Structured light illumination super-resolution fluorescence microscope imaging light path modification and construction method

Abstract

The invention relates to the technical field of optical imaging, and discloses a structured light illumination super-resolution fluorescence microscope imaging light path modification and construction method, which comprises the following steps: S1, an optical filter, a quarter-wave plate, a linear polarizer and a camera are sequentially arranged, and the camera is arranged on an emergent light path behind the linear polarizer and is used for obtaining an imaging result of a sample; and S2, on the basis of an original structured light illumination super-resolution fluorescence microscope imaging light path, a quarter-wave plate and a linear polarizer are added between a light path from a light filter to a camera, and the specific implementation scheme of further light path refitting is that the light filter, the quarter-wave plate and the linear polarizer which are sequentially arranged are aligned with the focus of the camera. The method provided by the invention has certain innovativeness, and compared with a traditional chiral characterization means, the method can reveal more detailed information, such as chiral difference of individuals in a sample and the like.

Description

Technical Field

[0001] The present invention relates to the field of optical imaging technology, and in particular to a method for modifying and building an imaging optical path of a structured light illumination super-resolution fluorescence microscope. Background Art

[0002] Since the 21st century, the Nobel Prize has been awarded twice in the field of chiral materials. So far, the development scale of the field of chiral materials in my country has reached the billion-yuan level, and it has broad application prospects in optoelectronic devices, selective adsorption, asymmetric synthesis, biomedicine and other fields. However, at present, the chiral properties of chiral materials are mainly studied through spectroscopy (circular dichroism, vibrational circular dichroism, etc.), x-ray single crystal diffraction, nuclear magnetic resonance hydrogen spectrum, etc. These chiral characterization methods of chiral materials are often subject to certain limitations due to the properties of the materials themselves. For example, x-ray single crystal diffraction requires that the material has good crystallinity, otherwise it is impossible to obtain good results. At the same time, the above chiral characterization methods mostly obtain the chiral results of a macroscopic whole material, reflecting the chiral information of the whole material, which is also very limited. Structured light illumination super-resolution fluorescence microscopy is based on conventional fluorescence microscopy. By improving its illumination method, it uses specific structured light imaging to break through the diffraction limit to obtain high-resolution sample information, and then obtains the sample microscopic image by Fourier transform. It is an optical microscope that can realize real-time dynamic imaging of the sample. It has the characteristics of easy operation, low material requirements (autofluorescence or staining), stability and high resolution, and has been widely used in the field of biomedical imaging. Since its imaging information comes from the fluorescence emitted by the sample when it is excited by laser, it is possible to use it to identify the information of chiral samples by simply modifying the imaging optical path. However, there are currently no relevant reports on this aspect. Therefore, it is very necessary to develop and assemble the imaging optical path of chiral samples for structured light illumination super-resolution fluorescence microscopy.

[0003] To this end, we proposed a method for modifying and building the imaging optical path of a structured light illumination super-resolution fluorescence microscope to solve the problem. Summary of the invention

[0004] The purpose of the present invention is to provide a method for modifying and building an imaging optical path of a structured light illumination super-resolution fluorescence microscope to solve the problems raised in the above-mentioned background technology.

[0005] In order to solve the above technical problems, the present invention provides the following technical solution: a method for modifying and building an imaging optical path of a structured light illumination super-resolution fluorescence microscope, comprising the following steps:

[0006] S1: A filter, a quarter wave plate, a linear polarizer and a camera are sequentially arranged, and the camera is arranged on the outgoing light path after the linear polarizer to obtain the imaging result of the sample;

[0007] S2: Based on the original structured light illumination super-resolution fluorescence microscope imaging optical path, a quarter-wave plate and a linear polarizer are added between the optical path from the filter to the camera. The specific implementation plan for further optical path modification is: align the filter, quarter-wave plate and linear polarizer arranged in sequence and the focus of the camera, wherein the quarter-wave plate has a 45° phase difference with the incident light axis, the short axis direction of the linear polarizer forms an angle of 45° with the incident light axis direction, and the camera is located at the end of the outgoing light path of the linear polarizer for imaging;

[0008] S3: Utilizing the intensity difference of left / right circularly polarized fluorescence emitted by the chiral sample to be tested, the emitted fluorescence is filtered through an optical filter, and the obtained circularly polarized light is converted into two mutually perpendicular linear polarized light beams under the action of a quarter wave plate. One of the linear polarized light beams is parallel to the optical axis of the light path, and the other is perpendicular to the optical axis of the light path. When these two light beams pass through the linear polarizer, only light in one direction is allowed to pass through. Finally, the light beams in different directions generated by the sample to be tested are obtained through a camera, thereby realizing imaging of the sample.

[0009] According to the above technical solution, the filter has a fluorescence wavelength range of 585-615 nm and reflects fluorescence of other wavelengths.

[0010] According to the above technical solution, the quarter wave plate converts the circularly polarized fluorescence into two mutually perpendicular linear polarized lights by introducing a 45° phase difference with the incident light axis, wherein one linear polarized light is parallel to the optical axis of the light path, and the other linear polarized light is perpendicular to the optical axis of the light path.

[0011] According to the above technical solution, the short axis of the linear polarizer forms an angle of 45° with the optical axis direction, and by adjusting the angle of the linear polarizer, only linear polarized light in a certain direction passes through.

[0012] Compared with the prior art, the beneficial effects achieved by the present invention are:

[0013] 1. The method provided by the present invention is simple to operate and has certain stability and sensitivity.

[0014] 2. The method provided by the present invention can be used for real-time monitoring of chiral samples to reveal some specific information, such as the differences between individuals in the sample, the fluorescence differences between different regions in the same individual, etc.

[0015] 3. The method provided by the present invention is innovative to a certain extent. Compared with traditional chirality characterization methods, it can reveal more detailed information, such as the chirality differences of individuals in the sample, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0017] Figure 1 The original optical path diagram and the modified optical path diagram of the structured light illumination super-resolution fluorescence microscope of the present invention;

[0018] Figure 2 It is a schematic flow chart of the method for using the optical path of the structured light illumination super-resolution fluorescence microscope of the present invention;

[0019] Figure 3 This is a diagram showing the stability imaging effect of the present invention;

[0020] Figure 4 It is an imaging diagram of the non-chiral sample of the present invention under different polarizer angle conditions;

[0021] Figure 5 This is an imaging diagram of the D chiral sample of the present invention under different polarizer angle conditions;

[0022] Figure 6 This is a summary diagram of fluorescence values ​​of the D chiral sample of the present invention under different polarizer angle conditions;

[0023] Figure 7 It is an imaging diagram of the L chiral sample of the present invention under some polarizer angle conditions;

[0024] Figure 8 It is a summary diagram of the fluorescence values ​​of the L chiral sample of the present invention under different polarizer angle conditions. DETAILED DESCRIPTION

[0025] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] Example 1: Reference Figure 1 Based on the original structured light illumination super-resolution fluorescence microscope imaging optical path, the optical path modification can be completed by simply adding a quarter glass slide and a linear polarizer between the filter and the camera.

[0027] The specific implementation plan for further optical path modification is: the filters, quarter glass, linear polarizer and camera are aligned in sequence. The quarter glass has a 45° phase difference with the incident light axis, the short axis direction of the linear polarizer is at an angle of 45° with the incident light axis, and the camera is located at the end of the outgoing light path of the linear polarizer.

[0028] The filter described above transmits the fluorescence emitted by the sample, specifically the fluorescence wavelength range is 585-615nm, and reflects the light of other wavelengths.

[0029] The quarter wave plate described above converts circularly polarized fluorescence into two mutually perpendicular linear polarized lights by introducing a 45° phase difference with the incident light axis, wherein one linear polarized light is parallel to the optical axis of the light path and the other linear polarized light is perpendicular to the optical axis of the light path.

[0030] The minor axis of the linear polarizer mentioned above forms an angle of 45° with the optical axis. By adjusting the angle of the linear polarizer, only linear polarized light in a certain direction can pass through.

[0031] The camera described above is located at the end of the outgoing light path behind the linear polarizer, and captures polarized light in a certain direction after passing through the linear polarizer to obtain imaging results of the sample.

[0032] Experimental Example 1: (1) Reference Figure 2 The method of using this optical path is basically the same as that of the traditional structured light illumination super-resolution fluorescence microscope. The difference is that this optical path needs to rotate the linear polarizer at different angles to obtain light beams in different directions for imaging. The further method of using the optical path for imaging chiral samples in structured light illumination super-resolution fluorescence microscope is as follows:

[0033] 1. Place the quarter wave plate and linear polarizer according to Figure 1 As shown, it is loaded onto the imaging light path from the filter to the camera, wherein the distance from the filter to the quarter-wave plate is about 5 cm, the distance from the quarter-wave plate to the linear polarizer is about 5 cm, and the distance from the linear polarizer to the camera is about 8 cm. Turn on the light source and use a light baffle to align the focus of the imaging light path and place them on the same horizontal plane.

[0034] 2. Prepare the sample according to the properties of the sample. Generally speaking, 1 mg of the sample can be ultrasonically dispersed in 2 ml of water or ethanol, 5 microliters of the sample can be dropped on a glass slide, covered with a coverslip, and then observed.

[0035] 3. Open the corresponding structured light illumination super-resolution fluorescence microscope control software and set the parameters. Generally speaking, you only need to adjust it until the imaging area is clearly observed.

[0036] 4. Sample shooting: You can find suitable area samples for shooting according to the test conditions or test content. Specific cases can be referred to as follows.

[0037] (2)Reference Figure 3, the stability imaging effect diagram of the optical path. As shown in the figure, under the same optical path conditions, as time changes, the characteristics of the standard sample (morphology, fluorescence) do not change much over time (0-10min), and the relative value of the change is ±5%, which is due to the error of the instrument itself. The specific fluorescence value of each ball is as follows Figure 3 As shown in the table, it proves the difference of individual fluorescence in the same sample.

[0038] (3)Reference Figure 4 , Imaging of non-chiral samples under different polarizer angles. As shown in the figure, under the same optical path conditions, the fluorescence intensity of the sample does not change with the change of angle.

[0039] (4)Reference Figure 5 , D is the imaging diagram of the chiral sample under different polarizer angle conditions. As shown in the figure, under the same parameter conditions, the intensity of the chiral sample will change visibly with the change of the polarizer angle. For specific values, please refer to Figure 6 , Summary of fluorescence values ​​of D chiral samples under different polarizer angles. For D chiral samples, the fluorescence intensity at 45° is less than that at 135°, and the fluorescence intensity at 225° is less than that at 315°. The corresponding relationship of fluorescence intensity at other angles, such as 0°, 90°, 180°, and 270°, remains basically unchanged.

[0040] (5)Reference Figure 7 , L chiral sample imaging under different polarizer angle conditions. As shown in the figure, under the same parameter conditions, the intensity of the chiral sample will change visibly with the change of the polarizer angle. For specific values, please refer to Figure 8 , Summary of fluorescence values ​​of L chiral samples under different polarizer angles. For L chiral samples, the fluorescence intensity at 45° is less than that at 135°, and the fluorescence intensity at 225° is less than that at 315°. The corresponding relationship of fluorescence intensity at other angles, such as 0°, 90°, 180°, and 270°, remains basically unchanged.

[0041] In summary, this optical path has a certain analytical effect on chiral samples, so as to observe the individual differences of samples, including factors such as morphology and fluorescence intensity, and see more information that traditional chiral analysis methods cannot see. In addition, this optical path can identify the chiral properties of samples, can distinguish between chiral and non-chiral samples, left-handed and right-handed samples, has good sensitivity and stability, and has the potential to become a new chiral characterization technology.

[0042] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0043] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for modifying and building the imaging optical path of a structured light illumination super-resolution fluorescence microscope, characterized in that: The following steps are involved: S1: A filter, a quarter wave plate, a linear polarizer and a camera are sequentially arranged, and the camera is arranged on the outgoing light path after the linear polarizer to obtain the imaging result of the sample; S2: Based on the original structured light illumination super-resolution fluorescence microscope imaging optical path, a quarter-wave plate and a linear polarizer are added between the optical path from the filter to the camera. The specific implementation plan for further optical path modification is: align the filter, quarter-wave plate and linear polarizer arranged in sequence and the focus of the camera, wherein the quarter-wave plate has a 45° phase difference with the incident light axis, the short axis direction of the linear polarizer forms an angle of 45° with the incident light axis direction, and the camera is located at the end of the outgoing light path of the linear polarizer for imaging; S3: Utilizing the intensity difference of left / right circularly polarized fluorescence emitted by the chiral sample to be tested, the emitted fluorescence is filtered through an optical filter, and the obtained circularly polarized light is converted into two mutually perpendicular linear polarized light beams under the action of a quarter wave plate. One of the linear polarized light beams is parallel to the optical axis of the light path, and the other is perpendicular to the optical axis of the light path. When these two light beams pass through the linear polarizer, only light in one direction is allowed to pass through. Finally, the light beams in different directions generated by the sample to be tested are obtained through a camera, thereby realizing imaging of the sample.

2. The method for modifying and building the imaging optical path of a structured light illumination super-resolution fluorescence microscope according to claim 1, characterized in that: The filter has a fluorescence wavelength range of 585-615 nm and reflects fluorescence of other wavelengths.

3. The method for modifying and building the imaging optical path of a structured light illumination super-resolution fluorescence microscope according to claim 2, characterized in that: The quarter wave plate converts the circularly polarized fluorescence into two mutually perpendicular linear polarized lights by introducing a 45° phase difference with the incident light axis, wherein one linear polarized light is parallel to the optical axis of the light path and the other linear polarized light is perpendicular to the optical axis of the light path.

4. The method for modifying and building the imaging optical path of a structured light illumination super-resolution fluorescence microscope according to claim 3, characterized in that: The short axis of the linear polarizer forms an angle of 45° with the optical axis direction. By adjusting the angle of the linear polarizer, only linear polarized light in a certain direction can pass through.

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