A total internal reflection module for a microscope and its usage method

Abstract

The present invention provides a total internal reflection module for a microscope and a method for using the same. The total internal reflection module for a microscope mainly consists of a sample stage and a rotator. The sample stage includes a sample holder, a prism holder, a semi-cylindrical prism, and a spring pressing plate. The rotator includes a driving motor, a motor protection box, a right-angle rotating arm, a collimator fixing bracket, a collimator, and a reflector. It can be commonly used for various types of microscopes, electrically calibrates the incident angle of the light beam, and has a simple operation. Only sample loading, turning on the excitation light, and adjusting the excitation optical path are required to perform total internal reflection microscopy imaging. The relative positions of the collimator, the reflector, and the semi-cylindrical prism are fixed, and the excitation light always perpendicularly enters the semi-cylindrical prism to the center position to excite the sample to be measured. The driving motor is used to electrically adjust the angle of the excitation light, achieving extremely small adjustment of the incident angle, which not only saves the sample testing time but also facilitates coupling with various microscopes to realize total internal reflection microscopy imaging on an ordinary microscope.

Description

Technical Field

[0001] The present invention discloses a total internal reflection module for a microscope, belonging to the technical field of optical microscopy imaging, specifically a total internal reflection fluorescence microscopy imaging module that is applicable to various types of microscopes and is used for performing total internal reflection fluorescence microscopy imaging on a sample to be measured. Background Art

[0002] A large number of facts in life sciences indicate that the dynamic characteristics of cells originate from the aggregation and interaction of individual protein molecules, and many crucial life activities in cells are completed on the cell membrane surface, such as signal transduction, protein transport, pathogen invasion, etc. Therefore, directly observing the life activity process on the cell surface at the molecular scale without being interfered by signals from the deep regions inside the cell is of great significance for exploring the complex life activities in cells. Total internal reflection fluorescence microscopy is one of the internationally recognized most promising single-molecule optical imaging techniques on the membrane. When light travels from an optically denser medium to an optically less dense medium, refraction occurs. When the incident angle of the light is the critical angle θ c , the refraction angle is exactly 90°. At this time, the excitation light is completely reflected at the interface between the two media, which is total reflection. When total reflection occurs, due to the wave effect, a part of the light energy will penetrate through the interface into the optically less dense medium and propagate along the interface. This part of the penetrated energy field is called the "evanescent wave". The principle of total internal reflection fluorescence microscopy is to use the evanescent wave generated during total internal reflection to illuminate the sample, so that the illumination area is limited within a thin layer on the sample surface, effectively controlling the excitation volume and having a high signal-to-noise ratio and contrast that cannot be compared with other optical imaging techniques. Currently, total internal reflection fluorescence microscopy imaging systems mainly include the objective type and the prism type. In the objective type total internal reflection fluorescence microscope system, a microscope equipped with a special objective lens is required. This objective lens serves both as a receiver for collecting the fluorescence signal of the sample and as an optical device for generating total internal reflection. Since the typical refractive index of cells is 1.33 - 1.38, to achieve total internal reflection, an objective lens with a relatively high numerical aperture (NA) is required. Therefore, only a special high-numerical-aperture total internal reflection objective lens can achieve this, and the cost is high. The prism method only requires an excitation light source, a prism, and a microscope. The excitation light enters the optically dense / less dense interface through the prism, and the microscope objective lens on the other side collects the fluorescence emitted by the fluorophore, which is relatively simple and has a lower cost. However, the manual adjustment of the total reflection angle for different media is cumbersome. Therefore, in order to obtain a simple, universal, and low-cost total internal reflection fluorescence microscopy imaging solution, the present invention designs an electric total internal reflection module that can be applicable to various upright and inverted microscopes. Summary of the Invention

[0003] In view of the above problems existing in the prior art, the present invention provides a total internal reflection module for a microscope, which can be commonly used in various types of microscopes to realize electric calibration of the incident angle of a light beam, so as to perform total internal reflection fluorescence microscopy imaging on a sample to be tested, meeting the requirements of cell biology research.

[0004] A total internal reflection module for a microscope proposed by the present invention mainly consists of a sample stage and a rotator. The sample stage includes a sample holder, a prism holder, a semi-cylindrical prism, and a spring pressing plate. The sample holder 1 is provided with an irregular groove for installing the rotator and the other components of the sample stage, and coupling with a microscope stage; the prism holder 5 is installed in the irregular groove opened on the sample holder 1, and a rectangular groove is opened in the middle of the prism holder for installing the semi-cylindrical prism, and the semi-cylindrical prism is used to carry the sample to be tested for testing, and the spring pressing plate is installed on the sample holder for tightly fixing the prism holder. The rotator includes a driving motor and a motor protection box, a right-angle rotating arm, a collimator fixed frame, a collimator, and a reflector. The driving motor has an angle closed-loop feedback function; the motor protection box is installed on the sample holder and connected to the driving motor for supporting and protecting the driving motor; one right-angle side of the right-angle rotating arm is installed on the output shaft of the driving motor, and the other right-angle side is fixed with the reflector and the collimator fixed frame, and the collimator fixed frame is used to fix the collimator.

[0005] Specifically, the center line of the semi-cylindrical prism is coaxial with the center line of the output shaft of the driving motor, and the semi-cylindrical prism and the reflector are located in the same vertical plane, so that the reflector fixed on the right-angle rotating arm always rotates around the center line of the semi-cylindrical prism; the light beam emitted by the collimator is always perpendicularly incident on the tangent plane of the semi-cylindrical prism after being reflected by the reflector and reaches the center position of the semi-cylindrical prism.

[0006] The present invention also provides a usage method of the total internal reflection module for a microscope, which can be used for performing total internal reflection fluorescence microscopy imaging on a sample to be tested.

[0007] Step 1) Loading the sample: After installing the semi-cylindrical prism carrying the sample to be tested on the prism holder, use the spring pressing plate to fix the prism holder with the semi-cylindrical prism installed on the sample holder;

[0008] Step 2) Turning on the excitation light: The excitation light beam is collimated by the collimator and then enters the reflector, and after being reflected by the reflector, it is perpendicularly incident on the tangent plane of the semi-cylindrical prism and reaches the center position of the semi-cylindrical prism, where reflection and refraction occur at the interface between the semi-cylindrical prism and the sample to be tested;

[0009] Step 3) Adjust the excitation optical path for total internal reflection imaging: Start the driving motor, and drive the mirror fixed on the right-angle rotating arm to rotate by an angle until the excitation light only reflects at the interface between the semi-cylindrical prism and the sample to be measured, without refraction. At this time, the excitation light undergoes total internal reflection at the interface between the semi-cylindrical prism and the sample to be measured, generating an evanescent wave to illuminate and excite the thin layer region of the sample to be measured close to the upper surface of the semi-circular prism, for total internal reflection microscopy imaging.

[0010] Advantages of the present invention:

[0011] (1) A total internal reflection module for a microscope disclosed by the present invention has a delicate structure and can be coupled to various upright and inverted microscopes to achieve total internal reflection microscopy imaging on an ordinary microscope.

[0012] (2) In the total internal reflection module for a microscope disclosed by the present invention, the relative positions of the collimating mirror, the reflecting mirror, and the semi-cylindrical prism are fixed. No matter what angle the reflecting mirror rotates to, the excitation light always perpendicularly enters the semi-cylindrical prism to the center position, exciting the sample to be measured for total internal reflection microscopy imaging, greatly saving the sample testing time.

[0013] (3) A total internal reflection module for a microscope disclosed by the present invention uses a driving motor to electrically adjust the angle of the excitation light, achieving extremely small incident angle adjustments that are difficult to achieve by manual calibration. This not only saves the sample testing time but also further improves the total internal reflection microscopy imaging.

[0014] (4) A total internal reflection module for a microscope disclosed by the present invention simplifies the total internal reflection microscopy imaging process. Compared with ordinary microscopy imaging, only by turning on the driving motor can the angle of the excitation light be adjusted to achieve total internal reflection microscopy imaging. The operation is simple and practical, with great application value. Description of the Drawings

[0015] Figure 1 It is an external view of a total internal reflection module for a microscope

[0016] Figure 2 It is a top view of a total internal reflection module for a microscope

[0017] Figure 3 It is a schematic diagram of the angle adjuster of a total internal reflection module for a microscope

[0018] Figure 4 It is a schematic diagram of the total internal reflection imaging optical path of a total internal reflection module for a microscope

[0019] Figure 5 It is a schematic diagram of the total internal reflection imaging working state of a total internal reflection module for a microscope coupled to an upright microscope

[0020] Figure 6 Schematic diagram of total internal reflection imaging of a microscope using a total internal reflection module coupled to an inverted microscope

[0021] In the figure: 1. Sample rack; 2. Motor protection box; 3. Drive motor; 4. Right-angle rotating arm; 5. Prism rack; 6. Spring pressure plate; 7. Semi-cylindrical prism; 8. Collimator fixing frame; 9. Collimator; 10. Reflector; 11. Glass slide DETAILED DESCRIPTION

[0022] The technical scheme of the present invention will be further described in detail below in conjunction with the accompanying drawings and specific implementation cases. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0023] As attached Figure 1 , 2 As shown in Figure 3, a total internal reflection module for a microscope provided by the present invention mainly includes a sample stage and a goniometer. The sample stage includes a sample holder 1, a prism holder 5, a semi-cylindrical prism 7 and a spring pressure plate 6. The sample holder 1 is provided with an irregular groove for installing the goniometer and other components of the sample holder, and coupled with the microscope stage; the prism holder 5 is installed in the irregular groove provided on the sample holder 1, and a rectangular groove is provided in the middle of the prism holder 5 for installing the semi-cylindrical prism 7. The semi-cylindrical prism 7 is used to carry the sample to be tested for testing, and the spring pressure plate 6 is installed on the sample holder 1 for pressing and fixing the prism holder 5. The goniometer includes a driving motor 3, a motor protection box 2, a right-angle rotating arm 4, a collimator fixing frame 8, a collimator 9 and a reflector 10. The motor protection box 2 is installed on the sample rack 1 and connected to the drive motor 3, so as to support and protect the drive motor 3; the right-angle rotating arm 4 is installed on the output shaft of the drive motor 3 at a right angle, and the other right angle is fixed with the reflector 10 and the collimator lens fixing frame 8, and the collimator lens 9 fixing frame 8 is used to fix the collimator lens 9.

[0024] Specifically, the center line of the semi-cylindrical prism 7 is coaxial with the center line of the output shaft of the driving motor 3, and the semi-cylindrical prism 7 and the reflector 10 are located on the same vertical plane, so that the reflector 10 fixed on the right-angle rotating arm 4 always rotates and moves around the center line of the semi-cylindrical prism 7; the outgoing light beam of the collimator 9 is always vertically incident on the tangent surface of the semi-circular prism after being reflected by the reflector 10, and reaches the center position of the semi-circular prism.

[0025] The following uses specific implementation cases to illustrate the usage method of a total internal reflection module for a microscope provided by the present invention. It is applicable to various types of microscopes and realizes total internal reflection microscopy imaging on a common microscope.

[0026] Example 1

[0027] As shown in the appendix Figure 5 is a schematic diagram of the total internal reflection imaging operation of a total internal reflection module for a microscope provided by the present invention coupled to an upright microscope. The sample holder 1 equipped with a rotator and the remaining components of the sample stage is coupled to the microscope sample stage. The rotator is adjusted to be below the sample holder 1 for total internal reflection microscopy imaging of the sample to be tested. First, load the sample. Drop the sample to be tested on the upper surface of the semi-cylindrical prism 7. After ensuring that the sample to be tested adheres well to the wall, install the semi-cylindrical prism 7 on the prism holder 5. By moving the spring pressing plate 6, the prism holder 5 equipped with the semi-cylindrical prism 7 is fixed in the irregular groove opened on the sample holder 1. Then turn on the excitation light source to emit excitation light. The excitation light is first collimated by the collimating mirror 9 and then vertically incident on the reflecting mirror 10. After being reflected by the reflecting mirror 10, it is vertically incident on the tangent plane of the semi-cylindrical prism 7 to the center position. At the interface between the semi-cylindrical prism 7 and the sample to be tested, a part of the excitation light is reflected at the interface, and a part of the excitation light refracts into the medium of the sample to be tested, exciting the sample to be tested to emit light. Finally, adjust the excitation optical path for total internal reflection imaging. Start the drive motor 3. The output shaft of the drive motor 3 drives the right-angle rotating arm 4 fixed thereon to rotate. The reflecting mirror 10 fixed on the right-angle rotating arm 4 rotates in a circular motion around the center line of the semi-cylindrical prism 7 and stops rotating when the refracted light beam above the interface between the semi-cylindrical prism 7 and the sample to be tested disappears. At this time, the excitation light undergoes total internal reflection at the interface between the semi-cylindrical prism 7 and the sample to be tested (as shown in the appendix Figure 4 ), generating an evanescent wave to illuminate and excite the thin layer area of the sample to be tested closely adhering to the upper surface of the semi-cylindrical prism 7. The objective lens collects the light emitted from the thin layer area of the sample to be tested closely adhering to the upper surface of the semi-cylindrical prism 7 to achieve total internal reflection microscopy imaging.

[0028] Example 2

[0029] As shown in the appendix Figure 6The figure shows a schematic diagram of the total internal reflection imaging operation of a total internal reflection module for a microscope coupled to an inverted microscope. The sample holder 1 equipped with a rotator and the remaining components of the sample stage is coupled to the microscope sample stage. The rotator is adjusted above the sample holder 1, and a glass slide 11 is used to replace the prism holder 5 and installed on the sample holder 1 for total internal reflection microscopy imaging of the sample to be measured. First, load the sample. Drop the sample to be measured on the surface of the clean glass slide 11. After ensuring that the sample to be measured adheres well to the wall, by moving the spring pressure plate 6, the glass slide 11 carrying the sample to be measured is fixed in the irregular groove opened on the sample holder 1, and the semi-cylindrical prism 7 is covered on the sample to be measured. Then, turn on the excitation light source and adjust the incident angle so that the refracted light disappears below the interface between the semi-cylindrical prism 7 and the sample to be measured. At this time, total internal reflection occurs at the interface between the semi-cylindrical prism 7 and the sample to be measured (as shown in the appendix Figure 4 ), and the evanescent wave generated illuminates and excites the thin layer region of the sample to be measured close to the upper surface of the semi-cylindrical prism 7. The objective lens collects the light emitted from the thin layer region of the sample to be measured close to the upper surface of the semi-cylindrical prism 7 for total internal reflection microscopy imaging.

[0030] The above describes the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for using a total internal reflection module for a microscope, It is characterized in that The total internal reflection module for microscope includes a sample stage and a goniometer; the sample stage includes a sample holder, a prism holder, a semi-cylindrical prism and a spring pressure plate, the sample holder is used to install the goniometer and other components of the sample stage; the prism holder is installed on the sample holder, a rectangular groove is opened in the middle of the prism holder for installing the semi-cylindrical prism, the semi-cylindrical prism is used to carry the sample to be tested for testing, the spring pressure plate is installed on the sample holder, and is used to press and fix the prism holder; the goniometer includes a driving motor, a motor protection box, a right-angle rotating arm, a collimating mirror fixing frame, a collimating mirror and a reflecting mirror, the driving motor has an angle closed-loop adjustment function, the motor protection box is installed on the sample holder and connected to the driving motor; a right-angle side of the right-angle rotating arm is installed on the output shaft of the driving motor, and the other right-angle side is fixed with the reflecting mirror and the collimating mirror fixing frame, and the collimating mirror fixing frame is used to fix the collimating mirror; The center line of the semi-cylindrical prism is coaxial with the center line of the output shaft of the driving motor, and the semi-cylindrical prism and the reflector are located on the same vertical plane, so that the reflector fixed on the right-angle rotating arm always rotates and moves around the center line of the semi-cylindrical prism; the light beam emitted by the collimator always vertically enters the tangent surface of the semi-circular prism after being reflected by the reflector, and reaches the center position of the semi-circular prism; The method of use includes three steps: loading the sample, turning on the excitation light, and adjusting the excitation light path for total internal reflection imaging; When the excitation light is turned on, the excitation light beam is collimated by the collimator and then incident on the reflector, and then vertically incident on the tangent surface of the semi-cylindrical prism after being reflected by the reflector, and reaches the center position of the semi-cylindrical prism; When adjusting the excitation light path for total internal reflection imaging, the drive motor is started to drive the reflector fixed on the right-angle rotating arm to rotate until the excitation light is totally reflected at the critical surface between the semi-cylindrical prism and the sample to be measured.

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