A dark-field microscope in an interference-scattering enhancement mode

Abstract

The present invention discloses a dark-field microscope in an interference-scattering enhancement mode, which includes a light source for emitting light, a reflection optical path composed of a plurality of mirrors, and a detection device disposed above a sample to be measured. The parallel light beam emitted by the light source enters the reflection optical path after passing through the sample to be measured, and then passes through the sample to be measured again as a parallel light beam through the reflection optical path. The sample to be measured scatters and is detected by the detection device. Compared with the prior art, the technical solution of the present invention has the advantages of simple structure, convenient operation, high detection sensitivity and high reliability. Compared with the existing dark-field microscopes, the present invention has the advantages of significantly improving the resolution and application range of the microscope, meeting the observation and imaging requirements at the nanoscale, low construction cost, and being able to be popularized and applied in ordinary laboratories, etc.

Description

Technical Field

[0001] The present invention relates to the technical field of dark-field microscopes, and particularly to a dark-field microscope in an interference-scattering enhancement mode. Background Art

[0002] In the field of life science and technology, in order to study the interaction between nano-drugs and tumor cells and the nano-scale extracellular vesicles secreted by cells, microscopic imaging technology is an essential technical means. The existing technology usually adopts spectral analysis means represented by molecular fluorescence, which has advantages such as real-time, in-situ, and non-invasive, and thus is widely used in the field of life analysis technology. However, since fluorescence analysis requires fluorescent labeling of the research object, the labeling process is not only cumbersome and time-consuming, but also inevitably interferes with the behavior of the research object, and even causes the death of the research object.

[0003] To solve the above technical problems existing in fluorescence analysis, dark-field microscopes have emerged. Dark-field microscopes have advantages such as stable optical signals, label-free, and fast imaging, and have unique advantages in the field of life analysis technology. In recent years, dark-field microscopic imaging technology has received increasing attention in the fields of single-cell and single-nanoparticle analysis.

[0004] The resolution of a conventional optical microscope is limited by the wavelength of light and generally does not exceed 0.3 micrometers. Moreover, the condenser of a dark-field microscope is different from that of a conventional microscope. Because in the existing technology, the dark-field microscope forms a conical light beam and irradiates the sample to be observed. At the same time, in the illumination of the dark-field microscope, in order to achieve the purpose of only allowing the light to irradiate the sample to be observed from the side, a light-shielding plate is usually used to filter the vertically incident light, so that only the side light passes through. If there is no sample on the stage, the numerical aperture of the condenser is larger than that of the objective lens, then the hollow conical light beam cannot enter the objective lens, and the observation background under the dark-field microscope is black. At this time, if the sample to be observed is placed on the stage, the conical light beam irradiates the object to be detected, and reflection, diffraction, and refraction occur on the surface and inside of the sample, and the scattering signal of the sample will be collected and imaged by the objective lens located at the center of the incident light axis.

[0005] In order to overcome the problems existing in the illumination light of dark-field digital holographic microscopy in the prior art, such as poor controllability of the ring diameter ratio of the inner and outer radii of the annular illumination light, uneven light waves, weak imaging contrast, and a large amount of light energy loss, and to improve the system resolution and enhance the contrast of the reconstructed image, Chinese Patent (Publication No. CN107907983A) discloses a digital holographic microscope device based on the illumination of a localized hollow beam and its working method. First, a plane wave is irradiated onto a conical mirror to generate a quasi-non-diffracting Bessel beam. The Bessel beam is focused by a lens to form a localized hollow beam. Then, the generated localized hollow beam enters a dark-field condenser to form an annular light cone to illuminate the experimental sample to obtain the scattered light of the object. Then, digital holography interferes the scattered light of the object with a spherical wave reference light, and the interference fringes are recorded into a computer through a photoelectric coupling device. However, the technical solution of this patent still has technical problems such as uneven light waves of the illumination beam in the dark-field microscope, large background signals, a large amount of light energy loss, and poor imaging contrast. Moreover, the technical solution of this patent uses a conical mirror to form a quasi-non-diffracting Bessel beam and forms a localized hollow beam through lens focusing, which cannot further improve the resolution through the interference of multiple optical paths. After multiple optical path interferences, serious imaging distortion will occur. Summary of the Invention

[0006] The main object of the present invention is to overcome the problems of the prior art and propose a dark-field microscope in an interference-scattering enhancement mode with a simple structure, convenient operation, and sensitive and reliable detection, aiming to make the scattered signal of the sample to be measured be greatly enhanced through the interference phenomenon of the incident light and the reflected light of the sample to be measured, and finally the imaging of the sample to be measured is clearly and highly sensitively captured by the detection device.

[0007] To achieve the above object, a dark-field microscope in an interference-scattering enhancement mode proposed by the present invention includes a light source for emitting light, a reflection optical path composed of several reflectors, and a detection device disposed above the sample to be measured. The parallel beam emitted by the light source enters the reflection optical path after passing through the sample to be measured, and passes through the sample to be measured several times as a parallel beam through the reflection optical path and is detected by the detection device.

[0008] Preferably, the reflection optical path includes a first reflector, a second reflector, a third reflector, and a fourth reflector that are arranged in sequence along the optical path in the same plane. A first collimating lens is provided between the light source and the sample to be measured to couple the light emitted by the light source and pass through the sample to be measured for the first time. A light attenuator is provided between the second reflector and the third reflector. A second collimating lens is provided between the third reflector and the sample to be measured to couple the light reflected by the third reflector and pass through the sample to be measured again. The fourth reflector is disposed in front of the light after the second collimating lens passes through the sample to be measured and continuously reflects the light to the sample to be measured.

[0009] Preferably, a rotating mechanism for adjusting the angular position of the spatial mirror surface is provided inside the first reflector, the second reflector, the third reflector, and the fourth reflector, and at least one mirror surface is included inside the first reflector, the second reflector, the third reflector, and the fourth reflector.

[0010] Preferably, the light source, the first collimating lens, the first reflector, the second reflector, the third reflector, the second collimating lens, and the fourth reflector are simultaneously connected to a first three-dimensional adjustment stage, the sample to be measured is connected to a second three-dimensional adjustment stage, and the first three-dimensional adjustment stage and the second three-dimensional adjustment stage are separated from each other.

[0011] Preferably, the transmittance of the light attenuation sheet is 0-100%.

[0012] Preferably, the light source is any one or more of a laser light source, an LED light source, a metal halide light source, and a high-pressure sodium light source, and at least one beam of light is emitted by the light source.

[0013] Preferably, the sample to be measured is a solid sample, a gaseous sample, or a liquid sample.

[0014] Preferably, the detection device is an objective lens and an industrial camera, and the detection device is further connected to a computer and a display screen.

[0015] The technical solution of the present invention has the following advantages over the prior art:

[0016] In the technical solution of the present invention, the parallel light generated by the light source is first irradiated on the sample to be measured, and then the reflected light is irradiated on the sample to be measured again through a reflection optical path system composed of a plurality of reflectors, so that there is an optical path difference in the optical path between the incident light first irradiated on the surface of the sample to be measured and the reflected light passing through the reflection optical path system, thereby correspondingly generating an interference phenomenon. Under the condition of interference, the scattering signal of the particles is greatly enhanced, and the detection signal is collected by the objective lens located at the center of the incident optical axis and imaged in the camera.

[0017] Therefore, by using parallel light to replace the traditional annular light beam and adopting the interference-scattering enhancement mode, the technical solution of the present invention can significantly improve the uniformity of light waves, reduce the background signal, improve the resolution and application range of the dark-field microscope, meet the observation and imaging requirements at the nanoscale, and greatly reduce the manufacturing cost of the overall device through a simple optical path reflection system, and has the advantages of being popularized and widely applied in ordinary laboratories. Description of the Drawings

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on the structures shown in these drawings.

[0019] Figure 1 It is a top view structural schematic diagram of the dark-field microscope in the interference-scattering enhancement mode of the present invention;

[0020] Figure 2 It is an observation effect diagram of the dark-field microscope of the prior art;

[0021] Figure 3 It is an observation effect diagram of the dark-field microscope in the interference-scattering enhancement mode of the present invention.

[0022] Explanation of the reference numerals in the drawings:

[0023] Label Name Label Name 1 Light source 7 Third reflector 2 First collimating lens 8 Second collimating lens 3 Sample to be measured 9 Fourth reflector 4 First reflector 10 First three-dimensional adjustment stage 5 Second reflector 11 Second three-dimensional adjustment stage 6 Neutral density filter

[0024] The realization, functional characteristics and advantages of the object of the present invention will be further described in conjunction with the embodiments with reference to the drawings. Specific embodiments

[0025] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0026] It should be noted that if there are directional indications (such as up, down, left, right, front, back...) involved in the embodiments of the present invention, the directional indications are only used to explain the relative position relationship and movement conditions between components in a specific posture (as shown in the drawings). If the specific posture changes, the directional indications will also change accordingly.

[0027] In addition, if the descriptions such as "first" and "second" are involved in the embodiments of the present invention, the descriptions of "first", "second", etc. are for descriptive purposes only, and should not be construed as indicating or implying their relative importance or implicitly indicating the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include at least one such feature. In addition, the technical solutions between various embodiments may be combined with each other, but it must be based on the fact that those of ordinary skill in the art can implement it. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0028] The present invention provides a dark-field microscope in an interference-scattering enhancement mode.

[0029] Please refer to Figure 1 , the dark-field microscope in the interference-scattering enhancement mode of the embodiment of the present invention includes a light source 1 for emitting light, a reflection optical path composed of a plurality of reflectors, and a detection device (not marked in the figure) disposed above a sample to be measured 3. The parallel light beam emitted by the light source 1 enters the reflection optical path after passing through the sample to be measured 3, and then passes through the sample to be measured again as a parallel light beam through the reflection optical path and is detected by the detection device.

[0030] Specifically, the reflection optical path of this embodiment includes a first reflector 4, a second reflector 5, a third reflector 7, and a fourth reflector 9 in the same plane. A first collimating lens 2 is provided between the light source 1 and the sample to be measured 3 to couple the light emitted by the light source 1 and pass through the sample to be measured 3 for the first time. A light attenuator 6 is provided between the second reflector 5 and the third reflector 7. A second collimating lens 8 is provided between the third reflector 7 and the sample to be measured 3 to couple the light reflected by the third reflector 7 and pass through the sample to be measured 3 again. The fourth reflector 9 is disposed in front of the light after the second collimating lens 8 passes through the sample to be measured 3 and continuously reflects the light to the sample to be measured 3.

[0031] Therefore, when the dark-field microscope in the interference-scattering enhancement mode of this embodiment works, first, light is emitted from the light source 1 to the sample 3 to be measured. After passing through the first collimating lens 2, the light becomes a parallel beam and enters the sample 3 to be measured. Part of the light is absorbed by the sample 3 to be measured, causing the sample 3 to scatter the incident light, and the other part of the light passes through the sample 3 to be measured and is reflected by the first reflector 4. The light is then reflected by the first reflector 4 to the second reflector 5, and then reflected by the second reflector 5 to the third reflector 7. When the light passes through the second collimating lens 8 between the third reflector 7 and the sample 3 to be measured, it forms parallel light. Then, after passing through the sample 3 to be measured, the light is irradiated onto the fourth reflector 9, and the fourth reflector 9 continuously reflects the light to the sample 3 to be measured. Therefore, there is an optical path difference between the incident light and the reflected light in the technical solution of this embodiment, resulting in an interference phenomenon. Finally, the scattering signal of the sample 3 to be measured is detected by the detection device arranged above the sample 3 to be measured. Due to the interference phenomenon between the incident light and the reflected light of the sample 3 to be measured, the scattering signal of the sample 3 to be measured is greatly enhanced, and finally the imaging of the sample to be measured is clearly and highly sensitively captured by the detection device, as Figure 2 and Figure 3 shown, the dark-field microscope in the interference-scattering enhancement mode of this embodiment has a more obvious scattering effect than the prior art. And through the interference phenomenon between the incident light and the reflected light, the scattering signal of the particles in the sample to be measured is enhanced, which can significantly improve the detection sensitivity.

[0032] Preferably, a rotating mechanism for adjusting the spatial angle of the mirror surface is provided inside the first reflector 4, the second reflector 5, the third reflector 7, and the fourth reflector 9 in this embodiment. The rotating mechanism in this embodiment includes a mounting groove seat provided on the back of the reflector and a ball head convex block connected to the first three-dimensional adjustment table 10. Through the tight fitting of the mounting groove seat and the ball head convex block, and then by tightening the inner-threaded tightening collar, the mounting groove seat and the ball head convex block are tightly connected. In other embodiments of the present invention, the rotating mechanism includes a ball head hinge connecting the reflector and the first three-dimensional adjustment table 10. Therefore, by adjusting the spatial angle between each reflector and the first three-dimensional adjustment table 10, a reliable reflection optical path can be formed by the four reflectors. Preferably, there is at least one mirror surface inside the first reflector 4, the second reflector 5, the third reflector 7, and the fourth reflector 9 in this embodiment, that is, the first reflector 4, the second reflector 5, the third reflector 7, and the fourth reflector 9 contain one plane mirror, or two plane mirrors, or multiple plane mirrors. Moreover, the reflection optical path formed by the plane mirrors of the multiple reflectors in this embodiment can cause the reflected light beam to interfere with the incident light beam, enhancing the scattering signal of the particles in the sample to be measured 3 and significantly improving the detection sensitivity. More importantly, the fourth reflector 9 in this embodiment is arranged in front of the reflected light after passing through the sample to be measured 3, so that the light reflected by the fourth reflector 9 continues to be reflected in the direction of the sample to be measured 3 and continues to interfere, resulting in the continuous enhancement of the scattering signal, thereby effectively improving the detection sensitivity.

[0033] In addition, the light source 1, the first collimating lens 2, the first reflector 4, the second reflector 5, the third reflector 7, the second collimating lens 8, and the fourth reflector 9 in this embodiment are simultaneously connected to the first three-dimensional adjustment table 10, and the sample to be measured 3 is connected to the second three-dimensional adjustment table 11. The first three-dimensional adjustment table 10 and the second three-dimensional adjustment table 11 are separated from each other. Therefore, the spatial installation positions of the reflectors and the collimating lenses are adjusted through the first three-dimensional adjustment table 10, and the spatial angle of the sample to be measured 3 is adjusted through the second three-dimensional adjustment table 11 to adaptively adjust the spatial relationship between the sample to be measured 3 and the reflection optical path.

[0034] Preferably, the transmittance of the light reduction film 6 in this embodiment is 0-100%. The reflection intensity of the reflected light beam is adaptively adjusted through the light reduction film 3 to adjust to the optimal reflection-scattering signal. The light source 1 in this embodiment can be any one or more of a laser light source, an LED light source, a metal halide light source, and a high-pressure sodium light source working simultaneously. The light beam emitted by the light source 1 is at least one beam. The sample to be measured 3 is a solid sample, a gaseous sample, or a liquid sample. The detection device is an objective lens with a small numerical aperture and an industrial camera for collecting the interference-scattering signal of the sample to be measured and imaging, which is arranged above the sample to be measured. The detection device is also connected to a computer and a display screen for convenient observation. Therefore, the interference-scattering signal of the sample to be measured 3 collected by the objective lens is recorded in real time by the industrial camera and displayed on the display screen for the user to observe conveniently.

[0035] The above are only the preferred embodiments of the present invention, and do not limit the patent scope of the present invention. Any equivalent structural transformation made under the concept of the present invention by using the content of the specification and drawings of the present invention, or direct / indirect application in other related technical fields, is included in the patent protection scope of the present invention.

Claims

1. A dark-field microscope in an interference-scattering enhancement mode, characterized in that, It includes a light source for emitting light, a reflection optical path composed of several reflectors, and a detection device arranged above the sample to be measured. The light source emits a parallel light beam, which enters the reflection optical path after passing through the sample to be measured, and passes through the sample to be measured several times as a parallel light beam and is detected by the detection device; The reflection optical path includes a first reflector, a second reflector, a third reflector, and a fourth reflector that are arranged in sequence along the optical path in the same plane. A first collimating lens is arranged between the light source and the sample to be measured to couple the light emitted by the light source and pass through the sample to be measured for the first time. A light attenuator is arranged between the second reflector and the third reflector. A second collimating lens is arranged between the third reflector and the sample to be measured to couple the light reflected by the third reflector and pass through the sample to be measured again. The fourth reflector is arranged in front of the light beam after the second collimating lens passes through the sample to be measured and continuously reflects the light beam to the sample to be measured.

2. The dark-field microscope in the interference-scattering enhancement mode according to claim 1, characterized in that, The first reflector, the second reflector, the third reflector, and the fourth reflector are internally provided with a rotating mechanism for adjusting the angle of the spatial mirror surface, and the first reflector, the second reflector, the third reflector, and the fourth reflector internally include at least one mirror surface.

3. The dark-field microscope in the interference-scattering enhancement mode according to claim 2, wherein The light source, the first collimating lens, the first reflector, the second reflector, the third reflector, the second collimating lens, and the fourth reflector are simultaneously connected to a first three-dimensional adjustment stage, and the sample to be measured is connected to a second three-dimensional adjustment stage. The first three-dimensional adjustment stage and the second three-dimensional adjustment stage are separated from each other.

4. The dark-field microscope in the interference-scattering enhancement mode according to claim 3, characterized in that, The transmittance of the light attenuator is 0-100%.

5. The dark-field microscope in the interference-scattering enhancement mode according to claim 4, characterized in that, The light source is any one or more of a laser light source, an LED light source, a metal halide light source, and a high-pressure sodium light source, and the light beam emitted by the light source is at least one beam.

6. The dark-field microscope in the interference-scattering enhancement mode according to claim 5, characterized in that, The sample to be measured is a solid sample, a gaseous sample, or a liquid sample.

7. The dark-field microscope in the interference-scattering enhancement mode according to claim 6, characterized in that, The detection device is an objective lens and an industrial camera, and the detection device is also connected to a computer and a display screen.

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