阅读说明：本技术 一种显微镜用全内反射模块及其使用方法 (Total internal reflection module for microscope and use method thereof ) 是由 何秀芳 于 2021-08-11 设计创作，主要内容包括：本发明提供了一种显微镜用全内反射模块及其使用方法。所述一种显微镜用全内反射模块主要由载样台和转角器组成,所述载样台包括样品架、棱镜架、半圆柱棱镜和弹簧压板,所述转角器包括驱动电机、电机保护箱、直角旋转臂、准直镜固定架、准直镜和反射镜。可通用于各种类型的显微镜,电动校准光束入射角度,操作简单,仅需装样、打开激发光和调整激发光路即可进行全内反射显微成像。准直镜、反射镜和半圆柱棱镜的相互位置固定,激发光始终垂直入射所述半圆柱棱镜至圆心位置激发待测样品；利用驱动电机实现电动调整激发光角度,实现极微小入射角度调整,不仅节省样品测试时间,并且方便耦合各类显微镜,在普通显微镜上实现全内反射显微成像。(The invention provides a total internal reflection module for a microscope and a using method thereof. The utility model provides a total internal reflection module mainly comprises year appearance platform and corner ware for microscope, it includes sample holder, prism frame, semi-cylindrical prism and spring clamp plate to carry the appearance platform, the corner ware includes driving motor, motor guard box, right angle swinging boom, collimating mirror mount, collimating mirror and speculum. The device can be universally used for various types of microscopes, the incident angle of the light beam is electrically calibrated, the operation is simple, and the total internal reflection microscopic imaging can be carried out only by loading samples, opening the excitation light and adjusting the excitation light path. The mutual positions of the collimating mirror, the reflecting mirror and the semi-cylindrical prism are fixed, and the exciting light is always vertically incident to the semi-cylindrical prism to the circle center position to excite the sample to be detected; utilize driving motor to realize electronic adjustment exciting light angle, realize the adjustment of minimum incident angle, not only save sample test time to convenient all kinds of microscopes of coupling realize the microscopic formation of image of total internal reflection on ordinary microscope.)

1. A total internal reflection module for a microscope is characterized by mainly comprising a sample carrying table and a corner device; the sample carrying table comprises a sample frame, a prism frame, a semi-cylindrical prism and a spring pressing plate, wherein the sample frame is used for mounting the corner device and other components of the sample carrying table; the prism frame is arranged on the sample frame, a rectangular groove is formed in the middle of the prism frame and used for installing the semi-cylindrical prism, the semi-cylindrical prism is used for bearing a sample to be tested to test, and the spring pressing plate is arranged on the sample frame and used for pressing and fixing the prism frame; the angle turning device comprises a driving motor, a motor protection box, a right-angle rotating arm, a collimating mirror fixing frame, a collimating mirror and a reflecting mirror, wherein the driving motor has an angle closed-loop adjusting function, and the motor protection box is arranged on the sample frame and is connected with the driving motor; the right-angle rotating arm is arranged on an output shaft of the driving motor at one right-angle edge, the reflector and the collimating mirror fixing frame are fixed at the other right-angle edge, and the collimating mirror fixing frame is used for fixing the collimating mirror.

2. The total internal reflection module for a microscope according to claim 1, wherein the semi-cylindrical prism center line is coaxial with the drive motor output shaft center line, and the semi-cylindrical prism and the mirror are located on the same vertical plane, so that the mirror fixed to the right angle rotary arm always moves rotationally about the semi-cylindrical prism center line.

3. The method of claim 1, comprising the steps of loading, opening the excitation light path, and adjusting the excitation light path for total internal reflection imaging.

4. The use method of the total internal reflection module for a microscope according to any one of claims 1 to 3, wherein when the excitation light is turned on, the excitation light beam is collimated by the collimating mirror and then enters the reflecting mirror, and after being reflected by the reflecting mirror, the excitation light beam perpendicularly enters the phase section of the semi-cylindrical prism and reaches the center position of the semi-cylindrical prism.

5. The use method of the total internal reflection module for a microscope according to any one of claims 1 to 3, wherein when the excitation light path is adjusted for total internal reflection imaging, the driving motor is started to drive the mirror fixed on the right-angle rotating arm to rotate by an angle until the excitation light is totally reflected at the critical surface of the semi-cylindrical prism and the sample to be measured.

Technical Field

The invention discloses a total internal reflection module for a microscope, belongs to the technical field of optical microscopic imaging, and particularly relates to a total internal reflection fluorescence microscopic imaging module which is generally used for various microscopes and used for carrying out total internal reflection fluorescence microscopic imaging on a sample to be detected.

Background

The great number of facts in life sciences indicate that the kinetic characteristics of cells are derived from the polymerization and interaction of individual protein molecules, and many vital processes of life activities in cells are performed on the surface of cell membranes, such as signal transduction, protein transport, pathogen invasion, etc. Therefore, the process of the vital movement on the cell surface is directly observed on the molecular scale without being interfered by signals from deep regions in the cell, which has great significance for exploring complex vital movement in the cell. Total internal reflection fluorescence microscopy is one of the most promising on-film single-molecule optical imaging techniques recognized internationally. When light enters the light thinner medium from the optically dense medium, the light is refracted, and the incident angle of the light is the critical angle theta_cWhen the refraction angle is just 90 degrees, the exciting light is totally reflected at the critical surface of the two media, namely total reflection; when total reflection occurs, due to the wave effect, a part of light energy permeates into the optically thinner medium through the critical surface and propagates along the critical surface, and this part of the transmitted energy field is called an "evanescent wave". The principle of the total internal reflection fluorescence microscopy is that an evanescent wave generated during total internal reflection is used for illuminating a sample, so that an illumination area is limited in the range of a sample surface thin layer, an excitation volume is effectively controlled, and the total internal reflection fluorescence microscopy has high signal-to-noise ratio and contrast ratio which are incomparable with other optical imaging technologies. At present, the total internal reflection fluorescence microscopic imaging system mainly comprises an objective lens type and a prism type, wherein a microscope provided with a special objective lens is required in the objective lens type total internal reflection fluorescence microscope system, the objective lens is used as a receiver for collecting a sample fluorescence signal and is also used as an optical device for generating total internal reflection, and the objective lens with higher Numerical Aperture (NA) is required to be used for realizing total internal reflection because the typical refractive index of a cell is 1.33-1.38, so that the objective lens can be realized only by the special total internal reflection objective lens with high numerical aperture, and the cost is high. WhileThe prism method only needs an excitation light source, a prism and a microscope, the excitation light enters the dense/sparse light critical surface through the prism, the microscope objective lens on the other side collects fluorescence emitted by the fluorophore, and the method is relatively simple and low in cost, but manual adjustment and calibration for total reflection angles of different media are tedious. Therefore, in order to obtain a simple, universal and low-cost scheme for total internal reflection fluorescence microscopic imaging, the invention designs the electric total internal reflection module which can be universally used for various forward microscopes and inverted microscopes.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides the total internal reflection module for the microscope, which can be universally used for various microscopes, realize the electronic calibration of the incident angle of the light beam, perform the total internal reflection fluorescence microscopic imaging on a sample to be detected and meet the requirement of cell biology research.

The invention provides a total internal reflection module for a microscope, which mainly comprises a sample carrying table and a corner device. The sample loading table comprises a sample frame, a prism frame, a semi-cylindrical prism and a spring pressing plate. The sample holder 1 is provided with an irregular groove for mounting the corner device and the rest parts of the sample carrying table and coupling the sample carrying table of the microscope; the prism frame 5 is arranged in an irregular groove formed in the sample frame 1, a rectangular groove is formed in the middle of the prism frame and used for installing the semi-cylindrical prism, the semi-cylindrical prism is used for bearing a sample to be tested to test, and the spring pressing plate is arranged on the sample frame and used for pressing and fixing the prism frame. The corner device comprises 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 feedback function; the motor protection box is arranged on the sample rack, is connected with the driving motor and is used for supporting and protecting the driving motor; the right-angle rotating arm is arranged on an output shaft of the driving motor at one right-angle edge, the reflector and the collimating mirror fixing frame are fixed at the other right-angle edge, and the collimating mirror fixing frame is used for fixing the collimating mirror.

Specifically, the central line of the semi-cylindrical prism is coaxial with the central line of the output shaft of the driving motor, and the semi-cylindrical prism and the reflector are positioned on the same vertical plane, so that the reflector fixed on the right-angle rotating arm always rotates around the central line of the semi-cylindrical prism; and the emergent light beam of the collimating mirror is reflected by the reflector and then vertically incident to the phase section of the semicircular prism all the time to reach the position of the center of the semicircular prism.

The invention also provides a using method of the total internal reflection module for the microscope, which can be used for performing total internal reflection fluorescence microscopy imaging on a sample to be detected.

Step one), sample loading: after the semi-cylindrical prism bearing the sample to be detected is arranged on the prism frame, the prism frame provided with the semi-cylindrical prism is fixed on the sample frame by the spring pressing plate;

step two) turning on the exciting light: the excitation light beam is collimated by the collimating mirror and then enters the reflecting mirror, is reflected by the reflecting mirror and then vertically enters the phase section of the semi-cylindrical prism to reach the circle center position of the semi-cylindrical prism, and is reflected and refracted on the critical surface of the semi-cylindrical prism and the sample to be measured;

step three), adjusting an excitation light path to perform total internal reflection imaging: and starting the driving motor, driving a reflector fixed on the right-angle rotating arm to rotate by an angle, so that the exciting light is only reflected on the critical surface of the semi-cylindrical prism and the sample to be detected, and no refraction phenomenon occurs, the exciting light is totally reflected on the critical surface of the semi-cylindrical prism and the sample to be detected, the generated evanescent wave illuminates and excites the sample to be detected to cling to the thin layer area on the upper surface of the semi-cylindrical prism, and total internal reflection microscopic imaging is performed.

The invention has the beneficial effects that:

(1) the total internal reflection module for the microscope has a delicate structure, can be coupled on various upright microscopes and inverted microscopes, and realizes total internal reflection microscopic imaging on a common microscope.

(2) According to the total internal reflection module for the microscope, disclosed by the invention, the mutual positions of the collimating mirror, the reflecting mirror and the semi-cylindrical prism are fixed, no matter the reflecting mirror rotates to any angle, exciting light is always vertically incident to the semi-cylindrical prism to the circle center position, a sample to be tested is excited to carry out total internal reflection microscopic imaging, and the test time of the sample is greatly saved.

(3) The total internal reflection module for the microscope realizes electric adjustment of the angle of exciting light by using the driving motor, realizes the adjustment of a tiny incident angle which is difficult to realize by manual calibration, saves the test time of a sample, and further improves total internal reflection microscopic imaging.

(4) The total internal reflection module for the microscope simplifies the total internal reflection microscopic imaging process, can adjust the angle of exciting light to realize total internal reflection microscopic imaging only by starting the driving motor compared with common microscopic imaging, is simple and practical to operate, and has great application value.

Drawings

FIG. 1 is an external view of a total internal reflection module for a microscope

FIG. 2 is a top view of a total internal reflection module for a microscope

FIG. 3 is a schematic view of a corner device of a total internal reflection module for a microscope

FIG. 4 is a schematic diagram of a total internal reflection imaging optical path of a total internal reflection module for a microscope

FIG. 5 is a schematic diagram of the total internal reflection imaging operation of the total internal reflection module coupled with an upright microscope for a microscope

FIG. 6 is a schematic diagram of the total internal reflection imaging operation of the total internal reflection module coupled with an inverted microscope for a microscope

In the figure: 1. a sample holder; 2. a motor protection box; 3. a drive motor; 4. a right angle rotating arm; 5. a prism frame; 6. a spring pressing plate; 7. a semi-cylindrical prism; 8. a collimator lens holder; 9. a collimating mirror; 10. a mirror; 11. glass slide

Detailed Description

The technical solution of the present invention will be described in further detail with reference to the accompanying drawings and specific embodiments. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.

As shown in fig. 1, 2 and 3, the total internal reflection module for a microscope provided by the invention mainly comprises a sample stage and a corner device. The sample loading table comprises a sample frame 1, a prism frame 5, a semi-cylindrical prism 7 and a spring pressing plate 6. The sample holder 1 is provided with an irregular groove for mounting the corner device and the rest parts of the sample carrying table and coupling the sample carrying table of the microscope; the prism frame 5 is arranged in an irregular groove formed in the sample frame 1, a rectangular groove is formed in the middle of the prism frame 5 and used for installing the semi-cylindrical prism 7, the semi-cylindrical prism 7 is used for bearing a sample to be tested to test, and the spring pressing plate 6 is arranged on the sample frame 1 and used for pressing and fixing the prism frame 5. The corner device comprises a driving motor 3, a motor protection box 2, a right-angle rotating arm 4, a collimating mirror fixing frame 8, a collimating mirror 9 and a reflecting mirror 10. The motor protection box 2 is arranged on the sample rack 1, is connected with the driving motor 3 and is used for supporting and protecting the driving motor 3; the right-angle rotating arm 4 is arranged on an output shaft of the driving motor 3 at a right-angle edge, the reflector 10 and the collimating mirror fixing frame 8 are fixed at the other right-angle edge, and the collimating mirror 9 fixing frame 8 is used for fixing the collimating mirror 9.

Specifically, the central line of the semi-cylindrical prism 7 is coaxial with the central line of the output shaft of the driving motor 3, and the semi-cylindrical prism 7 and the reflector 10 are positioned on the same vertical plane, so that the reflector 10 fixed on the right-angle rotating arm 4 always rotates around the central line of the semi-cylindrical prism 7; the light beam emitted by the collimating mirror 9 is reflected by the reflecting mirror 10 and then is always vertically incident to the phase section of the semicircular prism and reaches the position of the center of the semicircular prism.

The following description will explain, by means of specific embodiments, a method for using a total internal reflection module for a microscope, which is generally used for various types of microscopes to realize total internal reflection microscopy imaging on a common microscope.

Example 1

Fig. 5 is a schematic diagram of the total internal reflection imaging operation of the total internal reflection module coupled with an upright microscope according to the present invention. Coupling the sample holder 1 provided with the turning device and other parts of the sample carrying table on the sample stage of the microscope, adjusting the turning device to the lower part of the sample holder 1, and carrying out total internal reflection microscopic imaging on the sample to be detected. Firstly, loading a sample, dripping a sample to be tested on the upper surface of the semi-cylindrical prism 7, installing the semi-cylindrical prism 7 on the prism frame 5 after ensuring that the sample to be tested adheres well, and fixing the prism frame 5 provided with the semi-cylindrical prism 7 in an irregular groove formed in the sample frame 1 by moving the spring pressing plate 6; then, an excitation light source is opened to emit excitation light, and the excitation light is collimated by a collimating mirror 9 and then vertically enters a reflecting mirror 10; then the excitation light is reflected by the reflector 10 and vertically enters the phase section of the semi-cylindrical prism 7 to the circle center position, part of the excitation light is reflected at the critical surface of the semi-cylindrical prism 7 and the sample to be detected, and part of the excitation light is refracted into the sample medium to be detected to excite the sample to be detected to emit light; finally, adjusting an excitation light path to perform total internal reflection imaging, starting a driving motor 3, driving an output shaft of the driving motor 3 to drive a right-angle rotating arm 4 fixed on the right-angle rotating arm to rotate by an angle, then enabling a reflector 10 fixed on the right-angle rotating arm 4 to rotate circularly around the central line of a semi-cylindrical prism 7, rotating until excitation light disappears when refracted light beams above the interface of the semi-cylindrical prism 7 and a sample to be detected disappear, enabling the excitation light to be totally reflected at the critical surface of the semi-cylindrical prism 7 and the sample to be detected (shown in figure 4), illuminating and exciting the sample to be detected to cling to a thin layer area on the upper surface of the semi-cylindrical prism 7 by generated evanescent waves, and collecting light emitted by the sample to be detected to cling to the thin layer area on the upper surface of the semi-cylindrical prism 7 by an objective lens to realize total internal reflection microscopic imaging.

Example 2

Fig. 6 is a schematic diagram of the total internal reflection imaging operation of the total internal reflection module coupled with an inverted microscope according to the present invention. Coupling the sample holder 1 provided with the corner device and other parts of the sample carrying table on the sample stage of the microscope, adjusting the corner device to the upper part of the sample holder 1, replacing the prism frame 5 with the glass slide 11, and mounting on the sample holder 1 to carry out total internal reflection microscopic imaging of the sample to be measured. Firstly, loading a sample, namely dripping the sample to be detected on the surface of a clean glass slide 11, fixing the glass slide 11 bearing the sample to be detected in an irregular groove formed in a sample rack 1 by moving a spring pressing plate 6 after ensuring that the sample to be detected adheres well, and covering the semi-cylindrical prism 7 on the sample to be detected; and then, opening an excitation light source, adjusting an incidence angle to enable the refraction light of the excitation light to disappear below the critical surface of the semi-cylindrical prism 7 and the sample to be detected, totally reflecting the excitation light at the critical surface of the semi-cylindrical prism 7 and the sample to be detected (as shown in figure 4), illuminating and exciting a thin layer area of the sample to be detected, which is tightly attached to the upper surface of the semi-cylindrical prism 7, by the generated evanescent wave, and collecting light emitted by the sample to be detected, which is tightly attached to the thin layer area of the upper surface of the semi-cylindrical prism 7, by an objective lens to perform total internal reflection microscopic imaging.

The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.