Single-point rotation scanning dark-field optical microscopy imaging system and method

Abstract

The present invention discloses a single-point rotation scanning dark field optical microscopy imaging system and method, belonging to the field of optical microscopy imaging technology, comprising: a sample stage; an object environment for observing the sample stage; a camera for collecting the sample image through the object environment; a dark field illuminator for providing illumination light to illuminate the sample stage; the dark field illuminator comprises: an illumination light source for providing illumination light; a rotatable baffle for adjusting the illumination light of the illumination light source to illuminate the sample stage at a set angle; the rotatable baffle comprises: a first baffle and a second baffle that cooperates with the first baffle to form an adjustable gap; the single-point rotation scanning dark field optical microscopy imaging system and method reveal the variation law of scattering intensity between different orientations, establish the correlation between the scattering intensity and the morphology, construct a fast and high-throughput morphology recognition method for static particles, and provide a new solution for obtaining particle morphology distribution information.

Description

Technical Field

[0001] The invention belongs to the technical field of optical microscopic imaging, and in particular relates to a single-point rotation scanning dark-field optical microscopic imaging system and method. Background Art

[0002] Dark-field scattering microscopy is a label-free, fast-imaging optical imaging technique. The sample is acted on by an annular oblique illumination mode, which effectively prevents the illumination light from entering the objective lens, significantly reduces the background signal, and can detect weak scattering signals emitted by particles as small as 5 nm. It has been widely used in the detection of single nanoparticles. However, limited by the optical diffraction limit, this technology can only obtain the scattering signal of nanoparticles and cannot directly identify their morphological characteristics. However, according to the Mie scattering theory: the scattering intensity of a particle depends on the size of its scattering cross section at different orientations, and the scattering cross section of the particle is closely related to its microscopic morphology. Therefore, by collecting the scattering intensity of the particle at different orientations, its microscopic morphological information can be obtained. Existing studies have found that when the scattering signal of particles in a free solution changes, it is found that due to the Brownian motion of the particles, the particles flip and cause the orientation to change, and the recorded scattering signal also changes. The simple classification of spherical and rod-shaped particles can be preliminarily realized, providing a new idea for the rapid classification of particle morphology.

[0003] However, when the particles are stationary, the conventional dark-field microscopy technology cannot obtain the scattering intensity at different orientations because the particles cannot flip and cause orientation changes. Therefore, it is urgent to develop a new single-point rotation scanning dark-field optical microscopy system and method to solve the existing problems. Summary of the invention

[0004] The object of the present invention is to provide a single-point rotation scanning dark-field optical microscopy imaging system and method to solve the problem that traditional dark-field microscopy imaging technology cannot obtain the scattering intensity of static particles in different orientations.

[0005] To achieve the above object, the present invention provides the following technical solution: a single-point rotation scanning dark-field optical microscopy imaging system, comprising:

[0006] Sample table;

[0007] an objective lens for observing the sample stage;

[0008] a camera for collecting the sample image through the objective lens;

[0009] a dark field illuminator for providing illumination light to illuminate the sample stage;

[0010] The dark field illuminator comprises:

[0011] an illumination source for providing illumination light;

[0012] Adjusting the illumination light of the illumination light source to irradiate the rotatable baffle of the sample stage at a set angle;

[0013] The rotatable baffle comprises:

[0014] A first baffle plate and a second baffle plate that cooperates with the first baffle plate to form an adjustable gap.

[0015] Preferably, the dark field illuminator further comprises: an automatic rotator for controlling the angle of the rotatable baffle.

[0016] Preferably, the adjustable angle range of the notch is 1 degree to 30 degrees.

[0017] Preferably, the rotatable baffle has a circular structure with a radius of 2 cm to 10 cm.

[0018] Preferably, the automatic rotator comprises:

[0019] a drive gear connected to the rotatable baffle;

[0020] A servo motor for receiving control instructions to drive the driving gear to rotate according to a set speed and direction;

[0021] A controller for receiving preset program instructions and controlling the servo motor to run at a set speed and direction;

[0022] A power supply connected to the servo motor and the controller;

[0023] Fixture for fixing the servo motor.

[0024] Wherein, the serrated outer edge of the rotatable baffle is meshed with the driving gear.

[0025] Preferably, the fixing device includes a mounting groove for fixing the servo motor and a base fixed to the mounting groove, the output end of the servo motor is connected to the driving gear through a transmission shaft, a connecting rod is provided on the outside of the transmission shaft, the connecting rod is connected to the servo motor, and one end of a support rod is fixed to the connecting rod, and the other end of the support rod is connected to the base.

[0026] Preferably, the connecting rod is also connected to a chassis, a rotatable baffle is installed in the cavity of the chassis, the serrated outer edge of the rotatable baffle is meshed with the driving gear, the transmission shaft connected to the driving gear extends into the connecting rod and is connected to the output end of the servo motor, and the length of the connecting rod is less than the length of the transmission shaft.

[0027] The present invention further provides an imaging method of a single-point rotation scanning dark-field optical microscopic imaging system, which is characterized by comprising:

[0028] Starting the lighting source, the controller and the servo motor and rotating the rotatable baffle to an initial position;

[0029] According to the sample characteristics and experimental requirements, the rotation speed of 1-100 r / s is set in the controller. The controller drives the servo motor to drive the driving gear to rotate according to the preset parameters. The light emitted by the illumination light source passes through the gap of the rotatable baffle to form an illumination beam. The rotation of the rotatable baffle is controlled by the automatic rotator to change the incident angle of the single-point illumination beam, thereby scanning different areas of the sample. The particles in the sample generate scattered light signals under illumination at different incident angles. The scattered light signals are collected to obtain scattering signals of particles with different orientations.

[0030] According to the collected scattered light signal, the scanning parameters and scattering data are combined to display the particle information.

[0031] Preferably, the filter membrane is transparentized before use, comprising: preparing a high refractive index ZnCl2 solution according to the refractive index of the filter membrane material, adjusting its concentration to make it close to the refractive index of the filter membrane material, and soaking the filter membrane with a solution with a close refractive index.

[0032] Preferably, the method for regulating the concentration includes: the concentration range of the dilution solution is 8 M-12 M.

[0033] The technical effects and advantages of the present invention are as follows: the single-point rotation scanning dark field optical microscopy imaging system and method introduce a rotatable baffle in the dark field illuminator, which can directionally block the full-angle illumination and only allow the illumination light to pass through at a specific angle, so that the illumination light source is switched from the original annular full-angle illumination mode to the single-point rotation scanning illumination mode, thereby obtaining the scattering signal of the particles on the filter membrane at a specific orientation, and by rotating the baffle, the generated single-point rotation scanning illumination light source is directionally rotated and irradiated onto the sample, and the illumination intensity and wavelength of the light source are optimized to improve the scattering signal of the particles, and the scattering background of the filter membrane is reduced in combination with the transparent treatment of the filter membrane, the signal-to-noise ratio of the particle scattering signal is improved, the scattered signals generated by the particles on the filter membrane at different orientations are obtained, the variation law of the scattering intensity between different orientations is revealed, the correlation between it and the morphology is established, and a fast and high-throughput morphological recognition method for static particles is constructed, providing a new solution for obtaining particle morphological distribution information. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a working principle diagram of the single-point rotation scanning dark field scattering microscopic imaging system of the present invention;

[0035] Figure 2(a) is a cross-sectional view of the full-angle illumination structure of a conventional dark-field microscope of the present invention;

[0036] Figure 2 (b) is a schematic diagram of the structure of the rotatable baffle of the present invention;

[0037] Figure 2 (c) is a schematic diagram of the structure of the single-point rotation scanning dark field microscopic imaging system of the present invention;

[0038] Figure 2 (d) is a schematic diagram of the single-point rotating scanning dark field illuminator of the present invention scanning the particles on the filter membrane to obtain dark field scattering signals in different orientations;

[0039] Figure 3 (a) is a SEM image of the filter membrane of the present invention (5 × 5 µm);

[0040] Figure 3 (b) is a picture of the dry filter membrane of the present invention (top) and its dark field scattering background picture (bottom);

[0041] Figure 3 (c) is a picture of the filter membrane after soaking in ZnCl2 solution (top) and its dark field scattering background picture (bottom);

[0042] Figure 4 (a) is a single-point rotation scanning dark-field optical microscopy imaging system of the present invention;

[0043] Figure 4 (b) High-throughput dark-field imaging using a 5x objective lens (large field of view, 2.5 × 2.5 mm) for the present invention;

[0044] Figure 4 (c) is a partial enlarged view of Figure (b) of the present invention (the bright spot of a single particle is still clearly visible);

[0045] Figure 5 (a) is a SEM image (5 × 5 μm) of the filter membrane of the present invention and a dark field image (150 × 150 μm) of the filter membrane loaded with particles;

[0046] Figure 5 (b) is the dry state of the present invention;

[0047] Figure 5 (c) being infiltrated with pure water of the present invention;

[0048] Figure 5 (d) is the infiltration of ZnCl2 solution of the present invention; the bright spot is the dark field scattering imaging of 500 nm polystyrene (PS) nanospheres;

[0049] Figure 6A schematic diagram of the scattering signal intensity of spherical particles, rod-shaped particles and triangular flake particles obtained by the system of the present invention;

[0050] Figure 7 It is a structural schematic diagram of the rotatable baffle and the automatic rotator of the present invention;

[0051] Figure 8 It is a schematic structural diagram of the rotatable baffle, the automatic rotator and the objective lens of the present invention.

[0052] In the figure: 100, sample stage; 200, objective lens; 400, dark field illuminator; 410, illumination light source; 420, rotatable baffle; 421, first baffle; 422, second baffle; 423, serrated outer edge; 430, automatic rotator; 431, drive gear; 432, servo motor; 435, fixing device; 436, support rod; 437, connecting rod; 438, transmission shaft; 439, chassis. DETAILED DESCRIPTION

[0053] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0054] Embodiment 1

[0055] The present invention provides Figure 1 , Figure 2 , Figure 4 A single-point rotation scanning dark-field optical microscopy imaging system as shown in : comprises: a sample stage 100;

[0056] An objective lens 200 for observing the sample stage 100;

[0057] A camera for collecting the sample image through the objective lens 200;

[0058] A dark field illuminator 400 for providing illumination light to illuminate the sample stage 100;

[0059] The dark field illuminator 400 comprises:

[0060] an illumination light source 410 for providing illumination light;

[0061] Adjust the illumination light of the illumination light source 410 to irradiate the rotatable baffle 420 of the sample stage 100 at a set angle;

[0062] The rotatable baffle 420 includes:

[0063] A first baffle plate 421 and a second baffle plate 422 that cooperates with the first baffle plate 421 to form an adjustable gap.

[0064] The rotatable baffle 420 is circular with a radius of 2-10 cm. A notch is provided at one corner of the baffle for light to pass through, and the notch angle is 1-30 degrees.

[0065] Embodiment 2

[0066] The present invention improves the dark field illuminator 400 on the basis of the first embodiment. Figure 7 , Figure 8 As shown, the dark field illuminator 400 further includes: an automatic rotator 430 for controlling the angle of the rotatable baffle 420. The automatic rotator 430 includes:

[0067] a driving gear 431 connected to the rotatable baffle 420;

[0068] A servo motor 432 for receiving control instructions to drive the driving gear 431 to rotate according to a set speed and direction;

[0069] A controller for receiving preset program instructions and controlling the servo motor 432 to run at a set speed and direction;

[0070] A power supply connected to the servo motor 432 and the controller;

[0071] A fixing device 435 for fixing the servo motor 432;

[0072] The serrated outer edge 423 of the rotatable baffle 420 is meshed with the driving gear 431;

[0073] The fixing device 435 includes a mounting groove for fixing the servo motor 432 and a base fixed to the mounting groove. The output end of the servo motor 432 is connected to the driving gear 431 through a transmission shaft 438. A connecting rod 437 is arranged outside the transmission shaft 438. The connecting rod 437 is connected to the servo motor 432, and one end of a support rod 436 is fixed to the connecting rod 437. The other end of the support rod 436 is connected to the base.

[0074] The connecting rod 437 is also connected to a chassis 439, and a rotatable baffle 420 is installed in the cavity of the chassis 439. The serrated outer edge 423 of the rotatable baffle 420 is meshed with the driving gear 431. The transmission shaft 438 connected to the driving gear 431 extends into the connecting rod 437 and is connected to the output end of the servo motor 432, and the length of the connecting rod 437 is less than the length of the transmission shaft 438.

[0075] The outer edge of the rotatable baffle 420 is processed into a sawtooth shape and can be meshed and connected with the driving gear 431.

[0076] The controller is a Raspberry Pi controller. The driving gear 431 is engaged with the serrated outer edge 423 of the rotatable baffle 420 to realize the rotational drive of the rotatable baffle 420. The Raspberry Pi controller receives preset program instructions and controls the servo motor 432 to drive the gear 431 at a specific speed and direction, thereby accurately controlling the rotation angle of the rotatable baffle 420.

[0077] Embodiment 3

[0078] The present invention further provides a single-point rotation scanning dark-field optical microscopy imaging method, comprising:

[0079] Start the illumination light source 410, the Raspberry Pi controller and the servo motor 432 and rotate the rotatable baffle 420 to an initial position;

[0080] According to the sample characteristics and experimental requirements, a rotation speed of 1-100 r / s is set in the Raspberry Pi controller. The controller drives the servo motor 432 to drive the driving gear 431 to rotate according to the preset parameters. The light emitted by the illumination light source 410 passes through the gap of the rotatable baffle 420 to form an illumination beam. The rotation of the rotatable baffle 420 is controlled by the automatic rotator 430 to change the incident angle of the single-point illumination beam, thereby scanning different areas of the sample. The particles in the sample will generate scattered light signals under illumination at different incident angles. The scattered light signals are collected to obtain scattered signals of particles with different orientations.

[0081] According to the collected scattered light signal, the scanning parameters and scattering data are combined to display the particle information.

[0082] In this embodiment, a polycarbonate filter membrane is used to enrich the particles in the solution. The refractive index of the polycarbonate filter membrane is 1.52, which is significantly different from the refractive index of air 1.0. In addition, due to its uneven surface, Figure 3 As shown in (a), there is a large scattering effect on the illumination light, resulting in a strong dark field scattering background. Figure 3 As shown in (b), in order to solve this problem, the present application investigates electrolyte solutions with a relatively high refractive index, such as NaI and ZnCl2, and adjusts the solution concentration to a dilution concentration range of 8 M-12 M, preferably 10 M, and tests the refractive index to make it close to the refractive index of the polycarbonate filter membrane, and drops the solution on the filter membrane to test the effect of reducing the scattered background signal after the filter membrane is wetted, as shown in FIG. Figure 3 (c) shows that the most suitable electrolyte solution is determined to obtain a particle scattering signal with a high signal-to-noise ratio;

[0083] (1) Transparent treatment of filter membrane

[0084] The scattering behavior of light in a medium is closely related to the refractive index of the medium. Due to the problem of the manufacturing process, the surface of the commercial filter membrane is uneven, which has a strong scattering effect on light, resulting in a strong scattering background in the imaging process of the filter membrane, which is easy to cover up the scattering signal of the particles on the filter membrane. In order to solve this problem, the present application prepares a ZnCl2 solution with a high refractive index according to the refractive index of the filter membrane material, and adjusts its concentration to make it close to the refractive index of the filter membrane material, and uses a solution with a similar refractive index to soak the filter membrane, thereby significantly reducing the scattering effect of the filter membrane on light and achieving transparency of the filter membrane. Figure 5 (a)- Figure 5 As shown in (d), the scattering background of the filter membrane is reduced, and dark field imaging of particles on the filter membrane is achieved. This part of the preliminary research work provides a good solution for the sensitive detection of weak dark field scattering signals of particles on the filter membrane.

[0085] (2) Feasibility study of particle morphology classification using dark-field optical microscopy system

[0086] To further verify the feasibility of this scheme for particle morphology classification, the optical detection system was used to identify nanoparticles of known morphology on the filter membrane. Gold nanorods (100 nm length), silver nanotriangles (100 nm side length) and purchased PS nanospheres (500 nm diameter) of different morphologies were synthesized and evenly dispersed on the filter membrane. The scattering background of the filter membrane was reduced by adjusting the ZnCl2 concentration, and the scattering intensity changes of the particles under different irradiation angles were obtained using a single-point rotating scanning dark-field optical detection system. The relationship between the scattering signal and the scanning angle was used to distinguish nanoparticles of different morphologies, such as Figure 6 This shows that this scheme is feasible for morphological classification of particles.

[0087] The single-point rotation scanning dark field optical microscopy imaging system and method introduce a rotatable baffle in the dark field illuminator, which can directionally block the full-angle illumination and only allow the illumination light to pass through at a specific angle, so that the illumination light source is switched from the original annular full-angle illumination mode to the single-point rotation scanning illumination mode, thereby obtaining the scattering signal of the particles on the filter membrane at a specific orientation. By rotating the baffle, the generated single-point rotation scanning illumination light source is directionally rotated and irradiated onto the sample, and the illumination intensity and wavelength of the light source are optimized to improve the scattering signal of the particles. The scattering background of the filter membrane is reduced in combination with the transparent treatment of the filter membrane, the signal-to-noise ratio of the particle scattering signal is improved, the scattered signals generated by the particles on the filter membrane at different orientations are obtained, the variation law of the scattering intensity between different orientations is revealed, the correlation between the scattering intensity and the morphology is established, and a fast and high-throughput morphological recognition method for static particles is constructed, providing a new solution for obtaining particle morphological distribution information.

[0088] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An imaging method of a single-point rotation scanning dark-field optical microscopic imaging system, characterized in that: The system comprises: Sample table; an objective lens for observing the sample stage; a camera for collecting sample images through the objective lens; a dark field illuminator for providing illumination light to illuminate the sample stage; The dark field illuminator comprises: an illumination source for providing illumination light; Adjusting the illumination light of the illumination light source to irradiate the rotatable baffle of the sample stage at a set angle; an automatic rotator for controlling the angle of the rotatable baffle; The rotatable baffle comprises: A first baffle and a second baffle cooperating with the first baffle to form an adjustable notch; The automatic rotator comprises: a drive gear connected to the rotatable baffle; A servo motor for receiving control instructions to drive the driving gear to rotate according to a set speed and direction; A controller for receiving preset program instructions and controlling the servo motor to run at a set speed and direction; A power supply connected to the servo motor and the controller; A fixture for fixing the servo motor; wherein the serrated outer edge of the rotatable baffle is meshed with the driving gear; and the method comprises: The rotatable baffle is controlled to rotate by an automatic rotator to change the incident angle of the single-point illumination beam, thereby scanning different areas of the sample in the filter membrane. The particles in the sample generate scattered light signals under illumination at different incident angles. The scattered light signals are collected to obtain scattered signals of particles with different orientations. According to the collected scattered light signal, the scanning parameters and the scattered light data are combined to display the information of the particles; The filter membrane transparency treatment includes: preparing a high refractive index ZnCl2 solution according to the refractive index of the filter membrane material, adjusting the concentration so that the refractive index is close to that of the filter membrane material, and soaking the filter membrane with the solution having a close refractive index; The method for regulating the concentration includes: the concentration range of the dilution solution is 8 M-12 M.

2. The method according to claim 1, characterized in that: The adjustable angle range of the notch is 1 degree to 30 degrees.

3. The method according to claim 1, characterized in that: The rotatable baffle has a circular structure, and the length of its radius is 2 cm-10 cm.

4. The method according to claim 1, characterized in that: The fixing device includes a mounting groove for fixing the servo motor and a base fixed to the mounting groove. The output end of the servo motor is connected to the driving gear through a transmission shaft. A connecting rod is arranged outside the transmission shaft. The connecting rod is connected to the servo motor, and one end of a support rod is fixed to the connecting rod. The other end of the support rod is connected to the base.

5. The method according to claim 4, characterized in that: The connecting rod is also connected to a chassis, a rotatable baffle is installed in the cavity of the chassis, the serrated outer edge of the rotatable baffle is meshed with the driving gear, a transmission shaft connected to the driving gear extends into the connecting rod and is connected to the output end of the servo motor, and the length of the connecting rod is less than the length of the transmission shaft.

6. The method according to claim 1, characterized in that: Before the automatic rotator is used to control the rotation of the rotatable baffle, the following steps are further included: starting the illumination light source, the controller and the servo motor and rotating the rotatable baffle to an initial position; According to the sample characteristics and experimental requirements, the rotation speed of 1-100 r / s is set in the controller. The controller drives the servo motor to rotate the driving gear according to the preset parameters. The light emitted by the illumination light source passes through the gap of the rotatable baffle to form an illumination beam.

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