Prism-type total internal reflection imaging apparatus compatible with commercial inverted microscope, and method for using same

Abstract

Disclosed in the present invention are a prism-type total internal reflection imaging apparatus compatible with a commercial inverted microscope, and a method for using same. The method comprises the following steps: first injecting a sample into a sample cell; turning on a laser, and generated evanescent waves illuminating nearby fluorescent molecules to obtain an image; and processing the image to obtain the three-dimensional positions of all the fluorescent molecules in a visual field, obtaining the three-dimensional trajectories of the fluorescent molecules, calculating a mean square end-to-end distance and a diffusion factor of the trajectories, and obtaining a relative potential energy distribution curve on the basis of the relative positions of the molecules. The present invention can be compatible with a mainstream commercial inverted microscope, and does not need to carry out any refit on existing microscopes. A sample stage is mounted on the inverted microscope and an excitation light path module is mounted on the side edge of the sample stage, so that a total internal reflection light path having a specific wavelength can be generated. The apparatus can achieve single molecule positioning and three-dimensional tracking with a high signal-to-noise ratio, and the sample cell can be flexibly replaced before each test without influencing the total internal reflection light path. The apparatus is applied to the fields of life science, material analysis and the like.

Description

A prism-type total internal reflection imaging device compatible with commercial inverted microscopes and its use method Technical Field

[0001] The present invention relates to a prism-type total internal reflection imaging device compatible with a commercial inverted microscope, belongs to the field of optical microscopy, and particularly relates to a prism-type total internal reflection imaging device compatible with a commercial inverted microscope and a method for using the same. Background Art

[0002] Total internal reflection fluorescence microscopy (TIRF) is a high-precision near-field imaging technique. When the angle of incidence exceeds a certain value, total internal reflection (TIR) ​​occurs at interfaces between materials, such as glass-water, as the incident light passes from regions of higher refractive index to regions of lower refractive index. This causes an evanescent wave (EIW) to penetrate the interface. The intensity of the EIW decays exponentially with distance from the interface, and its penetration depth is typically only a few hundred nanometers. Because of this small penetration depth, the EIW only illuminates a very thin layer of the sample, giving TIR microscopy an extremely high signal-to-noise ratio. Furthermore, due to the exponential decay of the EIW, combined with fluorescence microscopy, by monitoring the fluorescence intensity changes of fluorescent molecules within the EIW illumination range, we can achieve high-precision three-dimensional imaging of substances and even molecules near the surface. Due to these characteristics, TIR is now widely used in fields such as life sciences, materials science, and surface physicochemical characterization, for example, in observing the transport of protein molecules.

[0003] Currently, total internal reflection microscopes include objective lens type and prism type. In the objective lens type total internal reflection microscope, the excitation and emission light paths share the same optical path and objective lens, resulting in a low signal-to-noise ratio, and a dedicated high numerical aperture objective lens is required to achieve this. The cost is high and the optical path is complex. More importantly, this method of generating total internal reflection requires reliance on angle control components within the optical path, and it is usually difficult to achieve quantitative control of the penetration depth of the evanescent wave generated by total internal reflection. This makes it impossible to accurately obtain the z-position of the object to be measured, and it can only be used to image objects near the surface, but not for accurate three-dimensional positioning. In the design of the prism type total internal reflection microscope, the excitation light path and the emission light path are separated from each other, the background light is less than 0.01%, and it has an extremely high signal-to-noise ratio. It can be used with various objective lenses, including air objective lenses, water immersion objective lenses, and oil immersion objective lenses, and the cost is low. More importantly, the prism type total internal reflection microscope has a certain penetration depth of the evanescent wave, which is a prerequisite for accurate three-dimensional positioning analysis. At the same time, the prism-type total internal reflection microscope is very flexible to build, has a variety of geometric structures, and is compatible with upright or inverted microscopes.

[0004] However, the design of prism-based total internal reflection microscopes still faces the following challenges: First, they are incompatible with commercial inverted microscopes. Traditional prism-based total internal reflection inverted microscopes require the construction of a complete microscope setup and cannot be used in conjunction with other microscopic imaging devices. Second, because a prism must be added to the sample reflection interface, the optical path must be readjusted each time the sample is changed, making its use extremely complex and hindering its further application. To address these issues, the present invention designs an excitation optical path independent of the inverted microscope, utilizing a prism-based total internal reflection optical path to achieve a high signal-to-noise ratio. The sample stage loaded with our device can be directly mounted on the sample stage of a commercial microscope. Furthermore, after the initial calibration of the total internal reflection angle, the device no longer requires optical path adjustment when replacing a new sample cell. This allows for high signal-to-noise ratio images and high-precision single-molecule three-dimensional localization and subsequent trajectory analysis. High-precision three-dimensional localization and analysis capabilities are of great significance in life sciences, materials science, and surface physicochemical characterization. Therefore, the present invention has the advantages of being compatible with commercial inverted microscopes, being easy to use, having a high signal-to-noise ratio, and being able to achieve three-dimensional positioning and dynamic behavior characterization of single molecules. Summary of the Invention

[0005] To address the above-mentioned problems, the present invention proposes a prism-type total internal reflection imaging device compatible with commercial inverted microscopes and a method for its use. The main purpose of the present invention is to solve the problems of complex construction process, complex operation, incompatibility, and high cost, while maintaining the high signal-to-noise ratio advantage of the total internal reflection optical path. It provides a total internal reflection imaging device that is compatible with any inverted microscope, simple to build and operate, low in cost, easy to calibrate the total internal reflection angle, and has a high signal-to-noise ratio. The device can be installed on any inverted microscope without interfering with the functional modules of other microscopes. The sample stage can be directly mounted on the inverted microscope translation stage. After the initial calibration of the total internal reflection angle, only the sample cell needs to be replaced, and total internal reflection imaging with a fixed angle and high signal-to-noise ratio can be achieved without adjusting the optical path. The advantages of this device are good compatibility, high signal-to-noise ratio image acquisition, high-precision three-dimensional positioning, and easy construction and use. A basic total internal reflection microscope single-molecule three-dimensional positioning algorithm is proposed, as well as an algorithm for characterizing the three-dimensional diffusion trajectory, diffusion behavior, diffusion coefficient, and single-molecule interactions of single molecules.

[0006] The technical solution adopted by the present invention to solve its technical problem is:

[0007] A prism-type total internal reflection imaging device compatible with a commercial inverted microscope comprises: an excitation light path and a sample stage; the excitation light path is placed on a horizontal substrate, the sample stage is placed on an inverted microscope, and the excitation light path and the sample stage are 30-60 cm apart horizontally and 20-40 cm apart vertically;

[0008] The excitation optical path includes a laser light source, an aperture, a first reflector, a first translation stage, a first six-axis optical adjustment frame, a second reflector, a second translation stage, a second six-axis optical adjustment frame, and a vertical base plate; the sample loading platform includes a prism, a sample cell, a prism fixing block, a sample fixing block, and a sample stage;

[0009] The excitation optical path is placed on the vertical substrate; the sample stage is placed on an inverted microscope; a laser light source is provided on one side of the aperture, and a first reflecting mirror, a first translation stage, and a first six-axis optical adjustment frame are provided on the other side of the aperture; a second reflecting mirror, a second translation stage, and a second six-axis optical adjustment frame are provided above the first reflecting mirror;

[0010] A sample pool is arranged above the sample stage; a prism is arranged above the sample pool; an objective lens is arranged below the sample pool for amplifying the signal; the sample fixing pressure block is arranged above the sample pool for fixing and pressing the sample pool; the prism fixing pressure block is arranged above the prism for fixing and pressing the prism.

[0011] In the excitation optical path, the laser light source outputs a spatially collimated light beam, the light beam passes through an aperture to reduce the beam diameter, the light beam reaches the first reflector and is reflected as a vertically upward light beam, the upward light beam reaches the second reflector and is reflected toward the prism surface;

[0012] On the sample stage, the light beam is incident vertically on the prism, and undergoes total internal reflection in the sample cell, thereby exciting the fluorescent substance to emit fluorescence.

[0013] Furthermore, in the excitation light path, the wavelength range of the laser light source is 100-2000 nm, and the wavelength is determined according to the excitation emission wavelength of the fluorescent dye.

[0014] Furthermore, in the excitation light path, the center installation height of the aperture must be consistent with the center of the laser light source's output beam. When the aperture is opened to the minimum, the laser light source's output light passes vertically through the aperture hole and outputs a horizontal collimated beam.

[0015] Furthermore, in the excitation light path, the center height of the first reflector is equal to the height of the aperture.

[0016] Furthermore, in the excitation light path, the horizontal collimated light output from the central hole of the aperture hits the center of the first reflector and is reflected vertically upward by the inclined first reflector.

[0017] Furthermore, in the excitation light path, the first translation stage is fixed on the vertical substrate, and the first translation stage can only move horizontally along the substrate; the first reflector is installed on the first six-axis optical adjustment frame, which can adjust the pitch and deflection angles. The first six-axis optical adjustment frame is installed on the first translation stage.

[0018] Further, in the excitation light path, the center of the second reflector is 40 to 50 cm above the center of the first reflector.

[0019] Furthermore, in the excitation light path, the light reflected from the first reflector hits the center of the second reflector, is reflected by the second reflector, and propagates toward the surface of the prism.

[0020] Furthermore, in the excitation light path, the second translation stage is fixed on the vertical substrate, and the second translation stage can only move vertically along the substrate; the second reflector is installed on the second six-axis optical adjustment frame, which can adjust the pitch and deflection angles. The second six-axis optical adjustment frame is installed on the second translation stage.

[0021] Furthermore, in the sample stage, the sample stage is a painted iron plate with a length of 10-100 cm and a width of 10-100 cm, which can be fixed by magnets. A through hole is opened in the middle for placing the objective lens. The sample stage is installed on an inverted microscope.

[0022] Furthermore, in the sample stage, the objective lens has a magnification of 10-100 times and a working distance of 0.2 mm to 10 mm.

[0023] Furthermore, in the sample stage, the prism fixing block is fixed on the sample stage with screws.

[0024] Furthermore, the sample cell on the sample stage consists of two thin glass slides and a double-sided adhesive spacer, which adheres the two slides together. The refractive index of the sample cell glass ranges from 1.47 to 1.53, the thickness of the glass slides is 0.10 to 0.5 mm, and the thickness of the spacer is 0.01 to 1 mm. The sample cell is 30 to 80 cm horizontally and 20 to 60 cm vertically from the center of the second reflector.

[0025] Furthermore, in the sample stage, the incident light enters the prism surface perpendicularly, and the light is incident on the lower surface of the glass slide above the sample cell, causing total internal reflection, thereby exciting the fluorescent molecules close to the glass slide.

[0026] Furthermore, the prism is designed with a Brewster angle, and the light reflected in the total internal reflection will enter the Brewster angle structure and continue to be totally internally reflected, and finally emerge from the Brewster angle, without interfering with the total internal reflection light path to cause background light.

[0027] Furthermore, the prism angle in the optical path is fixed, and the prism angle is between 62° and 85° (the angle between the prism surface and the bottom surface, as illustrated in FIG4 ).

[0028] Furthermore, the incident light in the excitation light path is reflected by the prism surface. After adjusting the incident light angle (adjusting the first and second six-axis optical adjustment mounts), the reflected light is reflected by the second reflector and the first reflector and returns to the center of the aperture along the original path. At this time, the incident light is perpendicular to the prism surface. According to the geometric relationship, the total internal reflection angle is equal to the prism angle (the angle between the prism surface and the bottom surface).

[0029] A method for using a prism-type total internal reflection imaging device compatible with a commercial inverted microscope, comprising the following steps:

[0030] Step 1) Sample loading: injecting the sample into the chamber of the sample cell, installing the sample cell under the prism, and fixing the sample cell with the sample fixing block;

[0031] Step 2) Turning on the laser to generate a total internal reflection optical path: the excitation light passes through the aperture and is then reflected by the first and second reflectors onto the prism surface. It is refracted and propagates to the interface between the lower surface of the upper glass slide in the sample cell and the sample. At this point, the total internal reflection angle equals the prism angle, and total internal reflection occurs at the interface between the lower surface of the upper glass slide in the sample cell and the sample. The resulting evanescent wave illuminates nearby fluorescent molecules, and the camera receives the signal, producing an image with an extremely high signal-to-noise ratio.

[0032] Step 3) Positioning, tracking and analysis: The image is deducted from the background average intensity, and Gaussian fitting is used to obtain the xy position of the centroid of the single molecule.

[0033] Furthermore, the background average light intensity is the average light intensity of all frames in the video.

[0034] Furthermore, the Gaussian fitting formula is defined as follows:

[0035]

[0036] Where I(x,y) is the light intensity, A, d, and b are Gaussian fitting parameters, and x0 and y0 represent the coordinates of a single molecule.

[0037] Furthermore, the average light intensity within a circle with a radius of d from the centroid position is extracted as the single molecule light intensity I(h) (d is adjusted according to the size of the single molecule), and the z-direction position is calculated using the formula of I(h) exponentially decaying with the separation distance.

[0038] Further, specifically, the formula is as follows:

[0039]

[0040] Where I0 is the maximum light intensity in the tracked single molecule. The single molecule with the maximum light intensity is located closest to the glass surface, and its z position is set to zero. I(h) is the average light intensity of any single molecule, h is the distance from the zero point (i.e., the z position of the single molecule), and β is the inverse of the penetration depth. l is the wavelength of the incident light, q is the angle of total internal reflection, and n1 and n2 are the refractive indices of the glass and sample.

[0041] Furthermore, the continuous three-dimensional trajectory of each molecule in the field of view is obtained based on the x, y, and z positions of each image (the distance between the upper and lower frames is less than 0.2-1.2μm and can be connected into a trajectory). The mean square displacement (MSD) in the three-dimensional direction is calculated based on the three-dimensional trajectory:

[0042]

[0043] Among them, r(t0) and r(t0+Dt) are the three-dimensional positions of the single molecule at time t0 and (t0+Dt), respectively.

[0044] Fit each trajectory with at least 100-500 points using the following formula:

[0045]

[0046] Where D is the equivalent diffusion factor, u is the MSD index, when u is greater than 1 it is active motion, when u is less than 1 it is restricted motion, and when u is equal to 1 it is diffusive motion and D is the true diffusion factor. In this case, the diffusion coefficient can be calculated using the Einstein-Stokes equation as follows:

[0047]

[0048] Among them, k B is the Boltzmann constant, T is the temperature, h is the fluid shear viscosity, σ is the molecular diameter, D is the diffusion coefficient,

[0049] Furthermore, the number of other single molecules in each image that are in a series of concentric spherical shells with increasing radius over a period of time is calculated, and the radial distribution function g(r) describing the change of density between single molecules with distance is calculated, which is defined as:

[0050]

[0051] Where f represents the local density within a sphere with a single molecule radius r, and f' represents the number density of single molecules in the system, which is defined as:

[0052]

[0053] Where N(r) is d r The number of molecules in the ring. For each molecule, the space surrounding it is calculated by counting the number of other molecules contained in a series of concentric spherical shells of increasing radius. When the concentration of the system is infinitely dilute, g(r) can be related to the interaction energy U(r) via the Boltzmann distribution.

[0054]

[0055] Furthermore, since infinite dilution is not possible, we can obtain the corresponding apparent potential energy U at a series of gradient concentrations: app (r):

[0056]

[0057] Among them, k B is the Boltzmann constant, T is the temperature, and we get U app (r), and then U(r) is obtained by mathematical fitting.

[0058] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0059] This invention adopts a prism-type total internal reflection optical path design, maintains the high signal-to-noise ratio of the total internal reflection mode, and builds an excitation optical path independent of the inverted microscope. The sample stage can be directly installed on the inverted microscope translation stage. After the initial calibration of the total internal reflection angle, only the sample cell needs to be replaced. Total internal reflection imaging with a certain angle and high signal-to-noise ratio can be achieved without adjusting the optical path. It is compatible with any inverted microscope. This invention can be used for simultaneous observation with other microscopes, such as in conjunction with a digital holographic microscope; this invention has a simple structure and low cost; the light of this invention is spatial light emitted directly from a laser, and does not require a collimated light source. It is easy to operate and has high light collimation, and the positioning results are more accurate; this invention can simply and accurately calibrate the total internal reflection angle, calculate the z-axis position of the molecule with high accuracy, and can also achieve total internal reflection at different angles by replacing prisms at different angles; this invention adopts a prism-type total internal reflection mode, and the background noise is the lowest among all total internal reflection modes. It has an extremely high signal-to-noise ratio and can achieve extremely high positioning accuracy; a basic total internal reflection microscope single-molecule three-dimensional positioning algorithm is proposed, as well as an algorithm to characterize the three-dimensional diffusion trajectory, diffusion behavior, diffusion coefficient, and single-molecule interaction of single molecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] FIG1 is a schematic diagram of an excitation optical path module of a prism-type total internal reflection imaging device;

[0061] FIG2 is a top view of a sample stage of a prism-type total internal reflection imaging device;

[0062] FIG3 is a front view of a sample stage of a prism-type total internal reflection imaging device;

[0063] FIG4 is a schematic diagram of a prism of a prism-type total internal reflection imaging device;

[0064] FIG5 is a schematic diagram of the total internal reflection optical path of a prism-type total internal reflection imaging device;

[0065] FIG6 is a three-dimensional diffusion diagram of Rhodamine B molecules at the glass-water interface;

[0066] FIG7 is a schematic diagram of the process of measuring single molecule interactions;

[0067] FIG8 is a radial distribution function curve of single molecule cross-linking points;

[0068] FIG9 is a curve showing the apparent potential energy distribution of single-molecule cross-linking points.

[0069] The components in the figure are as follows:

[0070] Laser light source 1, aperture 2, first reflecting mirror 3, first translation stage 4, first six-axis optical adjustment frame 5, second reflecting mirror 6, second translation stage 7, second six-axis optical adjustment frame 8, vertical base plate 9, prism 10, sample cell 11, prism fixing block 12, sample fixing block 13, sample stage 14. Modes for Carrying Out the Invention

[0071] The present invention is further described in detail below with reference to examples and drawings. It should be noted that the embodiments do not constitute a limitation to the protection claimed for the present invention.

[0072] Example 1

[0073] In this embodiment, a prism-type total internal reflection imaging device compatible with a commercial inverted microscope is provided, as shown in Figures 1 to 5, comprising: an excitation light path and a sample stage; the excitation light path comprises a laser light source 1, an aperture 2, a first reflector 3, a first translation stage 4, a first six-axis optical adjustment frame 5, a second reflector 6, a second translation stage 7, a second six-axis optical adjustment frame 8, and a vertical base plate 9; the sample stage comprises a prism 10, a sample cell 11, a prism fixing block 12, a sample fixing block 13, and a sample stage 14; the excitation light path is placed on the vertical base plate 9; the sample stage is placed on the inverted microscope; A laser light source 1 is provided on one side of the aperture 2, and a first reflector 3, a first translation stage 4, and a first six-axis optical adjustment mount 5 are provided on the other side of the aperture 2; a second reflector 6, a second translation stage 7, and a second six-axis optical adjustment mount 8 are provided above the first reflector 3; a sample cell 11 is provided above the sample stage 14; a prism 10 is provided above the sample cell 11; and an objective lens is provided below the sample cell 11; a sample fixing block 13 is provided above the sample cell 11 for fixing and pressing the sample cell 11; and a prism fixing block 12 is provided above the prism 10 for fixing and pressing the prism 10. In the excitation light path, the laser light source 1 outputs a spatially collimated light beam, which is reduced in diameter by the aperture 2. The light beam reaches the first reflector 3 and is reflected as a vertically upward light beam. The upward light beam reaches the second reflector 6 and is reflected toward the surface of the prism 10; on the sample stage, the light beam is vertically incident on the prism 10, undergoes total internal reflection in the sample cell 11, and excites the fluorescent substance to emit fluorescence.

[0074] In this embodiment, in the excitation light path, the wavelength of the laser light source is 100-2000nm, the center installation height of the aperture 2 must be consistent with the center of the output light beam of the laser light source 1, the height is 10-30cm, the hole diameter of the aperture 2 is 0.1-2.0mm, the output light of the laser light source 1 passes vertically through the aperture 2 hole, and outputs a horizontal collimated light beam; the center height of the first reflector 3 is equal to the height of the aperture 2; the horizontal collimated light output from the center hole of the aperture 2 hits the center of the first reflector 3, and is reflected vertically upward by the tilted first reflector 3; the first displacement stage 4 is fixed on the vertical substrate 9, and the first displacement stage 4 can only move horizontally along the substrate uniaxially, with a maximum stroke of 10-25cm; the first reflector 3 is installed on the first six-axis optical adjustment The first six-axis optical adjustment frame 5 is mounted on the first translation stage 4; the center of the second reflector 6 is 40-50 cm above the center of the first reflector 3; the light reflected from the first reflector 3 hits the center of the second reflector 6, is reflected by the second reflector 6, and propagates toward the surface of the prism 10; the second translation stage 7 is fixed on the vertical substrate 9, and the second translation stage can only move vertically along a single axis of the substrate, with a maximum stroke of 10-25 cm; the second reflector 6 is mounted on the second six-axis optical adjustment frame 8, and the pitch and deflection angles can be adjusted, with a maximum pitch and deflection angle of 10°, and the second six-axis optical adjustment frame 8 is mounted on the second translation stage 7.

[0075] In the sample loading platform, the sample stage 14 is a 10-100 cm long and 10-100 cm wide painted iron plate with a surface that can be fixed by magnets. A through hole with a diameter of 25-40 mm is opened in the middle for accommodating the objective lens. The objective lens has a magnification of 10-100 times and a working distance of 0.2 mm to 10 mm. The sample stage 14 is mounted on an inverted microscope. The prism fixing block 12 is fixed to the sample stage 14 with screws. The sample pool 11 consists of two glass slides and a 3M double-sided tape spacer. The double-sided tape adheres the two glass slides together. The thickness of the glass slide is 0.10-0.5mm, the refractive index range of the sample pool 11 glass is: 1.47-1.53, and the thickness of the gasket is 0.01-1mm; the sample pool 11 is 30-80cm away from the center of the second reflector 6 in the horizontal direction and 20-60cm away from the center of the second reflector 6 in the vertical direction; the incident light is perpendicular to the surface of the prism 10, and the light incident on the lower surface of the glass slide above the sample pool 11 causes total internal reflection, which excites the fluorescent molecules near the glass slide; the prism 10 is designed with a Brewster angle, and the light reflected in the total internal reflection will enter the Brewster angle structure of the prism 10 and continue to reflect completely Internal reflection, finally emitted from the Brewster angle, will not interfere with the total internal reflection light path to cause background light; the incident light of the excitation light path is reflected on the surface of the prism 10, and after adjusting the angle of the incident light, the reflected light is reflected by the second reflector 6 and the first reflector 3, and returns to the center of the aperture 2 along the original path. At this time, the incident light is perpendicular to the surface of the prism 10; according to the geometric relationship, the total internal reflection angle is equal to the prism angle of 62° to 85° (the angle between the prism surface and the bottom surface, as shown in Figure 4), completing the initial calibration of the total internal reflection angle. After that, you only need to replace the sample cell to directly cause total internal reflection when using it again.

[0076] The laser light source 1, aperture 2, first reflector 3, first displacement stage 4, first six-axis optical adjustment frame 5, second reflector 6, second displacement stage 7, second six-axis optical adjustment frame 8, and vertical substrate 9 are all purchased from Sorebo Optoelectronics Technology (Shanghai) Co., Ltd. The prism 10 can be replaced with prisms of other angles.

[0077] Example 2

[0078] A prism-based total internal reflection imaging setup compatible with a commercial inverted microscope is used to measure the three-dimensional diffusion behavior of rhodamine B molecules in a 1 nM rhodamine B solution at a glass-water interface, comprising the following steps:

[0079] Step 1: Prepare rhodamine solution, inject the sample into the chamber of the sample cell 11, install the sample cell 11 below the prism 10, and fix the sample cell 11 with the sample fixing block 13;

[0080] Step 2: Turn on the power of the laser light source 1 and preheat it for 10 minutes. The laser passes through the aperture 2 and is then reflected by the first reflector 3 and the second reflector 6 to the surface of the prism 10. After being refracted, it propagates to the interface between the lower surface of the upper glass slide of the sample cell and the sample. Total internal reflection occurs at the interface between the lower surface of the upper glass slide of the sample cell 11 and the sample. The generated evanescent wave illuminates the nearby fluorescent molecules. The camera receives the signal and obtains an image with an extremely high signal-to-noise ratio. Set the camera exposure time to 63ms, the shooting speed to 10fps, and the light source power to 100mW.

[0081] Step 3: Use a computer program to track the xy position of the fluorescent molecule, and then calculate the z position based on the total internal reflection light intensity attenuation formula to obtain the three-dimensional diffusion trajectory of the Rhodamine B fluorescent molecule, as shown in Figure 6, and further calculate its diffusion coefficient. The details are as follows:

[0082] The image was deducted from the background average light intensity, and Gaussian fitting was used to obtain the xy position of the center of mass of Rhodamine B.

[0083] Gaussian fitting formula:

[0084]

[0085] Where I(x,y) is the light intensity, A, d, and b are Gaussian fitting parameters, x0 and y0 represent the coordinates of the Rhodamine B single molecule, and then the average light intensity within a circle with a radius of d from the center of mass is extracted as the Rhodamine B light intensity I(h) (d is adjusted according to the size of the single molecule), and the z-direction position is calculated using the formula of I(h) exponentially decaying with separation distance.

[0086] Specifically, the formula is as follows:

[0087]

[0088] Where I0 is the maximum intensity of the tracked Rhodamine B. The Rhodamine B with the maximum intensity is located closest to the glass surface, and its z position is set to zero. I(h) is the average intensity of any Rhodamine B, h is the distance from zero (i.e., the z position of the Rhodamine B), and β is the inverse of the penetration depth. l is the incident light wavelength of 532 nm, q is the total internal reflection angle set to 73°, and n1 and n2 are the refractive indices of the glass and sample, which are 1.51 and 1.33 respectively.

[0089] Furthermore, the continuous three-dimensional trajectory of each rhodamine B in the field of view is obtained based on the x, y, and z positions of each image (the distance between the upper and lower frames is less than 0.2-1.2μm and can be connected into a trajectory). The mean square displacement (MSD) in the three-dimensional direction is calculated based on the three-dimensional trajectory:

[0090]

[0091] Among them, r(t0) and r(t0+Dt) are the three-dimensional positions of Rhodamine B at time t0 and (t0+Dt), respectively.

[0092] Fit each trajectory with at least 100-500 points using the following formula:

[0093]

[0094] Where D is the diffusion factor. When u is equal to 1, it is diffusion motion. In this case, the diffusion coefficient can be calculated using the Einstein-Stokes equation as follows:

[0095]

[0096] Among them, k B is the Boltzmann constant, T is the temperature, h is the fluid shear viscosity, σ is the molecular diameter, and D is the diffusion coefficient

[0097] Example 3

[0098] A prism-based total internal reflection imaging setup compatible with a commercial inverted microscope was used to measure the interactions at the crosslinking points of the interfacial gel (the calculation process is shown in Figure 7 ). The setup includes the following steps:

[0099] Step 1: Prepare a gel solution, inject the sample into the chamber of the sample pool 11, synthesize the gel with UV light, install the sample pool 11 under the prism 10, and fix the sample pool 11 with the sample fixing block 13;

[0100] Step 2: Turn on the power of the laser light source 1 and preheat it for 10 minutes. The laser passes through the aperture 2 and is then reflected by the first and second reflectors 3 and 6 to the surface of the prism 10. After being refracted, it propagates to the interface between the lower surface of the upper glass slide in the sample cell and the sample. Total internal reflection occurs at the interface between the lower surface of the upper glass slide in the sample cell 11 and the sample. The generated evanescent wave illuminates the nearby fluorescent molecules. The camera receives the signal and obtains an image with an extremely high signal-to-noise ratio.

[0101] Step 3: Use a computer program to track the xy position of the gel crosslinking point, calculate the z position using the total internal reflection light intensity attenuation formula, then calculate the radial distribution function of the crosslinking point (as shown in Figure 8), and then calculate the potential energy distribution to obtain the interaction data (as shown in Figure 9). The interaction calculation process is as follows:

[0102] Calculate the number of other single molecules in each image over time that are in a series of concentric spherical shells with increasing radius, and calculate the radial distribution function g(r) that describes the change in the density between single-molecule crosslinks as a function of distance, which is defined as:

[0103]

[0104] where f represents the local density of the crosslink within a sphere with a radius r, and f' represents the number density of crosslinks in the system, which is defined as:

[0105]

[0106] Where N(r) is d r The number of crosslinks in the ring. For each crosslink, the space surrounding it counts the number of other crosslinks that lie within a series of concentric spherical shells of increasing radius. When the concentration of the system is infinitely dilute, g(r) can be related to the interaction energy U(r) via a Boltzmann distribution.

[0107]

[0108] Furthermore, since infinite dilution is not possible, we can obtain the corresponding apparent potential energy U at a series of gradient concentrations: app (r):

[0109]

[0110] Among them, k B is the Boltzmann constant, T is the temperature, and we get U app (r), and then U(r) is obtained by mathematical fitting.

[0111] Of course, the invention is not limited to the above-mentioned embodiments. Those skilled in the art may make equivalent modifications or substitutions without violating the spirit of the invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. A prism-type total internal reflection imaging device compatible with a commercial inverted microscope, characterized in that: include: An excitation light path and a sample stage; the excitation light path is placed on a horizontal substrate, the sample stage is placed on an inverted microscope, the excitation light path and the sample stage are 30-60 cm apart horizontally and 20-40 cm apart vertically; The excitation optical path comprises a laser light source (1), an aperture (2), a first reflector (3), a first displacement stage (4), a first six-axis optical adjustment frame (5), a second reflector (6), a second displacement stage (7), a second six-axis optical adjustment frame (8) and a vertical substrate (9); The sample loading platform comprises a prism (10), a sample pool (11), a prism fixing block (12), a sample fixing block (13) and a sample platform (14); The excitation light path is placed on the vertical substrate (9); the sample stage is placed on an inverted microscope; the inverted microscope is located on an optical platform, and the excitation light path is on the side of the inverted microscope; a laser light source (1) is arranged on one side of the aperture (2), and a first reflecting mirror (3), a first displacement stage (4) and a first six-axis optical adjustment frame (5) are arranged on the other side of the aperture (2); the first displacement stage (4) is arranged on the vertical substrate (9), the first six-axis optical adjustment frame (5) is located on the first displacement stage (4), and the first reflecting mirror (3) is arranged on the first six-axis optical adjustment frame (5); the first reflecting mirror (3) is arranged on the first six-axis optical adjustment frame (5); A second reflector (6), a second displacement stage (7) and a second six-axis optical adjustment frame (8) are arranged on the side; the second displacement stage (7) is arranged on the vertical substrate (9), a second six-axis optical adjustment frame (8) is arranged on the second displacement stage (7), and the second reflector (6) is arranged on the second six-axis optical adjustment frame (8); a sample pool (11) is arranged above the sample stage (14); a prism (10) is arranged above the sample pool (11); an objective lens is arranged below the sample pool (11); the bottom of the prism fixing block (12) is fixed on the sample stage to press and fix the prism (10); the sample fixing block (13) fixes the sample pool; In the excitation light path, a laser light source (1) outputs a laser light beam, the light beam passes through an aperture (2) to reduce the beam diameter, the light beam reaches a first reflector (3) and is reflected as a vertically upward light beam, the upward light beam reaches a second reflector (6) and is reflected in a direction toward the surface of a prism (10); on the sample stage, the light beam vertically enters the prism (10), a total internal reflection phenomenon occurs in a sample pool (11), and the fluorescent substance is excited to emit fluorescence.

2. A prism-type total internal reflection imaging device compatible with an inverted microscope according to claim 1, characterized in that: In the excitation light path, the wavelength of the laser light source (1) is 100-2000 nm; the center installation height of the aperture (2) should be consistent with the center of the laser light source (1) output light beam, the height is 10-30 cm, the laser light source (1) output light vertically passes through the aperture (2) hole, the hole diameter is 0.1-2.0 mm, and outputs a horizontal collimated light beam; the center height of the first reflector (3) is equal to the height of the aperture (2); the horizontal collimated light output from the center hole of the aperture (2) hits the center of the first reflector (3), and is reflected vertically upward by the inclined first reflector (3).

3. The prism-type total internal reflection imaging device compatible with a commercial inverted microscope according to claim 1, characterized in that: In the excitation light path, the first displacement stage (4) moves horizontally along a vertical substrate (9) in a single axis, and the first reflector (3) is mounted on a first six-axis optical adjustment frame (5) to adjust the pitch and deflection angles, with a maximum adjustment angle of 10°; the light reflected from the first reflector (3) hits the center of the second reflector (6), is reflected by the second reflector (6), and propagates toward the surface of the prism (10).

4. The prism-type total internal reflection imaging device compatible with a commercial inverted microscope according to claim 1, characterized in that: In the excitation light path, the second displacement stage (7) moves vertically along a vertical substrate (9); the second reflector (6) is mounted on a second six-axis optical adjustment frame (8) and can adjust the pitch and deflection angles, with a maximum adjustment angle of 10°.

5. The prism-type total internal reflection imaging device compatible with a commercial inverted microscope according to claim 1, characterized in that: In the sample stage, the sample stage (14) is a lacquered iron plate with a length of 10-100 cm and a width of 10-100 cm. It is fixed by an adsorbing magnet and has a through hole in the middle for placing an objective lens. The objective lens has a magnification range of 10 to 100 times and a working distance of 0.2 mm to 10 mm. The sample stage (14) is installed on an inverted microscope. The prism fixing block (12) is fixed on the sample stage (14) with screws. The incident light is perpendicular to the surface of the prism (10). The light is incident on the upper surface of the sample pool (11) chamber to cause total internal reflection, thereby exciting fluorescent molecules close to the glass slide. The sample pool (11) is composed of two thin glass slides and a double-sided adhesive gasket. The double-sided adhesive bonds the two glass slides together. The thickness of the glass slide is 0.10-0.5 mm, and the thickness of the gasket is 0.01-1 mm. The sample pool is 30-80 cm away from the center of the second reflector in the horizontal direction and 20-60 cm away from the center of the second reflector in the vertical direction.

6. The prism-type total internal reflection imaging device compatible with a commercial inverted microscope according to claim 1, characterized in that: The prism (10) is made of quartz glass with a refractive index consistent with that of a commercial glass slide. The refractive index of the quartz glass is: 1.47-1.53, with a Brewster angle at the rear end. The light reflected in the total internal reflection light path will enter the Brewster angle structure and be continuously reflected back and forth, and finally all emitted from the Brewster angle, ensuring that this part of the reflected light will not reside in the prism to cause stray light and interfere with the collected image.

7. The prism-type total internal reflection imaging device compatible with a commercial inverted microscope according to claim 1, characterized in that: The incident angle of the prism (10) is fixed and is between 62° and 85°.

8. The prism-type total internal reflection imaging device compatible with a commercial inverted microscope according to claim 1, characterized in that: The incident light of the excitation light path is reflected on the surface of the prism (10). After the incident light angle is adjusted, the light reflected from the incident surface of the prism is reflected by the second reflector (6) and the first reflector (3), and returns to the center of the aperture (2) along the original path. At this time, the incident light is perpendicular to the surface of the prism (10). According to the geometric relationship, the total internal reflection angle is equal to the prism angle. This is the first calibration of the total internal reflection angle after the device is installed. After that, it only needs to replace the sample cell to directly cause the total internal reflection phenomenon.

9. A method for using a prism-type total internal reflection imaging device compatible with a commercial inverted microscope as described in any one of claims 1 to 8, characterized in that: The specific steps of using the device are as follows: Step 1) Sample loading: injecting the sample into the chamber of the sample pool, installing the sample pool under the prism, and fixing the sample pool with the sample fixing block; Step 2) Turn on the laser to generate a total internal reflection light path: the excitation light passes through the aperture, and is then reflected by the first reflector and the second reflector to the prism surface, and is refracted and propagates to the interface between the lower surface of the glass slide on the sample pool and the sample; at this time, the total internal reflection angle is equal to the prism angle, and a total internal reflection phenomenon occurs at the interface between the lower surface of the glass slide on the sample pool and the sample, and the generated evanescent wave illuminates nearby fluorescent molecules. The camera collects images at the set exposure time and shooting speed to obtain a total internal reflection fluorescence image with a high signal-to-noise ratio; Step 3) Positioning, tracking and analysis: Calculate the background average light intensity as the average of the light intensities of all the images taken; deduct the background average light intensity from the image at each moment, and use Gaussian fitting to obtain the xy position of the centroid of the single molecule. Extract the average light intensity within a circle with a radius of d from the centroid as the single molecule intensity I(h), where d is adjusted according to the size of the single molecule. Calculate the z position using the exponential decay formula of I(h) with separation distance. Obtain a continuous three-dimensional trajectory for each molecule in the field of view based on the x, y, and z positions of each image; Calculate the mean square end-to-end distance in the three-dimensional direction based on the three-dimensional trajectory, and further calculate the diffusion coefficient of the molecule based on the Einstein-Stokes equation. Count the number of other single molecules in each image that are in a series of concentric spherical shells with increasing radius over a period of time, calculate the radial distribution function g(r) that describes the change in density between single molecules with distance, and further calculate the apparent relative potential energy U(r) between molecules.

10. The method for using a prism-type total internal reflection imaging device compatible with a commercial inverted microscope according to claim 9, characterized in that: In step 3, the Gaussian fitting formula is defined as follows: Where I(x,y) is the light intensity, A, d, and b are Gaussian fitting parameters, and x0 and y0 represent the single-molecule coordinates; In step 3, the z position of a single molecule is calculated using the following formula: Where I0 is the maximum light intensity in the single molecule being tracked. The single molecule with the maximum light intensity is located closest to the surface of the glass sheet, and its z position is set to zero. I(h) is the average light intensity of any single molecule, h is the distance from the zero point, that is, the z position of the single molecule, and β is the inverse of the penetration depth. l is the wavelength of the incident light, q is the total internal reflection angle, n1 and n2 are the refractive indices of the glass and the sample, Calculate the mean square displacement (MSD) in the three-dimensional direction based on the three-dimensional trajectory: Among them, r(t0) and r(t0+Dt) are the three-dimensional positions of the single molecule at time t0 and (t0+Dt), respectively. Fit each trajectory with at least 100-500 points with the following formula: Where D is the equivalent diffusion factor, u is the MSD index, when u is greater than 1, it is active motion, when u is less than 1, it is restricted motion, when u is equal to 1, it is diffusion motion and D is the real diffusion factor; at this time, the Einstein-Stokes equation can be used to calculate the diffusion coefficient, the formula is as follows: Among them, k B is the Boltzmann constant, T is the temperature, h is the fluid shear viscosity, σ is the molecular diameter, and D is the diffusion coefficient; In step 3, the radial distribution function g(r) describing the density variation between single molecules with distance is calculated and is defined as: Where f represents the local density within the sphere of the single molecule radius r, and f' represents the number density of single molecules in the system, which is defined as: Where N(r) is d r The number of single molecules in the ring, for the space around each single molecule, counts the number of other single molecules in a series of concentric spherical shells with increasing radius; when the concentration of the system is infinitely dilute, g(r) can be related to the interaction energy U(r) through the Boltzmann distribution; U(r) is calculated using the following formula: Since infinite dilution is not possible, we can obtain the corresponding apparent potential energy U at a series of gradient concentrations: app (r): Among them, k B is the Boltzmann constant, T is the temperature, and we get U app (r), and then U(r) is obtained by mathematical fitting.

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