of the microscope objective 9.

[0024] The 4F system composed of the microscope objective 9 and the illumination tube lens 8 is used to adjust the conjugate relationship between the mask plate and the object plane, and to determine the conjugate position of the mask plate 73 through a large-size (millimeter level) positioning mask plate; the mask plate 73 is in a cross shape, a grid shape, or a regular hexagonal shape, and the size is customized according to the needs, with a diameter of 20-100μm; and the mask plate 73 adopts a soda lithography mask plate with a chrome-plated surface, and projection is performed by engraving different shapes.

[0025] See also Figure 7 The present application also discloses a microscopic illumination method based on mask projection layer section imaging, comprising: Step S1, assembling the first lens 2, the field stop 3, the second lens 4, the aperture stop 5, the third lens 6, the mask switching device 7, the illumination tube lens 8 and the microscope objective lens 9 in sequence along the set illumination light path; Step S2, placing the specimen sample, focusing the microscope objective lens 9 to a clear image under the bright field of the microscope, and then locking the focus of the microscope objective lens 9; Step S3, remove the specimen sample, add the light source 1 to input a parallel light beam in the forward direction along the illumination light path, place a light baffle at the rear end of the microscope objective 9, observe that the light spot on the light baffle is elliptical in distribution, then adjust the position of the illumination tube lens 8 on the illumination light path until the long axis direction of the light spot is about to change and stop the position adjustment; Step S4, reposition the specimen, switch the cross-shaped mask plate 73 on the rotating wheel 72 into the illumination light path, and adjust the position of the mask plate switching device 7 on the illumination light path to a clear stop position of the cross image observed by the microscope objective lens 9; Step S5, switching the mask plates 73 of different shapes to collect structured light images.

[0026] Example 1 In an embodiment of the present application, a method for positioning a mask plate is provided, comprising: a) Place the specimen, adjust the focus to a clear image under the bright field of the microscope objective 9, and lock the focus of the microscope objective 9; b) Remove the specimen, input a parallel beam forward in the illumination light path, place a light baffle behind the microscope objective 9, and observe that the light spot on the light baffle is distributed in an elliptical pattern, as shown in Figure 2. Figure 5 , adjust the front and rear positions of the illumination tube lens 8 until the major axis direction of the elliptical light spot is about to change. At this time, the illumination tube lens 8 and the microscope objective lens 9 form a 4F system; c) Reposition the specimen and switch the cross-shaped light-transmitting mask (cross slit width 1 mm) on the rotating wheel 72 into the illumination light path, as shown in Figure 6 As shown, under the Kohler illumination optical system, the position of the rotating wheel 72 is moved back and forth to find the image of the cross on the focal plane of the microscope objective 9. The mask plate 73 used for projection is generally at the micron level. Since the cross slit width is at the millimeter level, it is more suitable for positioning the rotating wheel 72. Until a clear cross image is seen, it can be determined that the rotating wheel 72 and the mask plate 73 are in a conjugate position with the sample surface. By switching the rotating wheel 72, the desired mask plate 73 can be switched.

[0027] Example 2 In the embodiment of the present application, a method for switching the illumination light field by using the rotating wheel 72 is provided, such as Figure 1As shown, first, according to the mask positioning method described in Example 1, the position of the rotating wheel 72 where the mask 73 is located is positioned. Assuming that the hole A of the rotating wheel 72 is in the illumination light path, the special mask 73 is installed on the rotating wheel 72, and a position of the rotating wheel 72 is left vacant. Then, the rotating wheel 72 is rotated so that the light beam passes through the hole A of the rotating wheel 72 and outputs a structured light or a parallel light beam, thereby completing the required wide field or structured light field switching requirements. The diameters of the holes A and B of the rotating wheel 72 and the diameter of the mask 73 are both 25.4 mm.

[0028] Example 3 In the embodiment of the present application, two special structured light mask plates 73 are designed. Figure 3 The mask plate 73 with a grid-shaped structure shown in the figure can achieve structured light effects in two directions D1 and D2 in the focal plane. Figure 4 The mask plate 73 with an alternating structure of regular hexagons shown can achieve structured light effects in three directions: D1, D2 and D3, where the white color is the light-transmitting area, the blue color is the light-shielding area, and the spacing of the grid shape is related to the actual diffraction limit, specifically at the level of 20-60μm.

[0029] In the description of the present application, the description with reference to the terms "one embodiment", "some embodiments", "in the present embodiment", "specific example", or "some examples" etc. means that the specific features, mechanisms, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, mechanisms, materials or characteristics described may be combined in a suitable manner in any one or more embodiments or examples. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0030] The above is only a specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed in the present application should be included in the protection scope of the present application. Therefore, the protection scope of the present application shall be based on the protection scope of the claims.

Claims

1. A microscopic illumination system based on mask projection layer section imaging, characterized in that: The invention comprises a light source (1), a first lens (2), a field stop (3), a second lens (4), an aperture stop (5), a third lens (6), a mask plate switching device (7), an illumination tube lens (8) and a microscope objective lens (9) which are sequentially arranged along an illumination light path, wherein the light source (1) is located at the front focal plane of the first lens (2), the field stop (3) is located at the back focal plane of the first lens (2) and the front focal plane of the second lens (4), the aperture stop (5) is located at the back focal plane of the second lens (4) and the front focal plane of the third lens (6), and the mask plate switching device (7) is located at the back focal plane of the second lens (4) and the front focal plane of the third lens (6). The plate switching device (7) is located at the rear focal plane of the third lens (6) and the front focal plane of the illumination tube lens (8). The mask plate switching device (7) comprises a base (71) and a rotating wheel (72). The rotating wheel (72) is mounted on the base (71) and is rotatable. A plurality of mask plates (73) of different shapes are embedded in the rotating wheel (72). The mask plates (73) are distributed in a circular array with the axis of the rotating wheel (72) as the center line. In an initial state, the center point of any one of the mask plates (73) coincides with the illumination light path.

2. The microscopic illumination system according to claim 1, characterized in that: The focal lengths of the first lens (2) and the second lens (4) are the same and are 60 mm, and the focal length of the third lens (6) is 30 mm.

3. The microscopic illumination system according to claim 1, characterized in that: The diameters of the first lens (2) and the second lens (4) are the same and are 50.8 mm, and the diameter of the third lens (6) is 25.4 mm.

4. The microscopic illumination system according to claim 1, characterized in that: Each of the mask plates (73) is a soda lithography mask plate and has a surface plated with chrome.

5. The microscopic illumination system according to claim 1, characterized in that: The shapes of each of the mask plates (73) include a cross shape, a grid shape and a regular hexagonal shape.

6. The microscopic illumination system according to claim 1, characterized in that: The diameter of each mask plate (73) is 20-100 μm.

7. The microscopic illumination system according to claim 1, characterized in that: The base (71) is mounted on a slide rail arranged along the illumination light path and the base (71) is slidably connected to the slide rail, and the slide rail is located between the third lens (6) and the illumination tube lens (8).

8. A microscopic illumination method based on mask projection layer section imaging, characterized in that: The microscopic illumination system as claimed in claim 1 or 5 comprises the following steps: Step S1, assembling the first lens (2), the field diaphragm (3), the second lens (4), the aperture diaphragm (5), the third lens (6), the mask switching device (7), the illumination tube lens (8), and the microscope objective lens (9) in sequence along a set illumination light path; Step S2, placing a specimen sample, focusing the microscope objective lens (9) to a clear image under a bright field of a microscope, and then locking the focus of the microscope objective lens (9); Step S3, removing the specimen sample, adding a light source (1) to input a parallel light beam in the forward direction along the illumination light path, placing a light baffle at the rear end of the microscope objective lens (9), observing that the light spot on the light baffle is elliptical in distribution, and then adjusting the position of the illumination tube lens (8) on the illumination light path until the long axis direction of the light spot is about to change and stopping the position adjustment; Step S4, repositioning the specimen sample, switching the cross-shaped mask plate (73) on the rotating wheel (72) into the illumination light path, and adjusting the position of the mask plate switching device (7) on the illumination light path to a clear stop position of the cross image observed through the microscope objective lens (9); Step S5, switching the mask plates (73) of different shapes to perform structured light image acquisition.

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