A coaxial interferometric scattering microscopy device and method based on dual-wavelength illumination

Abstract

The application discloses a coaxial interference scattering microscopic imaging device and method based on double-wavelength illumination, belongs to the technical field of optical detection and imaging of nanoparticles, and the laser output by two lasers is combined by a dichroic mirror after being collimated, expanded and spatially filtered by respective lens groups. The combined light is converged by a lens, passes through a beam splitter, reaches the focal plane of an objective lens placed on a vertical lifting displacement table, and illuminates a sample in the form of wide-field illumination. The scattered light and reflected light of sample particles are collected by the objective lens, pass through the beam splitter and the dichroic mirror, are separated into two beams according to wavelengths, and are respectively collected by a collection system to form images. The double-wavelength interference scattering microscope system adopted by the application can effectively improve the precision and measurement range of the observed particle axial position signal, and has the advantages of a large field of view, high sensitivity, a simple device and easy compatibility.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of optical detection and imaging of nanoparticles, and in particular to a coaxial interference scattering microscopic imaging device and method based on dual-wavelength illumination. BACKGROUND

[0002] In the field of material science and biomedical research, obtaining extremely weak signals such as single molecules and single particles at the nanoscale is the key to revealing the micro mechanism and the prerequisite for detecting ligand-receptor binding and elucidating the spatial regulation mechanism of cell processes. However, existing detection techniques generally have limitations in sensitivity, sample applicability or biological safety, making it difficult to meet the needs of precise and efficient nanoscale observation.

[0003] Traditional optical microscopes are limited by the diffraction limit, and the detection resolution is usually not less than 300 nm, which cannot meet the observation needs of nanoscale micro phenomena. Techniques such as scanning electron microscopy (SEM) and atomic force microscopy (AFM) can achieve nanoscale detection, but have significant defects: on the one hand, such techniques have extremely low detection efficiency and are difficult to achieve real-time observation of dynamic processes; on the other hand, the detection environment (such as high vacuum and strong probe force) can easily cause irreversible damage to biological samples, and cannot be applied to dynamic research under physiological conditions.

[0004] Fluorescence microscopy is the main tool for single molecule and single particle optical microscopy at the present stage. Fluorescence microscopic detection can effectively filter the background light interference of non-target areas through the matching screening of excitation light and fluorescence signals of specific wavelengths, and has the characteristics of strong background signal suppression and excellent imaging effect. However, the fluorescence probe relied on by fluorescence microscopic detection has problems such as photobleaching and photofluorescence, which can seriously damage sensitive measurement results and cannot meet the needs of long-term and high-precision micro process observation. Moreover, fluorescence microscopic detection requires fluorescence labeling of the detection target, and the complex labeling process not only increases the complexity of experimental operation, but also may interfere with the original properties and interactions of the target molecules or particles.

[0005] Unlike fluorescence signals, scattering signals can be linearly increased by increasing the illumination intensity, and will not have light saturation. Compared with fluorescence microscopy, scattering-based imaging has the advantages of label-free, non-toxicity, no photobleaching, and unlimited shooting time. Dark field microscopy uses oblique illumination principle to make only the sample scattering light enter the objective lens, forming an imaging effect of "dark background + bright sample", and the sample preparation is simple and the equipment modification cost is low. However, limited by the Rayleigh scattering limit, the intensity of particle scattering signal decreases by the sixth power with the diameter, and the resolution of dark field microscopic imaging is limited, and the shallow depth of field makes it difficult to present three-dimensional structures.

[0006] The interference scattering microscopic imaging technology is a microscopic imaging technology based on scattering and interference. The interference is generated on an imaging plane by introducing a same-path reflected reference light and particle scattering light of a sample, and the sensitivity of detection is greatly improved. Because the interference scattering microscopic imaging technology can amplify the weak scattering signal through the interference between the scattering light and the reflected light, the signal obtained by detection is mainly the interference term formed by the scattering light and the reflected light, and the interference term is proportional to the volume of the particle. Compared with a dark field microscope which is also used for detecting the micro particles, the scattering signal detected by the interference scattering microscope is proportional to the square of the volume of the particle. Therefore, as the particle size becomes smaller, the scattering signal detected by the interference scattering microscope attenuates less, and thus the interference scattering microscope is more suitable for detecting the micro particles. However, the interference scattering microscopic imaging needs the background light to be received by the detector together with the signal, the contrast of the signal light is low, and the current interference scattering microscope generally performs illumination observation at a single wavelength angle. The axial position range of the particle which can be detected by the single-wavelength interference scattering microscope is very narrow, only 100nm-200nm, and the axial position of the particle cannot be accurately positioned.

[0007] Based on this, the application designs a coaxial interference scattering microscopic imaging device and method based on double-wavelength illumination which can effectively suppress the interference scattering background light and realize the axial position detection of the nanoparticles to solve the above problems. SUMMARY

[0008] In view of the above-mentioned defects of the prior art, the application provides a coaxial interference scattering microscopic imaging device and method based on double-wavelength illumination. Based on the optical response of the nanoparticles to two different wavelengths, accurate positioning of the nanoparticles is realized. Meanwhile, combined with the advantages of interference scattering enhancement, high-contrast measurement and detection of the nanoparticles are realized.

[0009] To achieve the above object, the application is implemented by the following technical solutions:

[0010] A coaxial interference scattering microscopic imaging device based on double-wavelength illumination, comprising a light source module, a detection light path module, an imaging module and a control and processing module. The light source module generates and combines two different wavelengths of horizontal outgoing laser beams, realizes wide-field illumination of a sample in the detection light path module, and collects the scattering light of the sample particles and the substrate reflected light through the objective lens of the detection light path module, then performs interference imaging through the imaging module. The control and processing module collects and processes images to obtain particle interference scattering signals.

[0011] The light source module comprises a single-wavelength laser A, a collimating lens A, a first beam-expanding lens A, a spatial filter A and a second beam-expanding lens A arranged in sequence in one path, a single-wavelength laser B, a collimating lens B, a first beam-expanding lens B, a spatial filter B and a second beam-expanding lens B arranged in sequence in another path, and a first dichroic mirror for combining the parallel polarized light output by the second beam-expanding lens A and the second beam-expanding lens B.

[0012] The imaging module comprises a second dichroic mirror, a collection first lens A, a CMOS camera A, a collection first lens B and a CMOS camera B. The light after the beam splitter enters the second dichroic mirror and is separated into two independent color lights. One color light enters the collection first lens A and is collected by the CMOS camera A, and the other color light enters the collection first lens B and is collected by the CMOS camera B, so as to obtain the interference imaging results of the scattered light of the detected sample particles and the reflected light of the substrate surface at two wavelengths.

[0013] Further, the center wavelengths of the single-wavelength laser A and the single-wavelength laser B are 633 nm and 532 nm, respectively.

[0014] Further, the probe light path module comprises a first mirror, a variable diaphragm, an illumination lens, a second mirror, a beam splitter, a vertical lifting displacement table, an objective lens and a sample table arranged in sequence.

[0015] Further, the sample table is composed of a coarse sample table and a piezoelectric displacement table. The piezoelectric displacement table is fixed above the coarse sample table, and the detected sample particles are arranged on the substrate of the piezoelectric displacement table.

[0016] Further, the light after the combination enters the variable diaphragm through the reflection of the first mirror, is converged by the illumination lens, enters the second mirror vertically upward, enters the beam splitter, the transmitted light is converged on the back focal plane of the objective lens through the vertical lifting displacement table on which the objective lens is placed, and the parallel wide-field light spot after the objective lens illuminates the sample placed on the coarse sample table and the piezoelectric displacement table.

[0017] Further, the control and processing module comprises a computer system and a software control system for controlling the vertical lifting displacement table, the piezoelectric displacement table, the CMOS camera A and the CMOS camera B; the software control system is used for controlling the sample table to change the three-dimensional position of the detected sample in real time, adjusting the vertical lifting displacement table to facilitate the focusing of the objective lens, and controlling the CMOS camera A and the CMOS camera B to record the observation signals formed by the interference of the scattered light of the detected sample particles and the reflected light of the substrate surface.

[0018] Further, the metal mask plate A and the metal mask plate B are both composed of a metal layer and a non-metal layer. The metal layer is larger than the spot size of the reflected light of the substrate surface and smaller than the spot size of the scattered light of the detected sample particles.

[0019] Further, the imaging module further comprises a collecting second lens A, a metal mask plate A, a collecting third lens A, a collecting second lens B, a metal mask plate B and a collecting third lens B; the scattered light of the detected sample particles and the reflected light of the substrate surface reflected by the beam splitter are separated into two independent color lights by the second dichroic mirror, one color light enters the collecting first lens A, the collecting second lens A, the metal mask plate A and the collecting third lens A in sequence and is collected by the CMOS camera A, and the other color light enters the collecting first lens B, the collecting second lens B, the metal mask plate B and the collecting third lens B in sequence and is collected by the CMOS camera B, so that the interference imaging results of the scattered light of the detected sample particles and the reflected light of the substrate surface at two wavelengths are obtained.

[0020] Further, the beam splitter divides the light propagating vertically upward into reflected light and transmitted light with equal intensity.

[0021] In order to better achieve the purpose of the present application, the present application further provides an observation method of a coaxial interference scattering microscopic imaging device based on double-wavelength illumination, comprising the following steps:

[0022] Step one, the single-wavelength laser A and the single-wavelength laser B respectively emit linearly polarized light of two different wavelengths, the linearly polarized light emitted by the single-wavelength laser A sequentially passes through the collimating lens A, the beam expanding first lens A, the spatial filter A and the beam expanding second lens A, the linearly polarized light emitted by the single-wavelength laser B sequentially passes through the collimating lens B, the beam expanding first lens B, the spatial filter B and the beam expanding second lens B, the linearly polarized light of different wavelengths is adjusted into parallel polarized light with a suitable spot size, and then the parallel polarized light collimated and expanded by the beam expanding second lens A and the beam expanding second lens B is combined by the first dichroic mirror;

[0023] Step two, the combined light is reflected by the first mirror, enters the variable diaphragm and is converged by the illumination lens in sequence, and then vertically propagates upward into the beam splitter through the second mirror, the transmitted light is converged on the back focal plane of the objective lens through the vertical lifting displacement table on which the objective lens is placed, and parallel wide-field light spot illumination is generated after the objective lens to illuminate the sample placed on the coarse sample table and the piezoelectric displacement table;

[0024] Step three, the scattered light of the detected sample particles and the reflected light of the substrate surface enter the objective lens together, and then enter the beam splitter;

[0025] Step four, the scattered light of the detected sample particles reflected by the beam splitter and the reflected light of the substrate surface enter the second dichroic mirror and are separated into two independent color lights, one color light enters the collection first lens A, the collection second lens A, the metal mask plate A, the collection third lens A in turn and is collected by the CMOS camera A, the other color light enters the collection first lens B, the collection second lens B, the metal mask plate B, the collection third lens B in turn and is collected by the CMOS camera B, and then the interference imaging results of the scattered light of the detected sample particles and the reflected light of the substrate surface under two wavelengths are obtained;

[0026] Step five, the three-dimensional position of the detected sample is adjusted in real time through the adjustment and processing module, and the collected images are processed to obtain the interference scattering pattern.

[0027] Compared with the prior art, the present application has the following advantages: 1) the present application can observe the difference of the interference scattering pattern under two wavelengths, thereby accurately identifying the particles in a wider axial range.

[0028] 2) the present application device is simple, based on the working principle of the interference scattering microscope, and the dual-wavelength interference scattering microscopic imaging system has the advantages of rapid measurement, high sensitivity, etc. BRIEF DESCRIPTION OF DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creating laborious work.

[0030] Figure 1 The structure schematic diagram of the coaxial interference scattering microscopic imaging device based on dual-wavelength illumination of the present application embodiment one.

[0031] Figure 2 The structure schematic diagram of the coaxial interference scattering microscopic imaging device based on dual-wavelength illumination of the present application embodiment two.

[0032] Figure 3 It is a simulation comparison diagram of the interference scattering signal of the particle at the axial 0nm position under two different wavelength conditions.

[0033] Figure 4 It is a simulation comparison diagram of the interference scattering signal of the particle at the axial 200nm position under two different wavelength conditions.

[0034] Figure 5 It is a simulation comparison diagram of the interference scattering signal of the particle at the axial 400nm position under two different wavelength conditions.

[0035] Figure 6 The simulation contrast diagram of interference scattering signal of the particle at the axial 600nm position under two different wavelength conditions.

[0036] Figure 7 The simulation intensity change curve of interference scattering signal with the axial position change of the particle for the experiment using only a single wavelength.

[0037] Figure 8 The simulation intensity curve contrast result of interference scattering signal with the axial position change of the particle for the experiment using two wavelengths.

[0038] Figure 9 The experimental result diagram of interference scattering microscopic imaging of polystyrene nanoparticles with a particle size of 100nm.

[0039] The labels in the figure respectively represent: 1-single wavelength laser A, 2-single wavelength laser B, 3-collimating lens A, 4-collimating lens B, 5-expanding first lens A, 6-expanding first lens B, 7-space filter A, 8-space filter B, 9-expanding second lens A, 10-expanding second lens B, 11-first dichroic mirror, 12-first mirror, 13-variable diaphragm, 14-illumination lens, 15-second mirror, 16-beam splitter, 17-vertical lifting displacement table, 18- objective lens, 19-coarse adjustment sample table, 20-piezoelectric displacement table, 21-collecting first lens A, 22-collecting second lens A, 23-metal mask plate A, 24-collecting third lens A, 25-CMOS camera A, 26-second dichroic mirror, 27-collecting first lens B, 28-collecting second lens B, 29-metal mask plate B, 30-collecting third lens B, 31-CMOS camera B, 32-computer system. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are 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 labor fall within the scope of protection of the present application.

[0041] 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.

[0042] Embodiment one: please refer to the description of the accompanying drawings Figure 1 A coaxial interference scattering microscopic imaging device based on dual-wavelength illumination, comprising a light source module, a detection light path module, an imaging module, and a control and processing module; wherein the light source module generates and combines two different wavelength horizontal exit laser beams, the detection light path module realizes wide-field illumination of the sample, the scattered light and the substrate reflected light of the sample particles are collected by the objective lens 18 of the detection light path module, and then interference imaging is performed by the imaging module; the control and processing module collects and processes images to obtain particle interference scattering signals.

[0043] In this embodiment, the light source module includes a single-wavelength laser A1, a collimating lens A3, a first beam-expanding lens A5, a spatial filter A7, and a second beam-expanding lens A9 arranged in sequence in one path, a single-wavelength laser B2, a collimating lens B4, a first beam-expanding lens B6, a spatial filter B8, and a second beam-expanding lens B10 arranged in sequence in another path, and a first dichroic mirror 11 for combining the parallel polarized light output by the second beam-expanding lenses A9 and B10;

[0044] The single-wavelength lasers A1 and B2 respectively emit linearly polarized light of two different wavelengths, the linearly polarized light emitted by the single-wavelength laser A1 passes through the collimating lens A3, the first beam-expanding lens A5, the spatial filter A7, and the second beam-expanding lens A9 in sequence, and the linearly polarized light emitted by the single-wavelength laser B2 passes through the collimating lens B4, the first beam-expanding lens B6, the spatial filter B8, and the second beam-expanding lens B10 in sequence, so as to adjust the linearly polarized light of different wavelengths into parallel polarized light with a suitable spot size, and then the first dichroic mirror 11 combines the parallel polarized light output by the second beam-expanding lenses A9 and B10, and then enters the detection light path module.

[0045] In this embodiment, the center wavelengths of the single-wavelength lasers A1 and B2 are 633 nm and 532 nm respectively, and the output is linearly polarized light, which passes through the respective collimating and beam-expanding lenses, and the spatial filter has a constraining effect on the mode of the output light.

[0046] In the embodiment, the probe light path module comprises a first mirror 12, a variable diaphragm 13, an illumination lens 14, a second mirror 15, a beam splitter 16, a vertical lifting displacement table 17, an objective lens 18 and a sample table arranged in sequence.

[0047] The objective lens 18 is mounted on the vertical lifting displacement table 17, and the vertical lifting displacement table 17 is controlled by a computer system 32, so that the lifting of the vertical lifting displacement table 17 can be precisely adjusted to facilitate focusing of the objective lens 18.

[0048] In the embodiment, the numerical aperture of the objective lens 18 is 1.45, the magnification is 100, and the working medium is refractive index matching oil.

[0049] The sample table is composed of a coarse sample table 19 and a piezoelectric displacement table 20, and the piezoelectric displacement table 20 is fixed above the coarse sample table 19. A substrate for testing is placed on the piezoelectric displacement table 20, and the substrate is provided with a sample particle to be detected.

[0050] In the embodiment, the substrate is an experimental glass sheet carrying the sample particle to be detected, and the substrate reflection light is the reflection light reflected by the lower surface of the glass sheet after the light vertically incident through the objective lens.

[0051] The coarse sample table 19 can manually adjust the X and Y direction movement of the sample, and the piezoelectric displacement table 20 is controlled by the computer system 32, so that the X, Y and Z three-axis movement of the sample can be realized, and the movement precision is nanometer level.

[0052] After the light is combined, it is reflected by the first mirror 12 and then enters the variable diaphragm 13, the illumination lens 14, and then vertically upwardly propagates into the beam splitter 16 through the second mirror 15. At this time, the light beam is divided into two beams according to a certain intensity ratio, one of which is reflected, and the other is transmitted to continue to propagate to form wide-field illumination on the sample. The transmitted light converges at the back focal plane of the objective lens 18 through the vertical lifting displacement table 17 on which the objective lens 18 is placed, and then produces a parallel wide-field light spot through the objective lens 18 to irradiate the surface of the coarse sample table 19 and the piezoelectric displacement table 20 to generate a wide-field illumination light spot. The scattered light of the sample particle to be detected and the reflection light of the substrate surface enter the objective lens 18 together, then enter the imaging module after passing through the beam splitter 16.

[0053] In the embodiment, the beam splitter 16 divides the vertically upwardly propagating light into reflected light and transmitted light with equal intensity. The reflected light can be monitored by a power meter for power change, and the transmitted light vertically upwardly propagates and converges at the focal point of the objective lens 18, and then passes through the objective lens 18 to illuminate the sample table.

[0054] In the embodiment, the imaging module comprises a second dichroic mirror 26, a collection first lens A 21, a CMOS camera A 25, a collection first lens B 27 and a CMOS camera B 31.

[0055] The light after the beam splitter 16 enters the second dichroic mirror 26 and is separated into two independent color lights, one color light enters the collection first lens A21 and is collected by the CMOS camera A25, and the other color light enters the collection first lens B27 and is collected by the CMOS camera B31, and the CMOS camera A25 and the CMOS camera B31 are controlled by the computer system 32, and then the interference imaging results of the scattered light of the detected sample particles and the reflected light of the substrate surface at two wavelengths are obtained.

[0056] As shown in Figure 9 , the polystyrene nanoparticles with a particle size of 100 nm are selected to prepare the sample, and the experimental results of the interference scattering microscopic imaging of the sample can be observed, and the interference ring formed by the particle scattering light and the substrate reflection light can be clearly observed.

[0057] In this embodiment, the regulation and processing module includes the computer system 32 and the software control system for controlling the vertical lifting displacement table 17, the piezoelectric displacement table 20, the CMOS camera A25 and the CMOS camera B31. The software control system is used to control the sample table to change the three-dimensional position of the detected sample in real time, adjust the vertical lifting displacement table 17 to focus the objective lens 18, and control the CMOS camera A25 and the CMOS camera B31 to record the observation signals formed by the interference of the scattered light of the detected sample particles and the reflected light of the substrate surface.

[0058] As shown in Figures 3-6 , the FDTD and Matlab are used to write simulation codes, and the images of the scattered light and the substrate reflection light generated by the interference of the polystyrene nanoparticles with a particle size of 100 nm at different axial heights when irradiated by 532 nm and 633 nm respectively are simulated. It can be observed that under different wavelength conditions, the center intensity and the pattern shape of the interference signals generated by the particles at the same height are different.

[0059] As shown in Figure 7 , by processing the simulated interference scattering signals, the intensity change curves of the simulated interference scattering signals with the change of the axial position of the particles are extracted, which are obtained by using a single wavelength for the experiment; as shown in Figure 8 , the intensity curves of the simulated interference scattering signals with the change of the axial position of the particles are compared by using two wavelengths for the experiment. It can be found that if a single wavelength is used for the experiment, the interference scattering signals of the particles will change periodically with the axial height of the particles, and therefore the axial height of the particles cannot be effectively determined; but by comparing two different wavelength signals, it can be found that the introduction of another wavelength of light can provide an effective reference for the observation of the axial height of the particles. As shown in Figures 3-6As shown, by observing the difference between the interference scattering images corresponding to two different wavelengths at the same height, and the range of the difference being the least common multiple of the two wavelengths, the measurement range is greatly improved to the micrometer level, thus enabling the effective inference of the axial position of the particles.

[0060] In summary, unlike traditional interferometric scattering microscope systems, the dual-wavelength interferometric scattering microscope system used in this invention can effectively improve the accuracy and measurement range of particle axial position signals, while also possessing advantages such as a large field of view, high sensitivity, and simple and compatible device.

[0061] Example 2: Figure 2 As shown, the imaging module in this embodiment differs from that in Embodiment 1, but the rest is the same as in Embodiment 1. The imaging module in this embodiment includes a second dichroic mirror 26, a collection lens A21, a collection lens A22, a metal mask A23, a collection lens A24, and a CMOS camera A25 arranged sequentially in one path, and a collection lens B27, a collection lens B28, a metal mask B29, a collection lens B30, and a CMOS camera B31 arranged sequentially in another path.

[0062] In this embodiment, metal mask A23 is placed at the focal point where the second collecting lens A22 and the third collecting lens A24 coincide. Metal mask B29 is placed at the focal point where the second collecting lens B28 and the third collecting lens B30 coincide. At this focal plane, the scattered light from the sample particles being detected is parallel light, while the reflected light from the substrate surface converges into a very small spot.

[0063] In this embodiment, both metal masks A23 and B29 are composed of metal and non-metal layers. The design aims to attenuate the reflected light from the substrate surface to the same order of magnitude as the scattered light from the sample particles being detected, thereby improving the contrast of the collected signal. The metal layer is formed by vapor deposition using magnetron sputtering. The deposited metal needs to have strong reflectivity in the laser's operating wavelength range to better attenuate the reflected light from the substrate surface; gold or silver are suitable examples. The thickness of the metal layer affects the beam transmittance and needs to be designed according to the incident light of different wavelengths and intensities.

[0064] In this embodiment, the sizes of metal mask A23 and metal mask B29 depend on the spot size of the scattered light from the sample particles being detected and the reflected light from the substrate surface at the focal plane of collecting second lens A22, collecting second lens B28, collecting third lens A24, and collecting third lens B30. The metal layer portion should be larger than the spot size of the reflected light from the substrate surface but smaller than the spot size of the scattered light from the sample particles being detected.

[0065] In the imaging module, the scattered light of the detected sample particles and the reflected light of the substrate surface reflected by the beam splitter 16 are separated into two independent color lights by the second dichroic mirror 26, one of the color lights is sequentially collected by the collection first lens A21, the collection second lens A22, the metal mask plate A23, the collection third lens A24, and then collected by the CMOS camera A25, and the other color light is sequentially collected by the collection first lens B27, the collection second lens B28, the metal mask plate B29, the collection third lens B30, and then collected by the CMOS camera B31, and then the interference imaging results of the scattered light of the detected sample particles and the reflected light of the substrate surface under two wavelengths are obtained.

[0066] The scattered light of the detected sample particles and the reflected light of the substrate surface passing through the objective lens 18 are separated into two independent color lights by the second dichroic mirror 26, and then collected by the collection first lens A21 and the collection first lens B27, and then converged by the collection second lens A22 and the collection second lens B28 on the spectrum plane. The interference light field is modulated by the metal mask plate A23 and the metal mask plate B29 on the spectrum plane, so that the background reflected light can be effectively suppressed and the image contrast can be improved. The modulated light field passes through the collection third lens A24 and the collection third lens B30 and is imaged on the focal plane of the CMOS camera A25 and the CMOS camera B31, respectively.

[0067] The metal mask plate A23 and the metal mask plate B29 have certain distribution characteristics, the center is low transmittance, and the periphery is high transmittance, so that a high proportion of scattered light and a low proportion of background reflected light can be obtained.

[0068] The dual-wavelength interference scattering microscopic imaging system of the present application has the following technical effects relative to the conventional interference scattering microscope system:

[0069] 1) The present application can observe the difference of the interference scattering pattern under two wavelengths, so that the particles in a wider axial range can be accurately identified.

[0070] 2) The device of the present application is simple, based on the working principle of the interference scattering microscope, and the dual-wavelength interference scattering microscopic imaging system has the advantages of rapid measurement, high sensitivity, etc.

[0071] Embodiment three: based on the observation method of the coaxial interference scattering microscopic imaging device based on dual-wavelength illumination, comprising the following steps:

[0072] Step 1: Single-wavelength lasers A1 and B2 emit two linearly polarized lights of different wavelengths. The linearly polarized light emitted by single-wavelength laser A1 passes through collimating lens A3, beam expander A5, spatial filter A7, and beam expander A9 in sequence. The linearly polarized light emitted by single-wavelength laser B2 passes through collimating lens B4, beam expander B6, spatial filter B8, and beam expander B10 in sequence. The linearly polarized lights of different wavelengths are adjusted into parallel polarized lights of appropriate spot size. Then, the parallel polarized lights collimated and expanded by beam expander A9 and beam expander B10 are combined by first dichroic mirror 11.

[0073] Step 2: After the beam is combined, the light is reflected by the first reflecting mirror 12 and enters the variable aperture 13 in sequence. It is then focused by the illumination lens 14 and then propagates vertically upward through the second reflecting mirror 15 into the beam splitter 16. The transmitted light is focused on the back focal plane of the objective lens 18 by the vertical lifting and lowering stage 17 on which the objective lens 18 is placed. After passing through the objective lens 18, a parallel wide field light spot is generated to illuminate the sample placed on the coarse adjustment sample stage 19 and the piezoelectric displacement stage 20.

[0074] Step 3: The scattered light from the sample particles being tested and the reflected light from the substrate surface enter the objective lens 18 together, and then enter the beam splitter 16.

[0075] Step 4: The scattered light from the sample particles being tested, reflected by the beam splitter 16, and the reflected light from the substrate surface enter the second dichroic mirror 26 and are separated into two independent beams of light. One beam of light enters sequentially through the first collecting lens A21, the second collecting lens A22, the metal mask A23, and the third collecting lens A24 before being collected by the CMOS camera A25. The other beam of light enters sequentially through the first collecting lens B27, the second collecting lens B28, the metal mask B29, and the third collecting lens B30 before being collected by the CMOS camera B31. This process yields the interference imaging results of the scattered light from the sample particles being tested and the reflected light from the substrate surface at two wavelengths.

[0076] Step 5: The three-dimensional position of the sample under test is controlled in real time through the control and processing module, and the acquired image is processed to obtain the interference scattering pattern.

[0077] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions will not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A coaxial interferometric scattering microscopic imaging device based on dual-wavelength illumination, comprising a light source module, a probe optical path module, an imaging module, and a control and processing module; characterized in that: The light source module generates and combines two horizontally emitted lasers of different wavelengths, which are then used to illuminate the sample in a wide field in the detection optical path module. The scattered light from the sample particles and the reflected light from the substrate are collected by the objective lens (18) of the detection optical path module and then subjected to interference imaging by the imaging module. The control and processing module acquires and processes the image to obtain the particle interference scattering signal. The light source module includes a single-wavelength laser A (1), collimating lens A (3), beam expander first lens A (5), spatial filter A (7) and beam expander second lens A (9) arranged in sequence on one side; a single-wavelength laser B (2), collimating lens B (4), beam expander first lens B (6), spatial filter B (8) and beam expander second lens B (10) arranged in sequence on the other side; and a first dichroic mirror (11) that combines the parallel polarized light output from the beam expander second lens A (9) and the beam expander second lens B (10). The imaging module includes a second dichroic mirror (26), a first collecting lens A (21), a CMOS camera A (25), a first collecting lens B (27), and a CMOS camera B (31). After passing through the beam splitter (16), the light enters the second dichroic mirror (26) and is separated into two independent beams of light. One beam of light enters the first collecting lens A (21) and is collected by the CMOS camera A (25), while the other beam of light enters the first collecting lens B (27) and is collected by the CMOS camera B (31). The result is obtained by interfering the scattered light of the sample particles under two wavelengths with the reflected light of the substrate surface. The detection optical path module includes a first reflector (12), a variable aperture (13), an illumination lens (14), a second reflector (15), a beam splitter (16), a vertical lifting displacement stage (17), an objective lens (18), and a sample stage arranged in sequence; the sample stage consists of two parts: a coarse adjustment sample stage (19) and a piezoelectric displacement stage (20). The piezoelectric displacement stage (20) is fixed above the coarse adjustment sample stage (19), and the sample particles to be detected are provided on the base of the piezoelectric displacement stage (20); After the beam is combined, the light is reflected by the first reflecting mirror (12) and enters the variable aperture (13) in sequence. It is then focused by the illumination lens (14), and then passes through the second reflecting mirror (15) and propagates vertically upward into the beam splitter (16). The transmitted light passes through the vertical lifting displacement stage (17) on which the objective lens (18) is placed and converges on the back focal plane of the objective lens (18). After passing through the objective lens (18), a parallel wide field light spot is generated to illuminate the sample placed on the coarse adjustment sample stage (19) and the piezoelectric displacement stage (20).

2. The coaxial interferometric scattering microscopy imaging device based on dual-wavelength illumination according to claim 1, characterized in that, The center wavelengths of the single-wavelength laser A (1) and the single-wavelength laser B (2) are 633nm and 532nm, respectively.

3. The coaxial interferometric scattering microscopy imaging device based on dual-wavelength illumination according to claim 2, characterized in that, The control and processing module includes a computer system (32) and a software control system for controlling the vertical lifting stage (17), piezoelectric stage (20), CMOS camera A (25), and CMOS camera B (31); for controlling the sample stage to change the three-dimensional position of the sample under test in real time, adjusting the vertical lifting stage (17) to facilitate the focusing of the objective lens (18), and controlling the CMOS camera A (25) and CMOS camera B (31) to capture and record the observation signal formed by the interference of the scattered light of the sample particles and the reflected light of the substrate surface.

4. The coaxial interferometric scattering microscopy imaging device based on dual-wavelength illumination according to claim 3, characterized in that, Metal mask A (23) and metal mask B (29) are both composed of metal and non-metal layers; the metal layer portion is larger than the spot size of the reflected light from the substrate surface and smaller than the spot size of the scattered light from the sample particles being detected.

5. The coaxial interferometric scattering microscopy imaging device based on dual-wavelength illumination according to any one of claims 1 to 3, characterized in that, The imaging module also includes a second collecting lens A (22), a metal mask A (23), a third collecting lens A (24), a second collecting lens B (28), a metal mask B (29), and a third collecting lens B (30). The scattered light of the sample particles being tested reflected by the beam splitter (16) and the reflected light from the substrate surface enter the second dichroic mirror (26) and are separated into two independent beams of light. One beam of light enters the first collecting lens A (21), the second collecting lens A (22), the metal mask A (23), and the third collecting lens A (24) in sequence and is then collected by the CMOS camera A (25). The other beam of light enters the first collecting lens B (27), the second collecting lens B (28), the metal mask B (29), and the third collecting lens B (30) in sequence and is then collected by the CMOS camera B (31). Thus, the result of interference imaging of the scattered light of the sample particles being tested and the reflected light from the substrate surface at two wavelengths is obtained.

6. The coaxial interferometric scattering microscopy imaging device based on dual-wavelength illumination according to claim 5, characterized in that, The beam splitter (16) splits the vertically upward-propagating light into reflected light and transmitted light of equal intensity.

7. An observation method for a coaxial interferometric scattering microscopic imaging device based on dual-wavelength illumination according to claim 6, comprising the following steps: Step 1: Single-wavelength laser A (1) and single-wavelength laser B (2) emit two linearly polarized lights of different wavelengths respectively. The linearly polarized light emitted by single-wavelength laser A (1) passes through collimating lens A (3), beam expander first lens A (5), spatial filter A (7), and beam expander second lens A (9) in sequence. The linearly polarized light emitted by single-wavelength laser B (2) passes through collimating lens B (4), beam expander first lens B (6), spatial filter B (8), and beam expander second lens B (10) in sequence. The linearly polarized lights of different wavelengths are adjusted into parallel polarized lights of appropriate spot size. Then, the parallel polarized lights collimated and expanded by beam expander second lens A (9) and beam expander second lens B (10) are combined by the first dichroic mirror (11). Step 2: After the beam is combined, the light is reflected by the first reflecting mirror (12) and enters the variable aperture (13) in sequence. It is then focused by the illumination lens (14) and then propagates vertically upward through the second reflecting mirror (15) into the beam splitter (16). The transmitted light is focused on the back focal plane of the objective lens (18) by the vertical lifting displacement stage (17) on which the objective lens (18) is placed. After passing through the objective lens (18), a parallel wide field light spot is generated to illuminate the sample placed on the coarse adjustment sample stage (19) and the piezoelectric displacement stage (20). Step 3: The scattered light from the sample particles being tested and the reflected light from the substrate surface enter the objective lens (18) together, and then enter the beam splitter (16). Step 4: The scattered light of the sample particles being tested reflected by the beam splitter (16) and the reflected light from the substrate surface enter the second dichroic mirror (26) and are separated into two independent beams of light. One beam of light enters the first collecting lens A (21), the second collecting lens A (22), the metal mask plate A (23), and the third collecting lens A (24) in sequence and is then collected by the CMOS camera A (25). The other beam of light enters the first collecting lens B (27), the second collecting lens B (28), the metal mask plate B (29), and the third collecting lens B (30) in sequence and is then collected by the CMOS camera B (31). Thus, the result of interference imaging of the scattered light of the sample particles being tested and the reflected light from the substrate surface at two wavelengths is obtained. Step 5: The three-dimensional position of the sample under test is controlled in real time through the control and processing module, and the acquired image is processed to obtain the interference scattering pattern.

Images