CN117631248A - Construction method of optical path system of laser scanning copolymerization Jiao Daozhi fluorescence microscope - Google Patents
Construction method of optical path system of laser scanning copolymerization Jiao Daozhi fluorescence microscope Download PDFInfo
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- CN117631248A CN117631248A CN202311862160.2A CN202311862160A CN117631248A CN 117631248 A CN117631248 A CN 117631248A CN 202311862160 A CN202311862160 A CN 202311862160A CN 117631248 A CN117631248 A CN 117631248A
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- 230000003287 optical effect Effects 0.000 title claims abstract description 32
- 238000007334 copolymerization reaction Methods 0.000 title claims abstract description 15
- 238000010276 construction Methods 0.000 title claims description 7
- 238000005286 illumination Methods 0.000 claims abstract description 37
- 238000000034 method Methods 0.000 claims abstract description 27
- 230000008878 coupling Effects 0.000 claims abstract description 15
- 238000010168 coupling process Methods 0.000 claims abstract description 15
- 238000005859 coupling reaction Methods 0.000 claims abstract description 15
- 230000007935 neutral effect Effects 0.000 claims abstract 2
- 238000001514 detection method Methods 0.000 claims description 15
- 229920001577 copolymer Polymers 0.000 claims description 12
- 238000006073 displacement reaction Methods 0.000 claims description 3
- 238000012876 topography Methods 0.000 claims 1
- 238000012360 testing method Methods 0.000 description 4
- 238000003384 imaging method Methods 0.000 description 3
- 230000007547 defect Effects 0.000 description 2
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- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 230000001276 controlling effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000012634 optical imaging Methods 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
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- 239000011435 rock Substances 0.000 description 1
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Abstract
The invention provides a method for constructing a laser scanning copolymerization Jiao Daozhi fluorescence microscope optical path system, which comprises the following steps: turning on an illumination system for a bright field of the inverted fluorescence microscope; setting a magnification converter of the inverted fluorescence microscope objective lens as a neutral position; setting a light outlet of the inverted fluorescence microscope; connecting the laser scanning device to a light outlet of the inverted fluorescence microscope; adjusting the position of the sleeve lens, and coupling an illumination light spot into the center of the sleeve lens; setting the distance between the scanning lens and the image plane as the focal length of the scanning lens; coupling the illumination light spot passing through the scanning lens into the middle position of two reflectors in the galvanometer; coupling laser emitted by the laser source into the center of an illumination facula between two reflectors in the vibrating mirror, and collimating a laser path; and adding the light splitting flat sheet in front of the pinhole turntable. In the invention, the light path is established by adopting the way from the inverted fluorescence microscope to the optical scanning system and then to the laser system, so that the scanning light path and the laser light path can be quickly coupled together. The method for quickly constructing the optical path of the confocal laser scanning microscope can greatly reduce the use cost of the confocal laser scanning microscope, and the system is convenient to carry.
Description
Technical Field
The invention relates to the technical field of microscopes, in particular to a method for constructing a light path system of a laser scanning copolymer Jiao Daozhi fluorescence microscope.
Background
Compared with the traditional fluorescence microscope, the laser scanning confocal microscope has the characteristics of high resolution, high sensitivity and the like, so that the laser scanning confocal microscope is mainly applied to the field of life science and is an important tool for research of cyto-genomics, proteomics, oncology and the like. In addition, in the aspect of geological survey, the laser scanning confocal microscope uses laser as a light source to rapidly scan and image a sample point by point, line by line and face by face, images with different depth layers are obtained by Z-axis focusing, the three-dimensional structure of the sample is displayed through the three-dimensional composition, and further reservoir physical property analysis is carried out, so that the oil content distribution of a rock storage space or the relative content of light components and heavy components can be obtained.
However, the currently used confocal laser scanning microscope is expensive, has huge system, is not easy to carry, is easy to generate light path deviation during carrying, and is regulated by a professional tool, so that the cost of purchase and use is high.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a method for constructing a laser scanning copolymerization Jiao Daozhi fluorescent microscope optical path system.
In order to achieve the above and other objects, the present invention is achieved by the following technical solutions: the method for constructing the optical path system of the laser scanning copolymer Jiao Daozhi fluorescent microscope is provided, the optical path system of the laser scanning copolymer Jiao Daozhi fluorescent microscope comprises a laser scanning device and an inverted fluorescent microscope connected with the laser scanning device, the laser scanning device comprises a laser beam expanding assembly, a detection assembly, a pinhole turntable, a beam splitting flat sheet and a scanning assembly, wherein the laser beam expanding assembly comprises a laser source, and the scanning assembly comprises a sleeve lens, a scanning lens and a galvanometer; the construction method comprises the following steps: turning on an illumination system for a bright field of the inverted fluorescence microscope; setting a magnification converter of an objective lens of the inverted fluorescence microscope to be free; setting a light outlet of the inverted fluorescence microscope as a left side port or a right side port, wherein illumination spots can be seen from the light outlet; connecting the laser scanning device to a light outlet of the inverted fluorescence microscope; adjusting the position of the sleeve lens, and coupling an illumination light spot into the center of the sleeve lens; setting the distance between the scanning lens and the image plane as the focal length of the scanning lens; coupling the illumination light spot passing through the scanning lens into the middle position of two reflectors in the galvanometer; coupling laser emitted by the laser source into the center of an illumination facula between two reflectors in the vibrating mirror, and collimating a laser path; and adding the light splitting flat sheet in front of the pinhole turntable.
According to the invention, the light path can be built from the inverted fluorescent microscope to the optical scanning system and then to the laser system, so that the scanning light path and the laser light path of the system can be quickly coupled together, the system can be disassembled and assembled to be convenient to transport when the system needs to be carried, and the whole system can be quickly built when the system needs to be used. Meanwhile, in the testing process, accessories can be changed according to the characteristics of the sample, so that the expandability of the test is enhanced. In addition, the use cost is reduced.
Further, in the method for constructing the optical path system of the laser scanning copolymerization Jiao Daozhi fluorescent microscope, after the illumination system for the bright field of the inverted fluorescent microscope is turned on, the intermediate multiplying power converter is set to be 1X, the Z-axis displacement of the inverted fluorescent microscope is regulated, and the surface morphology of a specimen to be observed is seen in the inverted fluorescent microscope.
Further, in the method for constructing the optical path system of the laser scanning copolymer Jiao Daozhi fluorescence microscope, the distance between the scanning lens and the sleeve lens is the sum of the focal lengths of the two lenses.
Further, the method for constructing the optical path system of the laser scanning copolymerization Jiao Daozhi fluorescent microscope includes that the illumination light spots after scanning the scanning lenses are coupled into the middle positions of two reflectors in the galvanometer, and the positions of the galvanometer are adjusted, so that the illumination light spots can vertically exit through the galvanometer.
Further, according to the construction method of the optical path system of the laser scanning copolymerization Jiao Daozhi fluorescence microscope, an illumination light spot is coupled into the center of the sleeve lens, and a clear microscopic image of a specimen to be observed is seen at the focal length of the sleeve lens.
Further, in the method for constructing the optical path system of the laser scanning copolymerization Jiao Daozhi fluorescence microscope, the laser beam expanding assembly expands the laser beam according to phi=2na×f, wherein NA is the aperture of the objective lens of the inverted fluorescence microscope, f is the focal length of the objective lens, and phi is the diameter of the expanded laser beam.
Further, the method for constructing the optical path system of the laser scanning copolymerization Jiao Daozhi fluorescent microscope comprises the step of arranging a second apochromatic convergent lens and a third apochromatic convergent lens on the front side of the beam splitting flat sheet, wherein the third apochromatic convergent lens is arranged on the front side of the galvanometer.
Further, the method for constructing the optical path system of the laser scanning copolymerization Jiao Daozhi fluorescence microscope comprises the step of arranging the optical filter turntable at the rear of the light splitting flat plate, the step of arranging the first apochromatic convergent lens and the photomultiplier at the front of the first apochromatic convergent lens, and arranging the photomultiplier at the rear of the first apochromatic convergent lens.
Further, the method for constructing the optical path system of the laser scanning copolymerization Jiao Daozhi fluorescent microscope comprises the steps that the laser scanning device comprises a shell, the laser beam expanding assembly, the detection assembly, the pinhole turntable, the light splitting flat sheet and the scanning assembly are all located in the shell, a connector is arranged on the shell, and the connector is connected with the inverted fluorescent microscope in a detachable mode.
Further, the method for constructing the optical path system of the laser scanning copolymerization Jiao Daozhi fluorescence microscope comprises the step that the pinhole diameter of the pinhole turntable is 30 microns, or 40 microns, or 50 microns.
Drawings
FIG. 1 shows a schematic diagram of a laser scanning confocal inverted fluorescence microscope system according to an embodiment of the invention.
Fig. 2 is a perspective view of a laser scanning apparatus according to an embodiment of the invention.
Fig. 3 is a flow chart showing the steps of a method for constructing a light path system of a laser scanning copolymer Jiao Daozhi fluorescence microscope according to the present invention.
Detailed Description
Please refer to fig. 1 to 3. Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. It should be understood that numerous specific details are set forth in the following description in order to provide a thorough understanding of the present invention, however, that the present invention may be practiced in other ways than as described herein, and that the scope of the invention is therefore not limited to the specific embodiments disclosed below. Furthermore, the terms "first," "second," and the like, are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.
As shown in fig. 1, a laser scanning confocal inverted fluorescence microscope system a includes a laser scanning device 10 and an inverted fluorescence microscope 11 connected to the laser scanning device 10. The inverted fluorescence microscope 11 includes an objective lens. The laser scanning device 10 comprises a housing 101, a laser beam expanding assembly 102, a detection assembly 103, a pinhole turntable 104 and a beam splitting flat sheet 105, wherein the laser beam expanding assembly 102, the detection assembly 103, the pinhole turntable 104 and the beam splitting flat sheet are positioned in the housing 101. The laser beam expanding assembly 102 includes a laser source 1021, where the laser source 1021 is typically a blue-green laser, and has good monochromaticity, and can provide a beam with high intensity, high directivity, and high monochromaticity, for example, the laser may be 405nm. The light beam emitted by the laser source 1021 sequentially passes through the beam splitting flat plate 105, the pinhole of the pinhole turntable 104 and the objective lens, and then enters a sample to be observed, and after the sample receives the energy of the laser beam, the sample receives excitation, and the energy is radiated in the form of electromagnetic energy so as to generate fluorescence. The fluorescence reflected by the specimen sequentially passes through the objective lens, the pinhole of the pinhole turntable 104 and the light splitting flat sheet 105 and then is received by the detection component 103, the detection component 103 converts the fluorescence into a digital signal and transmits the digital signal to a computer, and finally, the digital signal is polymerized into a clear confocal image of the whole focal plane on a screen.
Both the emitted laser light and the reflected fluorescent light pass through the pinhole of the pinhole carousel 104, i.e. the pinhole of the pinhole carousel 104 is used as an illumination pinhole and a detection pinhole simultaneously, ensuring that the illumination pinhole and the detection pinhole are conjugated to the illuminated point or the detected point. In this way, light from the focal plane may be concentrated within the pinhole of the pinhole carousel 104, while scattered light from above or below the focal plane is blocked from imaging by the pinhole.
The utility model discloses in laser that laser source 1021 launched with the fluorescence of sample reflection all is through the pinhole of same pinhole carousel 104, and illumination pinhole and detection pinhole are same pinhole promptly, not only can guarantee that illumination pinhole and detection pinhole are conjugate to the detected point, compare the setting of the pinhole that laser that launches and fluorescence reflected respectively pass through different pinhole carousel 104 in prior art simultaneously, have reduced the quantity of system parts, have saved the space, have consequently reduced the volume of laser scanning confocal inversion fluorescence microscope system, convenient transport.
In this embodiment, the detection assembly 103 includes a filter turntable 1031, a first apochromatic convergent lens 1032, and a photomultiplier 1033, where the filter turntable 1031 is located in front of the first apochromatic convergent lens 1032, and the photomultiplier 1033 is located behind the achromatic lens, that is, fluorescence emitted by the specimen passes through a pinhole of the pinhole turntable 104, then passes through the filter turntable 1031, then passes through the first apochromatic convergent lens 1032, and then is received by the photomultiplier 1033, and finally, is subjected to signal processing to realize imaging. The filter carousel 1031 may be 500±50nm and 600±50nm in size.
For a clear imaging effect, the pinhole of the pinhole turntable 104 is located at the focus of the beam of the laser source 1021, and the pinhole turntable 104 has a pinhole diameter of 30 μm, or 40 μm, or 50 μm.
The confocal optical imaging process is essentially the illumination of a single point on a sample and the detection of that single point, requiring relative three-dimensional movement between the illuminated point and the sample, i.e., the laser light is scanned across the sample space to be detected, in order to image a three-dimensional region of a volumetric extent within the sample, and therefore the laser scanning apparatus 10 further includes a scanning assembly 106. The light beam of the laser tube source passes through the beam splitting flat plate 105, the pinhole turntable 104, the scanning assembly 106 and the objective lens and then enters the specimen to be observed. The scanning along the Z axis can realize optical fault scanning, form a two-dimensional optical slice at a focusing light spot of a sample to be observed, combine X-Y plane (focal plane) scanning with Z axis (optical axis) scanning, and obtain a three-dimensional image of the sample through accumulating continuous-level two-dimensional images and processing by special computer software. As a preferred embodiment, the scanning assembly 106 includes a galvanometer 1061, a scanning lens 1062, and a sleeve lens 1063. The sleeve lens 1063 is connected to the objective lens, the scanning lens 1062 is located between the galvanometer 1061 and the sleeve lens 1063, the galvanometer 1061 receives the beam of the laser source 1021 and reflects the beam, and then the reflected beam sequentially passes through the scanning lens 1062, the sleeve lens 1063 and the objective lens and reaches the specimen. The galvanometer 1061 includes two mirrors and also includes a driving part, such as a motor or an electromagnetic driver, which drives the two mirrors of the galvanometer 1061 to rotate or vibrate so that the laser light scans in space.
The laser beam expanding assembly 102 includes a second apochromatic converging lens 1022 and a third apochromatic converging lens 1023, the second apochromatic converging lens 1022 being located on the front side of the beam splitting plate 105, the third apochromatic converging lens 1023 being located on the front side of the galvanometer 1061.
And a control system connected with the inverted fluorescence microscope 11 and the laser scanning device 10 for controlling operation switching between the components.
In addition, in order to facilitate the transportation of the laser scanning copolymer Jiao Daozhi fluorescence microscope system a, the laser scanning device 10 is detachably connected to the inverted fluorescence microscope 11, the housing 101 is provided with a connector 1011, and the connector 1011 is detachably connected to the inverted fluorescence microscope 11. When the device is required to be carried, the laser scanning device 10 and the inverted fluorescence microscope 11 are detached, so that the operation is convenient.
The invention also provides a construction method of the optical path system of the laser scanning confocal inverted fluorescence microscope system, which is particularly shown in fig. 3. The light path can be built from the inverted fluorescence microscope, the optical scanning system and the laser system. Specifically, the construction method of the optical path system of the laser scanning copolymerization Jiao Daozhi fluorescence microscope system comprises the following steps:
step S10: turning on an illumination system for a bright field of the inverted fluorescence microscope;
step S20: setting a magnification converter of an objective lens of the inverted fluorescence microscope to be free;
step S30: setting a light outlet of the inverted fluorescence microscope as a left side port or a right side port, wherein illumination spots can be seen from the light outlet;
step S40: connecting the laser scanning device to a light outlet of the inverted fluorescence microscope;
step S50: adjusting the position of the sleeve lens, and coupling an illumination light spot into the center of the sleeve lens;
step S60: setting the distance between the scanning lens and the image plane as the focal length of the scanning lens;
step S70: coupling the illumination light spot passing through the scanning lens into the middle position of two reflectors in the galvanometer;
step S80: coupling laser emitted by the laser source into the center of an illumination facula between two reflectors in the vibrating mirror, and collimating a laser path;
step S90: and adding the light splitting flat sheet in front of the pinhole turntable.
In order to facilitate connection with the laser scanning device 10, the inverted fluorescent microscope is provided with a left light outlet and a right light outlet, and the left light outlet or the right light outlet can be flexibly selected according to the needs.
For step 10, in some embodiments, step 10 comprises: after the illumination system for the bright field of the inverted fluorescence microscope is turned on, the intermediate magnification converter is set to 1X, the Z-axis displacement of the inverted fluorescence microscope is adjusted, and the surface morphology of the specimen to be observed is seen in the inverted fluorescence microscope.
For step 50, in some embodiments, after coupling the illumination spot into the center of the sleeve lens, a clear microscopic image of the specimen to be observed may be seen at the focal length of the sleeve lens, step 50.
For step 60, in some embodiments, in step 60, the distance between the scan lens and the sleeve lens is the sum of the focal lengths of the two lenses, at which point a converging illumination spot is visible at the other end of the scan lens.
For step 70, in some embodiments, step 70 comprises: and after the illumination light spots passing through the scanning lens are coupled into the middle positions of the two reflectors in the vibrating mirror, the positions of the vibrating mirror are adjusted, so that the illumination light spots can vertically exit through the vibrating mirror.
According to the invention, the light path can be built from the inverted fluorescent microscope to the optical scanning system and then to the laser system, so that the scanning light path and the laser light path of the system can be quickly coupled together, the system can be disassembled and assembled to be convenient to transport when the system needs to be carried, and the whole system can be quickly built when the system needs to be used. Meanwhile, in the testing process, accessories can be changed according to the characteristics of the sample, so that the expandability of the test is enhanced. In addition, the use cost is reduced.
In addition, in the invention, the laser beam expanding assembly expands the laser beam according to phi=2na×f, wherein NA is the aperture of the objective lens of the inverted fluorescent microscope, f is the focal length of the objective lens, and phi is the diameter of the expanded laser beam.
Therefore, the invention effectively overcomes various defects in the prior art and has high industrial utilization value. The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.
Claims (10)
1. A method for constructing a laser scanning copolymerization Jiao Daozhi fluorescence microscope optical path system is characterized by comprising the following steps: the optical path system of the laser scanning copolymer Jiao Daozhi fluorescent microscope comprises a laser scanning device and an inverted fluorescent microscope connected with the laser scanning device, wherein the laser scanning device comprises a laser beam expanding assembly, a detection assembly, a pinhole turntable, a beam splitting flat plate and a scanning assembly, the laser beam expanding assembly comprises a laser source, and the scanning assembly comprises a sleeve lens, a scanning lens and a galvanometer; the construction method comprises the following steps: turning on an illumination system for a bright field of the inverted fluorescence microscope; setting a magnification converter of an objective lens of the inverted fluorescence microscope as a neutral position; setting a light outlet of the inverted fluorescence microscope as a left side port or a right side port, wherein illumination spots can be seen from the light outlet; connecting the laser scanning device to a light outlet of the inverted fluorescence microscope; adjusting the position of the sleeve lens, and coupling an illumination light spot into the center of the sleeve lens; setting the distance between the scanning lens and the image plane as the focal length of the scanning lens; coupling the illumination light spot passing through the scanning lens into the middle position of two reflectors in the galvanometer; coupling laser emitted by the laser source into the center of an illumination facula between two reflectors in the vibrating mirror, and collimating a laser path; and adding the light splitting flat sheet in front of the pinhole turntable.
2. The method for constructing an optical path system of a fluorescence microscope for laser scanning copolymerization Jiao Daozhi according to claim 1, further comprising setting an intermediate-magnification converter to 1X after the illumination system for a bright field of the inverted fluorescence microscope is turned on, adjusting a Z-axis displacement of the inverted fluorescence microscope in which a surface topography of a specimen to be observed is seen.
3. The method for constructing the optical path system of the laser scanning copolymer Jiao Daozhi fluorescent microscope according to claim 2, wherein the distance between the scanning lens and the sleeve lens is the sum of the focal lengths of the two lenses.
4. A method of constructing a light path system for a fluorescence microscope of a laser scanning co-polymer Jiao Daozhi as claimed in claim 3, wherein coupling the illumination spot after scanning the scanning lens into the intermediate position of two mirrors in the galvanometer comprises adjusting the position of the galvanometer so that the illumination spot can exit vertically through the galvanometer.
5. The method of claim 4, wherein coupling the illumination spot into the center of the sleeve lens comprises viewing a clear microscopic image of the specimen to be observed at the focal length of the sleeve lens.
6. The method for constructing a light path system of a fluorescence microscope according to any one of claims 1 to 5, wherein the laser beam expanding assembly expands the laser beam according to Φ=2na×f, where NA is an aperture of the objective lens of the inverted fluorescence microscope, f is a focal length of the objective lens, and Φ is a diameter of the expanded laser beam.
7. The method of constructing a light path system of a laser scanning co-polymer Jiao Daozhi fluorescence microscope of claim 6, wherein said laser beam expanding assembly comprises a second apochromatic converging lens and a third apochromatic converging lens, said second apochromatic converging lens being positioned on a front side of said beam splitting plate, said third apochromatic converging lens being positioned on a front side of said galvanometer.
8. The method of claim 1, wherein the detection assembly comprises a filter turret positioned behind the beam splitting plate, a first apochromatic converging lens positioned in front of the first apochromatic converging lens, and a photomultiplier positioned behind the first apochromatic converging lens.
9. The method for constructing the optical path system of the laser scanning copolymer Jiao Daozhi fluorescent microscope according to claim 1, wherein the laser scanning device comprises a shell, the laser beam expanding assembly, the detection assembly, the pinhole turntable, the beam splitting flat plate and the scanning assembly are all located in the shell, and a connector is arranged on the shell and is detachably connected with the inverted fluorescent microscope.
10. The method for constructing the optical path system of the laser scanning co-polymer Jiao Daozhi fluorescent microscope according to claim 1, wherein the pinhole diameter of the pinhole turntable is 30 μm, or 40 μm, or 50 μm.
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