CN114396888A - Confocal microscopic device capable of realizing bright field and dark field imaging simultaneously - Google Patents
Confocal microscopic device capable of realizing bright field and dark field imaging simultaneously Download PDFInfo
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- 238000003384 imaging method Methods 0.000 title claims abstract description 51
- 230000004888 barrier function Effects 0.000 claims abstract description 10
- 238000013519 translation Methods 0.000 claims abstract description 9
- 238000004624 confocal microscopy Methods 0.000 claims description 12
- 238000005259 measurement Methods 0.000 claims description 9
- 230000003287 optical effect Effects 0.000 claims description 3
- 238000011065 in-situ storage Methods 0.000 abstract description 7
- 230000006872 improvement Effects 0.000 abstract description 2
- 239000000523 sample Substances 0.000 description 31
- 238000001514 detection method Methods 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 5
- 238000005286 illumination Methods 0.000 description 5
- 239000000463 material Substances 0.000 description 4
- 238000000034 method Methods 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 230000005540 biological transmission Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000010226 confocal imaging Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000004573 interface analysis Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01B—MEASURING LENGTH, THICKNESS OR SIMILAR LINEAR DIMENSIONS; MEASURING ANGLES; MEASURING AREAS; MEASURING IRREGULARITIES OF SURFACES OR CONTOURS
- G01B11/00—Measuring arrangements characterised by the use of optical techniques
- G01B11/24—Measuring arrangements characterised by the use of optical techniques for measuring contours or curvatures
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/47—Scattering, i.e. diffuse reflection
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/55—Specular reflectivity
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/47—Scattering, i.e. diffuse reflection
- G01N2021/4735—Solid samples, e.g. paper, glass
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/55—Specular reflectivity
- G01N2021/556—Measuring separately scattering and specular
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2201/00—Features of devices classified in G01N21/00
- G01N2201/06—Illumination; Optics
- G01N2201/061—Sources
- G01N2201/06113—Coherent sources; lasers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2201/00—Features of devices classified in G01N21/00
- G01N2201/10—Scanning
- G01N2201/103—Scanning by mechanical motion of stage
- G01N2201/1035—3D motion
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A90/00—Technologies having an indirect contribution to adaptation to climate change
- Y02A90/10—Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation
Abstract
The invention relates to a confocal microscope device capable of realizing bright field and dark field imaging simultaneously, which comprises: the device comprises a continuous laser, a first photoelectric detector, a three-dimensional translation table and a second photoelectric detector; one side of the laser emitting end of the continuous laser is sequentially provided with a beam expanding system, a light splitting flat sheet, a double-cavity triple prism and a light barrier; the upper end of the first photoelectric detector is sequentially provided with a first confocal pinhole and a first collecting lens. The device effectively separates the reflected light and the scattered light of the sample on the space by using the difference of the reflected light and the scattered light in the space direction through the double-cavity triple prism, and respectively performs bright field imaging and dark field imaging, well ensures the in-situ performance of imaging, can perform bright field and dark field high-resolution microscopic imaging on the sample to be detected simultaneously, and can realize system improvement by using a conventional photoelectric device on the basis of the conventional confocal microscopic imaging system, thereby being convenient and rapid.
Description
Technical Field
The invention belongs to the technical field of microscopic imaging, and particularly relates to a confocal microscopic device capable of realizing bright field and dark field imaging simultaneously.
Background
The confocal microscopic imaging technology is used as a high-resolution optical probe, can perform non-contact measurement on the three-dimensional appearance of a measured sample, has the advantages of no damage, chromatography and the like, and is widely applied to the fields of material science, physical chemistry, biomedicine and the like. The confocal microscope compresses a three-dimensional point spread function by adopting a spatial filtering mode in a collection light path based on an imaging mode of point illumination and point detection, and improves the spatial resolution of three-dimensional imaging. Compared with the traditional microscopic imaging technology, the confocal microscopic imaging technology is improved by 1.4 times in the transverse resolution and the axial resolution.
With the rapid development of subject technologies such as low-dimensional materials, nanoparticles and biological cells, the confocal microscope also gradually shows the limitation of single measurement mode. Because the traditional confocal microscope only utilizes the reflected light or the transmitted light of a sample to carry out scanning imaging, only bright field images of the measured sample can be obtained, namely, only low-frequency information of the three-dimensional appearance of the sample can be detected, and dark field imaging can reflect more edge details of the sample and acquire high-frequency information of the appearance of the sample, thereby supplementing sample structure information which cannot be obtained by the bright field images. Therefore, a multi-mode confocal microscope capable of simultaneously performing bright field imaging and dark field imaging on a sample is developed, which can effectively make up for the defects of the current confocal microscope in the imaging mode so as to meet the urgent needs of the subject fields of material interface analysis, in-situ detection of living tissues, subcellular imaging, nano-structure material manufacturing and the like.
Disclosure of Invention
In order to solve the above problems, the present invention provides a confocal microscopy apparatus capable of simultaneously realizing bright field and dark field imaging, comprising: the device comprises a continuous laser, a first photoelectric detector, a three-dimensional translation table and a second photoelectric detector;
one side of the laser emitting end of the continuous laser is sequentially provided with a beam expanding system, a light splitting flat sheet, a double-cavity triple prism and a light barrier;
the upper end of the first photoelectric detector is sequentially provided with a first confocal pinhole and a first collecting lens;
the lower end of the second photoelectric detector is sequentially provided with a second confocal pinhole and a second collecting lens;
a sample to be measured is placed on the upper side of the three-dimensional translation table, a measuring objective lens is arranged right above the sample to be measured, and the double-cavity triple prism is positioned between the second collecting lens and the measuring objective lens;
the bevel edge surface of the double-cavity triple prism is plated with a high-reflection film, and the right-angle edge surface of the double-cavity triple prism is plated with an anti-reflection film.
In the imaging process, the device does not need to be switched, and high-precision and in-situ three-dimensional bright field and dark field imaging is realized in a mode of common light path illumination and light splitting path detection.
Preferably, the second collecting lens is located directly above the measurement objective.
Preferably, the cross section of the double-cavity triple prism is a right-angled triangle, the inclined plane of the double-cavity triple prism faces the light splitting flat sheet, and the two right-angled planes of the double-cavity triple prism respectively face the second collecting lens and the light barrier.
Preferably, the light splitting flat sheet is located right above the first collecting lens, and the light splitting flat sheet is obliquely arranged.
Preferably, first confocal pinhole is installed at first photoelectric detector's input, and the confocal pinhole of second is installed at second photoelectric detector's input, two-chamber triple prism is located between beam splitting plain film and the barn door, first collection lens is located first confocal pinhole directly over, measure objective position to be measured sample directly over, for eliminating system stray light, place the barn door in two-chamber triple prism's transmission direction to prevent that illuminating beam from leaking.
The technical scheme of the invention has the following advantages:
the device effectively separates the reflected light and the scattered light of the sample on the space by using the difference of the reflected light and the scattered light in the space direction through the double-cavity triple prism, and respectively performs bright field imaging and dark field imaging, well ensures the in-situ performance of imaging, can perform bright field and dark field high-resolution microscopic imaging on the sample to be detected simultaneously, and can realize system improvement by using a conventional photoelectric device on the basis of the conventional confocal microscopic imaging system, thereby being convenient and rapid.
The technical solution of the present invention is further described in detail by the accompanying drawings and embodiments.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1 is a diagram of a basic implementation of the present invention;
fig. 2 is a schematic structural view of the twin-chambered prism of the present invention.
The device comprises a 1-three-dimensional translation table, 2-a sample to be measured, 3-a measurement objective lens, 4-a light barrier, 5-a double-cavity triple prism, 6-a second collection lens, 7-a second confocal pinhole, 8-a second photoelectric detector, 9-a first collection lens, 10-a first confocal pinhole, 11-a first photoelectric detector, 12-a beam expansion system, 13-a continuous laser and 14-a beam splitting plain film.
Detailed Description
Technical problems, technical solutions and advantageous effects to be solved by the present invention will be more clearly understood, and the present invention will be further described in detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
It will be understood that when an element is referred to as being "secured to" or "disposed on" another element, it can be directly on the other element or be indirectly on the other element. When an element is referred to as being "connected to" another element, it can be directly or indirectly connected to the other element.
It will be understood that the terms "length," "width," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," and the like, as used herein, refer to an orientation or positional relationship indicated in the drawings that is solely for the purpose of facilitating the description and simplifying the description, and do not indicate or imply that the device or element being referred to must have a particular orientation, be constructed and operated in a particular orientation, and is therefore not to be construed as limiting the invention.
Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the present invention, "a plurality" means two or more unless specifically defined otherwise.
The invention provides a confocal microscopy device capable of realizing bright field and dark field imaging simultaneously, which is shown in figures 1-2 and comprises: the device comprises a continuous laser 13, a first photoelectric detector 11, a three-dimensional translation table 1 and a second photoelectric detector 8;
one side of the laser emitting end of the continuous laser 13 is sequentially provided with a beam expanding system 12, a light splitting flat sheet 14, a double-cavity triple prism 5 and a light barrier 4;
the upper end of the first photoelectric detector 11 is sequentially provided with a first confocal needle hole 10 and a first collecting lens 9;
the lower end of the second photoelectric detector 8 is sequentially provided with a second confocal pinhole 7 and a second collecting lens 6;
a sample 2 to be measured is placed on the upper side of the three-dimensional translation table 1, a measuring objective lens 3 is arranged right above the sample 2 to be measured, wherein the double-cavity triple prism 5 is positioned between the second collecting lens 6 and the measuring objective lens 3
In the embodiment, the bevel edge surface of the double-cavity triple prism 5 is plated with a high reflection film, the right-angle edge surface of the double-cavity triple prism 5 is plated with an antireflection film, the second collecting lens 6 is positioned right above the measuring objective lens 3, the section of the double-cavity triple prism 5 is a right-angled triangle, the inclined plane of the double-cavity triple prism 5 faces the light splitting flat sheet 14, two right-angle surfaces of the double-cavity triple prism 5 respectively face the second collecting lens 6 and the light barrier 4, the light splitting flat sheet 14 is positioned right above the first collecting lens 9, the light splitting flat sheet 14 is obliquely arranged, the first confocal pinhole 10 is arranged at the input end of the first photoelectric detector 11, the second confocal pinhole 7 is arranged at the input end of the second photoelectric detector 8, the double-cavity triple prism 5 is positioned between the light splitting flat sheet 14 and the light barrier 4, the first collecting lens 9 is positioned right above the first confocal pinhole 10, and the measuring objective lens 3 is positioned right above the sample 2 to be measured.
The working principle and the beneficial technical effects of the technical scheme are as follows: firstly, laser emitted by a continuous laser is expanded by a beam expanding system and is reflected by the high reverse side of a double-cavity triple prism after being expanded by a beam expanding system and a light splitting flat sheet to form an annular illuminating beam, the annular illuminating beam is focused by a measuring objective lens and then is converged at a sample to be measured, and meanwhile reflected light and scattered light are generated. Because the reflected light of the sample has good directivity (is symmetrical to the incident light about a normal), the reflected light beam and the illuminating light beam have the same annular shape, and in the collecting process, the reflected light is collected and detected through the first collecting lens, the first focusing pinhole and the first photoelectric detector after being reflected by the high back surface of the double-cavity triple prism and the light splitting flat sheet again, so that the confocal bright field detection is realized. Meanwhile, the scattered light generated by the sample has no directivity, namely can be collected and detected at any angle, so that in the collecting process, the scattered light passes through the hollow cavity of the double-cavity triple prism and is collected and detected by the second collecting lens, the second confocal pinhole and the second photoelectric detector, and the confocal dark field detection is realized. And finally, three-dimensional scanning is carried out on the sample through a three-dimensional translation table, and in-situ three-dimensional bright field and dark field imaging of the sample is realized.
It is noted that during the device construction, the diameter of the expanded beam needs to be in the range of 1.5 to 2.5 times the diameter of the double-cavity triple prism cavity to ensure sufficient signal-to-noise ratio and spatial resolution for dark field imaging and bright field imaging.
When the sample is scanned and imaged three-dimensionally, the expanded laser beam is reflected by the double-cavity triple prism to form an annular illumination beam, and the annular illumination beam is focused on the sample and simultaneously generates reflected light and scattered light. The two light beams are collected by a measuring objective lens and then are spatially separated at a double-cavity triple prism, wherein the reflected light of the sample is reflected by the double-cavity triple prism and then is used for bright field imaging; and after the sample scattered light passes through the hollow cavity of the double-cavity triple prism, the sample scattered light is used for dark field imaging. The two light intensity detection systems are provided with confocal pinholes, so that in-situ confocal imaging combining a bright field and a dark field can be formed. In addition, in order to eliminate system stray light, a light blocking plate is placed in the transmission direction of the double-cavity triple prism to prevent the illumination light beam from leaking.
The difference between the optical path structure of the device of the invention and the traditional confocal microscope is that the traditional confocal microscope generally adopts a light beam separation device such as a light splitting flat sheet or a light splitting prism, and weak scattered light cannot be stripped from reflected light, so dark field imaging cannot be realized by using sample scattered light. The device effectively separates the reflected light and the scattered light of the sample on the space by the double-cavity triple prism and by utilizing the difference of the reflected light and the scattered light on the space direction, and respectively performs bright field imaging and dark field imaging, and well ensures the in-situ performance of imaging. In addition, the device can also carry out three-dimensional dark-field tomography measurement on the measured sample, which cannot be realized by the traditional dark-field imaging technology.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (10)
1. A confocal microscopy device capable of simultaneously realizing bright field imaging and dark field imaging is characterized by comprising: the device comprises a continuous laser (13), a first photoelectric detector (11), a three-dimensional translation table (1) and a second photoelectric detector (8);
one side of the laser emitting end of the continuous laser (13) is sequentially provided with a beam expanding system (12), a light splitting flat sheet (14), a double-cavity triple prism (5) and a light barrier (4);
the upper end of the first photoelectric detector (11) is sequentially provided with a first confocal pinhole (10) and a first collecting lens (9);
the lower end of the second photoelectric detector (8) is sequentially provided with a second confocal pinhole (7) and a second collecting lens (6);
the sample (2) to be measured is placed to the upside of three-dimensional translation platform (1), has installed measurement objective (3) directly over sample (2) to be measured, and wherein, two-chamber prism (5) are located between second collection lens (6) and measurement objective (3).
2. The confocal microscopy device capable of realizing bright field and dark field imaging simultaneously as claimed in claim 1, wherein the hypotenuse face of the double-cavity triple prism (5) is coated with a high reflection film, and the right-angle side face of the double-cavity triple prism (5) is coated with an antireflection film.
3. Confocal microscopy apparatus for simultaneously bright-field and dark-field imaging according to claim 1, characterized in that the second collection lens (6) is located directly above the measurement objective (3).
4. The confocal microscopy apparatus capable of simultaneously realizing bright-field and dark-field imaging according to claim 1, characterized in that the section of the double-cavity triple prism (5) is a right triangle.
5. The confocal microscopy apparatus capable of simultaneously realizing bright-field and dark-field imaging according to claim 4, wherein the inclined plane of the doubly-curved triple prism (5) faces the light splitting flat sheet (14), and the two right-angle planes of the doubly-curved triple prism (5) respectively face the second collecting lens (6) and the light barrier (4).
6. The confocal microscopy apparatus capable of simultaneously realizing bright-field and dark-field imaging according to claim 1, wherein the beam splitter (14) is located right above the first collecting lens (9), and the beam splitter (14) is obliquely arranged.
7. The confocal microscopy apparatus for simultaneously realizing bright-field and dark-field imaging according to claim 1, wherein the first confocal pinhole (10) is installed at the input end of the first photodetector (11), and the second confocal pinhole (7) is installed at the input end of the second photodetector (8).
8. Confocal microscopy apparatus for simultaneous bright-field and dark-field imaging according to claim 1, characterized in that the twin-cavity triple prism (5) is located between the beam splitter (14) and the optical barrier (4).
9. The confocal microscopy apparatus capable of simultaneously realizing bright-field and dark-field imaging according to claim 1, wherein the first collection lens (9) is located right above the first confocal pinhole (10).
10. Confocal microscopy apparatus for simultaneously bright-field and dark-field imaging according to claim 1, characterized in that the measurement objective (3) is located directly above the sample (2) to be measured.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114966086A (en) * | 2022-05-11 | 2022-08-30 | 上海交通大学 | Pulse laser driven particle flight speed measuring device and method |
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