An integrated single molecule microscopy method and apparatus

Abstract

The application discloses a comprehensive single-molecule microscopic imaging method and device, which comprises a laser, a lens group, a beam splitter, a quarter-wave plate, a mirror, a three-dimensional displacement table, a sample to be detected, a long-focus tube lens, a push-pull filter plate, an imaging objective and an industrial camera. In the optical path, the angle of the mirror is first changed so that the incident light is incident at a plurality of angles corresponding to the supercritical angle or the surface plasmon resonance angle of the corresponding material; and secondly, a push-pull filter plate is used to adjust whether the sample surface reflected light is collected or not, so that four modes of total internal reflection-interference scattering microscopic imaging, surface plasmon resonance-interference scattering imaging, traditional interference scattering imaging and dark field imaging are realized on the same sample in a set of system. The application provides a multi-modal non-labeled imaging technology which can be verified with each other, and can greatly improve the utilization rate of the device and the reuse ability in the actual system.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of optical microscopic imaging technology, in particular to a comprehensive single-molecule microscopic imaging method and device. BACKGROUND

[0002] In recent years, the emergence of single-molecule detection methods has brought important development for determining molecular interactions in order to carry out drug screening, molecular diagnosis, biochemical analysis, etc., and has become a powerful tool for detecting small particles and quantifying molecular interaction dynamics. Specific technologies such as interference scattering (iSCAT), total (internal) reflection (TIR), surface plasmon resonance (SPR), dark field, optical waveguide, optical microcavity, etc. Each of these technologies has a unique working principle and independent operating system, resulting in that existing single-molecule microscopic devices are often difficult to have multiple functions at the same time. If multiple functions need to be realized, different modules need to be connected on the same microscopic platform, which greatly increases the system complexity, operation difficulty and assembly cost.

[0003] Total internal reflection technology utilizes the characteristic that evanescent waves are generated on the other side of the medium after total reflection of incident light, excites molecules to observe the near-field region of the sample, and the dynamic range of observation is usually below 200 nm. Because the excitation light decays exponentially, only the sample region close to the total reflection surface will produce reflection, greatly reducing the interference of background light noise, so this technology is widely used in dynamic observation of cell surface substances.

[0004] Surface plasmon resonance technology detects the density wave of electrons propagating on the metal interface through resonance. When the sample interacts with the metal film on the surface of the substrate, it will cause a change in the refractive index of the film surface, resulting in a change in the corresponding resonance angle (SPR angle). By detecting the change in the SPR angle, information such as the concentration, affinity, kinetic constant and specificity of the analyte can be obtained.

[0005] The common feature of these two methods is to generate an evanescent field within a few hundred nanometers of the sensor surface for measurement. The evanescent field strength decays exponentially in the axial direction, so the motion behavior of the analyte can be monitored and tracked by changes in signal strength, with a resolution as low as sub-nanometers. More importantly, the evanescent field can significantly reduce the illumination volume to enhance the interaction between light and analyte and reduce environmental noise, achieving high sensitivity detection. However, there are limitations. First, the heating effect accompanied by plasmonic enhancement limits its application in detecting small biological molecules under normal conditions. Second, the limited evanescent field allows it to observe only the shallow surface region within a few hundred nanometers of the sample. Third, the relatively stringent excitation conditions make the optical system structure tend to be complex.

[0006] Interference scattering optical microscopy (iSCAT) is a new type of far-field optical imaging technology based on light scattering, in which a sample is illuminated with coherent light (e.g. laser light), and the signal is generated by the interference between the light scattered by the sample and the reference light (usually light reflected from a nearby interface). The detected particle signal is proportional to the cube of the particle diameter, so as the size of the nanoparticle continues to decrease, the interference scattering microscopic imaging technology highlights higher detection sensitivity. However, due to the low signal-to-background ratio, the obtained image often needs to be post-processed, so there is still some difficulty in achieving real-time imaging.

[0007] Dark field microscopy usually utilizes direct blocking of the central light beam so that it cannot pass through the sample from bottom to top into the objective lens, so that the incident light changes its way and is obliquely illuminated on the observed sample, and the sample surface is then reflected or scattered into the objective lens, so that the entire field appears dark. Dark field imaging often has higher contrast than ordinary imaging mode, but the required incident light intensity is easy to cause glare and distortion.

[0008] In summary, by utilizing the advantages of various imaging modes, it is an urgent problem to provide an integrated imaging technology with multiple modalities that can complement and verify each other. SUMMARY

[0009] To overcome the above problems, the present application provides a comprehensive single molecule microscopic imaging method and device.

[0010] The first aspect of the present application provides a comprehensive single molecule microscopic imaging method, comprising the following steps:

[0011] (1) The laser beam is reflected by a beam expander after collimation, then passes through a quarter-wave plate to change the polarization form, and is projected onto the sample surface through the objective lens;

[0012] (2) The mirror is placed in front of the motorized three-dimensional translation stage, and the angle of the mirror is adjusted in three-dimensional space to make the incident light incident at different angles;

[0013] (3) The position of the motorized displacement stage in three-dimensional space is adjusted to match the sample focal plane with the objective lens used, and the detection light is further focused on the back focal plane of the objective lens;

[0014] (4) A push-pull type filter plate with a certain width is used as a mask, and the sliding filter module allows it to block or allow the reflected light beam and most of the interference formed detection light to enter the lens barrel lens, and be collected by the camera.

[0015] Further, in step (2), the position of the mirror is adjusted to make the incident light incident at a supercritical angle, inducing an evanescent wave;

[0016] In the step (4), a push-pull filter is used as a mask, which has a certain width to block the reflected light beam, i.e., background light, and allow most of the detection light to enter the lens barrel, which is collected by the camera, so as to realize total reflection-interference scattering imaging.

[0017] Further, in the step (2), the position of the mirror is adjusted so that the incident light is incident at the surface plasmon resonance angle, so as to induce an evanescent wave.

[0018] In the step (4), a push-pull filter is used as a mask, which has a certain width to block the reflected light beam, i.e., background light, and allow most of the detection light to enter the lens barrel, which is collected by the camera, so as to realize total reflection-interference scattering imaging.

[0019] Further, in the step (2), the position of the mirror is adjusted so that the incident light is incident at the surface plasmon resonance angle, so as to induce an evanescent wave.

[0020] In the step (4), the push-pull filter is pulled to the neutral position, so as to allow the sample scattered light, the substrate reflected light and the interference light of the two to enter the lens barrel, which is collected by the camera, so as to realize interference scattering imaging.

[0021] Further, in the step (2), the position of the mirror is adjusted so that the incident light is incident at the surface plasmon resonance angle, so as to induce an evanescent wave.

[0022] In the step (4), a push-pull filter is used as a mask, which has a certain width to block the reflected light beam, i.e., background light, and allow most of the detection light to enter the lens barrel, which is collected by the camera, so as to realize total reflection-interference scattering imaging.

[0023] The second aspect of the present application provides a reflection device for implementing a comprehensive single-molecule microscopic imaging method, which comprises a laser, a first focusing lens, a first collimating lens, a second focusing lens, a second collimating lens, a polarization beam splitter prism, a glass slide, a mirror, an objective lens, a sample to be measured, a push-pull filter, a sleeve lens and a camera.

[0024] The laser is arranged on a temperature control base and is driven by a table diode current controller and a temperature controller; the laser beam emitted by the laser enters the first focusing lens, the first collimating lens, the second focusing lens, the second collimating lens and the polarization beam splitter prism in sequence; the incident linearly polarized light is reflected by the polarization beam splitter prism and becomes circularly polarized light after passing through the glass slide.

[0025] The mirror is placed in front of the motorized three-dimensional translation stage, and the angle of the mirror is adjusted in three-dimensional space to make the incident light incident at a supercritical angle; the circularly polarized light formed by the interference of the evanescent wave scattered by the rough glass substrate surface and the scattered light of the sample to be measured becomes p-polarized light after passing through the glass, and then reaches the detection path through the polarizing beam splitter; the position of the motorized displacement stage in three-dimensional space is adjusted to match the sample focal plane with the imaging objective, and the detection light is further focused on the back focal plane of the objective; the push-pull filter plate is used as a mask, and the mask has a width to block the reflected light beam and allow most of the interference-formed detection light to enter the lens barrel lens and be collected by the camera.

[0026] Further, another embodiment of a reflective device for implementing a comprehensive single-molecule microscopic imaging method comprises a laser, a first focusing lens, a first collimating lens, a second focusing lens, a second collimating lens, a polarizing beam splitter, a glass, a mirror, an imaging objective, a sample to be measured, a push-pull filter plate, a sleeve lens, and a camera.

[0027] The laser is arranged on a temperature-controlled base and driven by a benchtop diode current controller and a temperature controller; the laser beam emitted by the laser sequentially enters the first focusing lens, the first collimating lens, the second focusing lens, the second collimating lens, and the polarizing beam splitter, and the incident linearly polarized light becomes circularly polarized light after being reflected by the polarizing beam splitter and passing through the glass.

[0028] The mirror is placed in front of the motorized three-dimensional translation stage, and the angle of the mirror is adjusted in three-dimensional space to make the incident light incident at a surface plasmon resonance angle; the circularly polarized light formed by the interference of the evanescent wave excited by the metal-coated substrate surface and the scattered light of the sample to be measured becomes p-polarized light after passing through the glass, and then reaches the detection path through the polarizing beam splitter; the push-pull filter plate is used as a mask, and the mask has a certain width to block the reflected light beam and allow most of the interference-formed detection light to enter the lens barrel lens and be collected by the camera.

[0029] Further, another embodiment of a reflective device for implementing a comprehensive single-molecule microscopic imaging method comprises a laser, a first focusing lens, a first collimating lens, a second focusing lens, a second collimating lens, a polarizing beam splitter, a glass, a mirror, an imaging objective, a sample to be measured, a push-pull filter plate, a sleeve lens, and a camera.

[0030] The laser is arranged on a temperature-controlled base and driven by a benchtop diode current controller and a temperature controller; the laser beam emitted by the laser sequentially enters the first focusing lens, the first collimating lens, the second focusing lens, the second collimating lens, and the polarizing beam splitter, and the incident linearly polarized light becomes circularly polarized light after being reflected by the polarizing beam splitter and passing through the glass.

[0031] The mirror is placed in front of the motorized three-dimensional translation stage to make the incident light perpendicular; the scattered light of the sample to be measured and the reflected light of the substrate surface become p-polarized light after passing through the glass sheet, and then reach the detection path through the polarizing beam splitter prism; the push-pull filter plate is used as a mask, and the mask has a certain width to block the reflected light beam and allow most of the scattered light and the interference formed detection light to enter the lens barrel lens, and be collected by the camera.

[0032] The third aspect of the application provides a transmission device for implementing a comprehensive single-molecule microscopic imaging method, comprising a laser, a first focusing lens, a first collimating lens, a second focusing lens, a second collimating lens, a mirror, an illumination objective lens, a sample to be measured, an imaging objective lens, a push-pull filter plate, a sleeve lens, and a camera.

[0033] The laser is fixed on a temperature-controlled base and driven by a benchtop diode current controller and a temperature controller; the laser beam emitted by the laser sequentially enters the first focusing lens, the first collimating lens, the second focusing lens, and the second collimating lens.

[0034] The mirror is placed in front of the motorized three-dimensional translation stage to make the incident light perpendicular; the scattered light of the sample to be measured and the reflected light of the substrate surface become p-polarized light after passing through the glass sheet, and then reach the detection path through the polarizing beam splitter prism; the push-pull filter plate is used as a mask, and the mask has a certain width to block the reflected light beam and allow most of the scattered light and the interference formed detection light to enter the lens barrel lens, and be collected by the camera.

[0035] Further, another transmission device for implementing a comprehensive single-molecule microscopic imaging method, comprising a laser, a first focusing lens, a first collimating lens, a second focusing lens, a second collimating lens, a mirror, an illumination objective lens, a sample to be measured, an imaging objective lens, a push-pull filter plate, a sleeve lens, and a camera.

[0036] The laser is fixed on a temperature-controlled base and driven by a benchtop diode current controller and a temperature controller; the laser beam emitted by the laser sequentially enters the first focusing lens, the first collimating lens, the second focusing lens, and the second collimating lens.

[0037] The mirror is placed in front of the electric three-dimensional translation stage, and the position of the mirror is adjusted in the three-dimensional space to make the incident light incident at the surface plasmon resonance angle. The light formed by the interference of the evanescent wave excited by the metal substrate surface coated with a certain thickness and the scattered light of the sample to be measured reaches the detection path through the imaging objective lens; the push-pull filter plate is used as a mask, and the push-pull filter plate has a certain width to block the reflected light beam and allow most of the interference formed detection light to enter the lens barrel lens, and is collected by the camera.

[0038] Further, another embodiment of the comprehensive single molecule microscopic imaging method is a transmission device, characterized by comprising a laser, a first focusing lens, a first collimating lens, a second focusing lens, a second collimating lens, a mirror, an illumination objective lens, a sample to be measured, an imaging objective lens, a push-pull filter plate, a sleeve lens, and a camera.

[0039] The laser is fixed on a temperature control base and is driven by a table diode current controller and a temperature controller; the laser beam emitted by the laser enters the first focusing lens, the first collimating lens, the second focusing lens, and the second collimating lens in sequence.

[0040] The mirror is placed in front of the electric three-dimensional translation stage to make the incident light vertically incident; the light formed by the interference of the reflected light of the substrate surface and the scattered light of the sample to be measured reaches the detection path through the imaging objective lens; the push-pull filter plate is in the neutral position, so that it allows the sample scattered light, the substrate reflected light, and the detection light formed by the interference of the two to enter the lens barrel lens, and is collected by the camera.

[0041] The present application has the following advantages:

[0042] (1) By simply adjusting the incident angle and the push-pull filter plate position, the device can realize total internal reflection-interference scattering imaging, surface plasmon resonance-interference scattering imaging, dark field-interference scattering imaging, and traditional interference scattering imaging, etc. multiple imaging modes, meet the user's demand under most conditions, effectively save cost, greatly improve the utilization rate of the device and the reuse ability in the actual system;

[0043] (2) The imaging areas before and after switching different imaging modes are consistent and can be mutually corroborated;

[0044] (3) Combined with the advantages of multiple imaging modes, switching can realize label-free, high-sensitivity, real-time online monitoring of the sample to be measured, effectively simplifying the experimental steps, and having important significance and great value in the development and practicality of single molecule detection technology, especially in the detection of small particles. BRIEF DESCRIPTION OF DRAWINGS

[0045] Figure 1 It is a reflective comprehensive single molecule microscopic imaging device.

[0046] Fig. 2 is a schematic diagram of the total reflection-interference scattering imaging mode (a) and the surface plasmon resonance-interference scattering imaging mode (b) of the present application;

[0047] Fig. 3 is a schematic diagram of the interference scattering imaging mode (a) and the dark field-interference scattering imaging mode (b) of the present application;

[0048] Figure 4 is a top view of the push-pull filter plate of the present application;

[0049] Figure 5 is a transmission-type comprehensive single-molecule microscopic imaging device of the present application;

[0050] BRIEF DESCRIPTION OF DRAWINGS 1-laser; 2-first focusing lens; 3-first collimating lens; 4-second focusing lens; 5-second collimating lens; 6-polarization beam splitter prism; 7-1 / 4 glass sheet; 8-mirror; 9-imaging objective lens; 10-sample to be measured; 11-push-pull filter plate; 12-sleeve lens; 13-camera; 14-illumination objective lens; 15-glass substrate; 16-metal film. DETAILED DESCRIPTION

[0051] The technical solutions of the present application will be described clearly and completely below in conjunction with the drawings. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application.

[0052] In the description of the present application, it should be noted that the orientation or positional relationship indicated by terms such as "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer" and the like is based on the orientation or positional relationship shown in the drawings, and is only for the convenience of describing the present application and simplifying the description, and therefore cannot be understood as indicating or implying that the device or element referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as limiting the present application. In addition, the terms "first", "second", "third" appear only for the purpose of description, and cannot be understood as indicating or implying relative importance.

[0053] In the description of the present application, it should be noted that, unless otherwise explicitly specified and limited, the terms "mounting", "connecting", "connection" should be understood in a broad sense, for example, it can be fixed connection, or detachable connection, or integrally connected; it can be mechanical connection, or electrical connection; it can be directly connected, or indirectly connected through an intermediate medium, or the communication inside two elements. For those skilled in the art, the specific meaning of the above terms in the present application can be understood according to the specific circumstances.

[0054] Embodiment One

[0055] Refer to the attached drawings Figures 1-5 A comprehensive single-molecule microscopic imaging method, comprising the following steps:

[0056] (1) The laser beam is reflected by a beam expander collimator, then passes through a quarter-wave plate to change the polarization form, and is projected to the sample surface through the objective lens;

[0057] (2) The mirror is placed in front of the electric three-dimensional translation stage, and the angle of the mirror is adjusted in three-dimensional space to make the incident light incident at different angles. The specific incident modes are as follows:

[0058] First, refer to Figure 2(a), by adjusting the angle of the mirror in three-dimensional space, the light is incident on the sample surface at an angle greater than or equal to the supercritical angle.

[0059] Second, refer to Figure 2(b), by adjusting the angle of the mirror in three-dimensional space, the light is incident on the sample surface at a surface plasmon resonance angle.

[0060] Third, refer to Figures 3(a) and (b), by adjusting the angle of the mirror in three-dimensional space, the light is incident on the sample surface perpendicularly.

[0061] (3) Adjust the position of the electric displacement stage in three-dimensional space to match the sample focal plane with the objective lens used, and further converge the detection light to the back focal plane of the objective lens;

[0062] (4) A push-pull filter plate with a certain width is used as a mask, and the sliding filter module is blocked to allow the reflected light beam and most of the interference to form detection light to enter the lens barrel lens, and is collected by the camera.

[0063] Embodiment Two

[0064] This embodiment provides a reflective comprehensive single-molecule microscopic imaging device, referring to Figure 1 , the device comprises a laser 1; a first focusing lens 2; a first collimating lens 3; a second focusing lens 4; a second collimating lens 5; a polarization beam splitter 6; a 1 / 4 glass 7; a mirror 8; an objective lens 9; a sample to be measured 10; a push-pull filter plate 11; a sleeve lens 12; a camera 13;

[0065] The reflective comprehensive single-molecule microscopic imaging device in this embodiment includes the following working modes, referring to Figures 2-3:

[0066] The full reflection-interference scattering imaging mode is:

[0067] The laser 1 is started and fixed on a temperature controlled base, which is driven by a benchtop diode current controller and temperature controller. The incident light is collimated by a lens group, which is composed of two short focal length achromatic doublet lenses 2 and 3, and then the converging form of the light is adjusted again by a second lens group composed of two long focal length lenses 4 and 5. The incident s-polarized light becomes circularly polarized light after being reflected by a polarizing beam splitter 6 and passing through a quarter wave plate 7. A mirror 8 is placed in front of the motorized three-dimensional translation stage, and the angle of the mirror 8 is adjusted in three-dimensional space to make the incident light to be incident at a supercritical angle. The circularly polarized light formed by the interference of the evanescent wave excited by the glass substrate surface with a certain roughness and the scattered light of the sample 10 to be measured becomes p-polarized light after passing through the quarter wave plate 7, and then reaches the detection path through the polarizing beam splitter 6. The position of the motorized displacement stage in three-dimensional space is adjusted to match the sample focal plane with the objective lens 9 used, and the detection light is further converged to the back focal plane of the objective lens 9. A push-pull filter 11 is used as a mask here, which has a certain width to block the reflected light beam (i.e. background light) and allow most of the detection light formed by interference to enter the lens barrel lens 12, and be collected by the camera 13.

[0068] The surface plasmon resonance-interference scattering imaging mode is:

[0069] The mirror 8 is placed in front of the motorized three-dimensional translation stage, and the angle of the mirror 8 is adjusted in three-dimensional space to make the incident light to be incident at a surface plasmon resonance angle. The circularly polarized light formed by the interference of the evanescent wave excited by the metal substrate surface coated with a certain thickness and the scattered light of the sample 10 to be measured becomes p-polarized light after passing through the quarter wave plate 7, and then reaches the detection path through the polarizing beam splitter 6. The push-pull filter 11 is used as a mask, which has a certain width to block the reflected light beam (i.e. background light) and allow most of the detection light formed by interference to enter the lens barrel lens 12, and be collected by the camera 13.

[0070] The interference scattering imaging mode is:

[0071] The mirror 8 is placed in front of the motorized three-dimensional translation stage to make the incident light to be incident perpendicularly. The circularly polarized light formed by the interference of the scattered light of the sample 10 to be measured and the reflected light of the substrate surface becomes p-polarized light after passing through the quarter wave plate 7, and then reaches the detection path through the polarizing beam splitter 6. The push-pull filter 11 is pulled to the empty position, which allows the sample scattered light and the substrate reflected light and the detection light formed by the interference of the two to enter the lens barrel lens 12, and be collected by the camera 13.

[0072] The dark field-interference scattering imaging mode is:

[0073] The mirror 8 is placed in front of the motorized three-dimensional translation stage to make the incident light perpendicular. The scattered light of the sample 10 and the reflected light of the substrate surface become p-polarized light after passing through the quarter-wave plate 7, and then reach the detection path through the polarizing beam splitter prism 6. The push-pull filter plate 11 is used as a mask, which has a certain width to block the reflected light beam and allow most of the scattered light and interference detection light to enter the lens barrel lens 12 and be collected by the camera 13.

[0074] The modes in this embodiment are described in a progressive manner, and each mode focuses on the differences from other modes. The same or similar parts between modes can be referred to each other.

[0075] Embodiment three

[0076] This embodiment provides a transmission type comprehensive single molecule microscopic imaging device, which is shown in Figure 5 , and specifically includes a laser 1, a first focusing lens 2, a first collimating lens 3, a second focusing lens 4, a second collimating lens 5, a mirror 8, an illumination objective 14, a sample 10 to be measured, an imaging objective 9, a push-pull filter plate 11, a lens barrel 12, and a camera 13.

[0077] The transmission type comprehensive single molecule microscopic imaging device in this embodiment includes the following working modes, as shown in FIGS. 2-3:

[0078] The total reflection-interference scattering imaging mode is:

[0079] The laser 1 is started, which is fixed on a temperature-controlled base and driven by a table diode current controller and a temperature controller. The incident light is collimated by a lens group composed of two short focal length achromatic doublet lenses 2 and 3, and then adjusted again by a second lens group composed of two long focal length lenses 4 and 5. The mirror 8 is placed in front of the motorized three-dimensional translation stage, and the angle of the mirror 8 is adjusted in three-dimensional space to make the incident light incident at a supercritical angle. The light formed by the interference of the evanescent wave excited by the rough glass substrate surface and the scattered light of the sample 10 to be measured reaches the detection path through the imaging objective 9. The illumination objective 14 and the imaging objective 9 are of the same type to keep the beam diameter and quality unchanged. The position of the motorized displacement stage in three-dimensional space is adjusted to match the sample focal plane with the imaging objective 9 used, and the detection light is further focused on the back focal plane of the imaging objective 9. A push-pull filter plate 11 is used as a mask, which has a certain width to block the reflected light beam (i.e. background light) and allow most of the interference detection light to enter the lens barrel lens 12 and be collected by the camera 13.

[0080] The surface plasmon resonance-interference scattering imaging mode is:

[0081] The mirror 8 is placed in front of the motorized three-dimensional translation stage, and the position of the mirror 8 is adjusted in three-dimensional space to make the incident light incident at the surface plasmon resonance angle. The light formed by the interference of the evanescent wave excited by the metal-coated substrate surface and the scattered light of the sample 10 to be detected passes through the imaging objective 9 to the detection path. The push-pull filter plate 11 is used as a mask, which has a certain width to block the reflected light beam (i.e. background light) and allow most of the interference-formed detection light to enter the lens barrel lens 12 and be collected by the camera 13.

[0082] The interference scattering imaging mode is:

[0083] The mirror 8 is placed in front of the motorized three-dimensional translation stage so that the incident light is perpendicular to the incident light. The light formed by the interference of the substrate surface reflected light and the scattered light of the sample 10 to be detected passes through the imaging objective 9 to the detection path. The push-pull filter plate 11 is pulled to the empty position, which allows the sample scattered light and the substrate reflected light and the detection light formed by the interference of the two to enter the lens barrel lens 12 and be collected by the camera 13.

[0084] The dark field-interference scattering mode is:

[0085] The mirror 8 is placed in front of the motorized three-dimensional translation stage so that the incident light is perpendicular to the incident light. The light formed by the interference of the substrate surface reflected light and the scattered light of the sample 10 to be detected passes through the imaging objective 9 to the detection path. The push-pull filter plate 11 is used as a mask, which has a certain width to block the reflected light beam and allow most of the interference-formed detection light to enter the lens barrel lens 12 and be collected by the camera 13.

[0086] The various modes in this embodiment are described in a progressive manner, and each mode focuses on the differences from other modes. The same and similar parts between modes can be referred to each other.

[0087] The present application mainly aims to solve the problem that the single interference scattering technology has poor signal-to-background ratio when detecting small particle samples and is limited in combination with other technologies. By simply adjusting the incident form of the laser, the total internal reflection-interference scattering microscopy, surface plasmon resonance-interference scattering imaging, dark field imaging and other modes are integrated in a set of system, providing a multi-modal label-free imaging technology for the same sample and the same region, which can be verified with each other, greatly improving the utilization rate of the device and the reuse ability in actual system, and having important significance and great value in the development and practicality of single molecule detection technology, especially in the detection technology of small particles.

[0088] The comprehensive single-molecule microscopic imaging method and device provided by the present application are described in detail above, and the principles and implementation manners of the present application are described by applying specific examples in the present document. The above description of the examples is only used to help understand the method of the present application and its core idea; meanwhile, for those skilled in the art, the specific implementation manners and application ranges will be changed according to the idea of the present application, and the content of the present description should not be understood as a limitation on the present application.

[0089] It should be noted that each of the embodiments in the present description is described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the embodiments can be understood by mutual reference. For the device disclosed in the embodiments, since it corresponds to the method disclosed in the embodiments, the description is relatively simple, and the relevant parts can be understood by referring to the description of the method.

[0090] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present application. Various modifications to the embodiments will be apparent to those skilled in the art, and the general principles defined in the present document can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown in the present document, but will conform to the widest scope consistent with the principles and novel features disclosed in the present document.

Claims

1. A comprehensive single-molecule microscopic imaging method, characterized in that, Includes the following steps: (1) The laser beam is collimated by beam expander, reflected by beam splitter, and then polarized by quarter-wave plate before being projected onto the sample surface through objective lens. (2) Place the reflector in front of the electric three-dimensional translation stage, and rotate and adjust the angle of the reflector in three-dimensional space so that the incident light enters at different angles. (3) Adjust the position of the electric displacement stage in three-dimensional space to match the sample focal plane with the objective lens used, and the detection light is further focused onto the back focal plane of the objective lens; (4) A push-pull filter plate with a certain width is used as a mask. The sliding filter module can block or allow the reflected beam and most of the interference formed detection light to enter the lens barrel and be collected by the camera.

2. The comprehensive single-molecule microscopic imaging method as described in claim 1, characterized in that: In step (2), the position of the reflector is adjusted so that the incident light is incident at a supercritical angle, thereby inducing an evanescent wave; In step (4), a push-pull filter is used as a mask. The mask has a certain width to block the reflected light beam, i.e., the background light, and allows most of the detection light to enter the lens barrel and be collected by the camera to achieve total internal reflection-interference scattering imaging.

3. The comprehensive single-molecule microscopic imaging method as described in claim 1, characterized in that: In step (2), the position of the reflector is adjusted so that the incident light is incident at the surface plasmon resonance angle, thereby inducing evanescent waves; In step (4), a push-pull filter plate is used as a mask. The mask has a certain width to block the reflected light beam, i.e., the background light, and allows most of the detection light to enter the lens barrel and be collected by the camera to realize surface plasmon resonance-interference scattering imaging.

4. The comprehensive single-molecule microscopic imaging method as described in claim 1, characterized in that: In step (2), the incident light can be made to be perpendicular to the incident light by adjusting the position of the reflector; In step (4), the push-pull filter plate is pulled to the neutral position, so that the sample scattered light, the substrate reflected light and their interference light can enter the lens barrel at the same time and be collected by the camera to realize interference scattering imaging.

5. The comprehensive single-molecule microscopic imaging method as described in claim 1, characterized in that: In step (2), the incident light can be made to be perpendicular to the incident light by adjusting the position of the reflector; In step (4), a push-pull filter plate is used as a mask. The mask has a certain width to block the reflected light beam, i.e., the background light, allowing most of the detection light to enter the lens barrel and be collected by the camera to achieve dark field interference scattering imaging.

6. A reflective device for implementing the comprehensive single-molecule microscopic imaging method according to claim 2, characterized in that: It includes a laser (1), a first focusing lens (2), a first collimating lens (3), a second focusing lens (4), a second collimating lens (5), a polarizing beam splitter (6), a quarter glass slide (7), a mirror (8), an objective lens (9), a sample to be tested (10), a push-pull filter (11), a sleeve lens (12), and a camera (13); The laser (1) is mounted on a temperature control base and driven by a desktop diode current controller and a temperature controller. The laser beam emitted from the laser (1) is sequentially incident on the first focusing lens (2), the first collimating lens (3), the second focusing lens (4), the second collimating lens (5), and the polarizing beam splitter (6). The incident linearly polarized light is reflected by the polarizing beam splitter 6 and then passes through a 1 / 4 glass plate (7) to become circularly polarized light. The reflector (8) is placed in front of the motorized three-dimensional translation stage. The angle of the reflector (8) is adjusted by rotating it in three-dimensional space so that the incident light is incident at a supercritical angle. The circularly polarized light formed by the interference of the evanescent wave excited by the scattering of the rough glass substrate surface and the scattered light of the sample (10) becomes p-polarized light after passing through the 1 / 4 glass plate (7), and then reaches the detection path through the polarizing beam splitter (6). The position of the motorized stage in three-dimensional space is adjusted so that the focal plane of the sample matches the imaging objective (9), and the detection light is further focused onto the back focal plane of the objective (9). The push-pull filter plate (11) is used as a mask. The mask has a width to block the reflected beam and allow most of the interference-formed detection light to enter the lens barrel (12) and be collected by the camera (13).

7. A reflective device for implementing the comprehensive single-molecule microscopic imaging method according to claim 3, characterized in that: It includes a laser (1), a first focusing lens (2), a first collimating lens (3), a second focusing lens (4), a second collimating lens (5), a polarizing beam splitter (6), a quarter glass slide (7), a mirror (8), an imaging objective lens (9), a sample to be tested (10), a push-pull filter plate (11), a sleeve lens (12), and a camera (13); The laser (1) is mounted on a temperature control base and driven by a desktop diode current controller and a temperature controller. The laser beam emitted from the laser (1) is sequentially incident on the first focusing lens (2), the first collimating lens (3), the second focusing lens (4), the second collimating lens (5), and the polarizing beam splitter (6). The incident linearly polarized light is reflected by the polarizing beam splitter 6 and then passes through a 1 / 4 glass plate (7) to become circularly polarized light. The reflector (8) is placed in front of the electric three-dimensional translation stage. The angle of the reflector (8) is adjusted by rotating it in three-dimensional space so that the incident light is incident at the surface plasmon resonance angle. The circularly polarized light formed by the interference between the evanescent wave excited by the surface of the metal substrate with a certain thickness and the scattered light of the sample to be tested (10) becomes p-polarized light after passing through the 1 / 4 glass plate (7), and then reaches the detection path through the polarization beam splitter (6). The push-pull filter plate (11) is used as a mask. The mask has a certain width to block the reflected beam and allow most of the detection light formed by the interference to enter the lens barrel (12) and be collected by the camera (13).

8. A reflective device for implementing the comprehensive single-molecule microscopic imaging method according to claim 4, characterized in that: It includes a laser (1), a first focusing lens (2), a first collimating lens (3), a second focusing lens (4), a second collimating lens (5), a polarizing beam splitter (6), a quarter glass slide (7), a mirror (8), an imaging objective lens (9), a sample to be tested (10), a push-pull filter plate (11), a sleeve lens (12), and a camera (13); The laser (1) is mounted on a temperature control base and driven by a desktop diode current controller and a temperature controller. The laser beam emitted from the laser (1) is sequentially incident on the first focusing lens (2), the first collimating lens (3), the second focusing lens (4), the second collimating lens (5), and the polarizing beam splitter (6). The incident linearly polarized light is reflected by the polarizing beam splitter 6 and then passes through a 1 / 4 glass plate (7) to become circularly polarized light. The reflector (8) is placed in front of the electric three-dimensional translation stage so that the incident light is perpendicular to the incident light; the scattered light of the sample (10) and the reflected light from the substrate surface become p-polarized light after passing through the 1 / 4 glass plate (7), and then reach the detection path through the polarization beam splitter 6. A push-pull filter plate (11) is used as a mask, which has a certain width to block the reflected light beam and allow most of the scattered light and interference-formed detection light to enter the lens barrel (12) and be collected by the camera (13).

9. A transmission-type device for implementing the comprehensive single-molecule microscopic imaging method according to claim 2, characterized in that: Includes a laser (1), a first focusing lens (2), a first collimating lens (3), a second focusing lens (4), a second collimating lens (5), a mirror (8), an illumination objective (14), a sample to be tested (10), an imaging objective (9), a push-pull filter (11), a sleeve lens (12), and a camera (13); The laser (1) is fixed on the temperature control base and driven by the desktop diode current controller and temperature controller; the laser beam emitted by the laser (1) is sequentially incident on the first focusing lens (2), the first collimating lens (3), the second focusing lens (4), and the second collimating lens (5); The reflector (8) is set in front of the electric three-dimensional translation stage. The angle of the reflector (8) is adjusted by rotating in three-dimensional space so that the incident light is incident at a supercritical angle. The light formed by the interference between the evanescent wave excited by the scattering of the glass substrate surface with a certain roughness and the scattered light of the sample to be tested (10) reaches the detection path through the imaging objective (9). The illumination objective (14) and the imaging objective (9) are of the same model so that the beam diameter and quality do not change significantly; the position of the electric displacement stage in three-dimensional space is adjusted so that the sample focal plane matches the imaging objective (9) used, and the detection light is further focused onto the back focal plane of the imaging objective (9); the push-pull filter (11) is used as a mask, and the push-pull filter (10) has a certain width to block the reflected beam and allow most of the interference-formed detection light to enter the barrel lens (12) and be collected by the camera (13).

10. A transmission-type device for implementing the comprehensive single-molecule microscopic imaging method according to claim 3, characterized in that: Includes a laser (1), a first focusing lens (2), a first collimating lens (3), a second focusing lens (4), a second collimating lens (5), a mirror (8), an illumination objective (14), a sample to be tested (10), an imaging objective (9), a push-pull filter (11), a sleeve lens (11), and a camera (12); The laser (1) is fixed on the temperature control base and driven by the desktop diode current controller and temperature controller; the laser beam emitted by the laser (1) is sequentially incident on the first focusing lens (2), the first collimating lens (3), the second focusing lens (4), and the second collimating lens (5); The reflector (6) is placed in front of the electric three-dimensional translation stage. The position of the reflector (6) is adjusted by rotating in three-dimensional space so that the incident light is incident at the surface plasmon resonance angle. The light formed by the interference between the evanescent wave excited by the surface of the metal substrate with a certain thickness and the scattered light of the sample 10 under test reaches the detection path through the imaging objective lens 9. The push-pull filter plate (11) is used as a mask. The push-pull filter plate (11) has a certain width to block the reflected beam and allow most of the detection light formed by the interference to enter the lens barrel (12) and be collected by the camera (13).

11. A transmission-type device for implementing the comprehensive single-molecule microscopic imaging method according to claim 4, characterized in that: Includes a laser (1), a first focusing lens (2), a first collimating lens (3), a second focusing lens (4), a second collimating lens (5), a mirror (8), an illumination objective (14), a sample to be tested (10), an imaging objective (9), a push-pull filter (11), a sleeve lens (12), and a camera (13); The laser (1) is fixed on the temperature control base and driven by the desktop diode current controller and temperature controller; the laser beam emitted by the laser (1) is sequentially incident on the first focusing lens (2), the first collimating lens (3), the second focusing lens (4), and the second collimating lens (5); The reflector (6) is placed in front of the electric three-dimensional translation stage so that the incident light is perpendicular to the surface. The light formed by the interference between the reflected light from the substrate surface and the scattered light from the sample (10) reaches the detection path through the imaging objective (9). The push-pull filter (11) is in the neutral position so that the sample scattered light, the substrate reflected light, and the detection light formed by their interference can enter the lens (12) and be collected by the camera (13).

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