CN107831589B - Focusing controllable super-resolution microscopic device based on spherical micro-nano liquid drop lens - Google Patents
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- G—PHYSICS
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- G02B21/00—Microscopes
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- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0052—Optical details of the image generation
- G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
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- 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
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
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- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
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- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
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- G—PHYSICS
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- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0052—Optical details of the image generation
- G02B21/0068—Optical details of the image generation arrangements using polarisation
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- G—PHYSICS
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- G02B21/00—Microscopes
- G02B21/33—Immersion oils, or microscope systems or objectives for use with immersion fluids
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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
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N2021/6463—Optics
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Abstract
The invention discloses a focusing controllable super-resolution microscopic device based on a spherical micro-nano liquid drop lens, which comprises: the system comprises a laser, a reflector, a first lens group, a dichroic mirror, a linear polarizer, a microscope objective, an encapsulation box, a spherical micro-nano droplet lens, a micro-flow tube, a micro-displacement table, a sample table, a second lens group, an imaging device and a computer control system. The computer control system controls the micro-flow pipe to release or absorb liquid drops with a certain volume in the packaging box, so as to regulate and control the radius of the spherical micro-nano liquid drop lens, and realize the zooming of the spherical micro-nano liquid drop lens. Aiming at the defects of uncontrollable focusing characteristics and the like of the existing micro-nano structure, the invention provides a focusing controllable super-resolution microscopic device based on a spherical micro-nano liquid drop lens, which utilizes the spherical micro-nano liquid drop lens to generate photon nano-jet, and realizes scanning of different depths of a sample under the condition of no mechanical structure vibration by regulating the focusing characteristics of the spherical micro-nano liquid drop lens; and can combine little displacement platform regulation and control sample platform to realize the scanning of horizontal direction, accomplish the three-dimensional super-resolution microscopic imaging to the sample.
Description
Technical Field
The invention relates to the field of optical microscopic imaging, in particular to a focusing controllable super-resolution microscopic device based on a spherical micro-nano liquid drop lens.
Background
Since Abbe in 1873 proposed the concept of optical diffraction limit, how to break through the optical diffraction limit and obtain higher quality high resolution images has been a hot spot of academic research. Until the super-resolution optical imaging technology appears, the resolution limit of a conventional optical microscope is broken through, and unprecedented solutions are provided for the research in the fields of bioscience, biological cytology and the like. Currently, the main super-resolution optical microscopy techniques mainly include: target switching and reading microscopy, as represented by fluorescence emission loss microscopy as proposed by s.w. hell et al, and random switching and reading microscopy, as represented by light-sensitive localization microscopy as proposed by e.betzig et al, and random optical reconstruction microscopy as proposed by x.zhuang et al. Both of the above-mentioned microscopic techniques, while capable of super-resolution microscopic imaging, still suffer from drawbacks such as: the system has complex structure and high cost, and the vibration of the mechanical structure is difficult to avoid when realizing three-dimensional scanning.
The method for realizing super-resolution imaging by utilizing photon nano jet effect generated by the micro-nano structure and sub-wavelength focusing has the characteristics of simple structure, lower cost, no vibration of a mechanical structure and the like, and has recently been paid more attention to by more researchers. In 2017, in the paper entitled "microsphere super-resolution microscopy effect based on near field optics" issued by the "physical school journal", zhou Rui et al, 66, photon nanojet generated by the microsphere is regulated by performing methods of circular concentric ring engraving, center shielding and surface coating on the surface of the microsphere, so that the diffraction limit is broken through, and super-resolution microscopy imaging is realized. However, the focusing characteristic generated by a single microsphere in the structure is not adjustable, so that super-resolution microscopic imaging cannot be performed on different depths of a sample, the flexibility is poor, and three-dimensional microscopic imaging of the sample cannot be realized.
Disclosure of Invention
Aiming at the defects of uncontrollable focusing characteristics and the like when the existing micro-nano structure realizes super-resolution microscopic imaging in the near field, the invention provides a focusing controllable super-resolution microscopic device based on a spherical micro-nano liquid drop lens. The device utilizes the spherical micro-nano liquid drop lens to generate photon nano injection, and realizes scanning of different depths of a sample under the condition of no vibration of a mechanical structure by regulating and controlling the focusing characteristic of the spherical micro-nano liquid drop lens; and can combine little displacement platform regulation and control sample platform to realize the scanning of horizontal direction, accomplish the three-dimensional super-resolution microscopic imaging to the sample.
A focusing controllable super-resolution microscopy device based on a spherical micro-nano droplet lens, comprising: the system comprises a laser, a reflector, a first lens group, a dichroic mirror, a linear polarizer, a microscope objective, a packaging box, a spherical micro-nano droplet lens, a micro-flow tube, a micro-displacement table, a sample table, a second lens group, an imaging device and a computer control system;
the light beam output by the laser is incident on a reflecting mirror, the reflecting mirror reflects the incident light beam to a first lens group, the light beam passing through the first lens group enters a dichroic mirror, the light beam passes through the dichroic mirror, and is incident on a linear polarizer, linearly polarized light emitted from the linear polarizer is incident on a rear focal plane of a microscope objective, and forms parallel light beam after passing through the microscope objective, and the parallel light beam is incident on a spherical micro-nano liquid drop lens, photon nano injection reaching super-resolution effect is generated at the other side of the spherical micro-nano liquid drop lens, and the injected nano photons excite a sample on a sample stage to generate fluorescence;
fluorescence is reversely collected by the microscope objective, the fluorescence collected by the microscope objective is emitted to the linear polarizer, the fluorescence is incident to the surface of the dichroic mirror after passing through the linear polarizer, and the fluorescence is focused to an imaging device through the second lens group after being reflected by the dichroic mirror, so that imaging is realized.
In the present invention, the first lens group includes two convex lenses for collimation.
In the present invention, the second lens group includes two convex lenses for focusing.
In the invention, the dichroic mirror shows high transmittance to the laser output beam and high reflection to the fluorescence emitted by the sample; the high transmittance is that the pointer outputs light beams to the laser, and the transmittance is more than 98%; the high reflection is the fluorescence emitted by the pointer to the sample, the reflectivity is more than 98 percent.
In the invention, the linear polarizer is used for modulating light beams into linearly polarized light, and the electric field vibration direction of the linearly polarized light is horizontal, namely, is vertical to the transmission direction of the light beams passing through the linear polarizer.
In the invention, the lower substrate of the packaging box is a glass slide, and the side substrate is a spacer bar; the sample platform is embedded in the micro-displacement platform; the micro-displacement platform is used for regulating and controlling the sample platform, so that the sample platform can move along the horizontal direction of the plane where the sample platform is located.
In the invention, the photon nanometer jet refers to that when the spherical micro-nano liquid drop is irradiated by a parallel light beam, one diameter generated at the other side of the spherical micro-nano liquid drop lens which is irradiated by the parallel light beam is a sub-wavelength or a half-sub-wavelength, the divergence angle is small, the focused light intensity is high, and the focal length is larger than 2Or less than 2->Is provided.
In the invention, the computer control system controls the micro-flow pipe to release or absorb liquid in the packaging box so as to regulate and control the radius of the spherical micro-nano liquid drop lens; the larger the radius of the spherical micro-nano liquid drop lens is, the larger the focal length of the corresponding spherical micro-nano liquid drop lens is, and the focal length of the spherical micro-nano liquid drop lens can be regulated and controlled by regulating and controlling the radius of the micro-nano liquid drop lens; the focal length of the spherical micro-nano liquid drop lens is the distance from the end face of the spherical micro-nano liquid drop lens to the photon nano jet focusing center.
In the invention, the super-resolution effect means that the half-width of photon nano injection generated by utilizing the spherical micro-nano liquid drop lens is less than half of the wavelength of the light beam emitted by the laser; the half-width refers to the width of photon nanometer injection when the light intensity is half of the maximum light intensity.
Preferably, the wavelength of the light beam output by the laser is in the range of 570 nm to 670 nm.
Preferably, the spherical micro-nano droplet lens is made of water.
Preferably, the numerical aperture of the microscope objective is 1.35.
Compared with the prior art, the invention has the following beneficial technical effects:
1. the invention adjusts and controls the focusing characteristic of the spherical micro-nano liquid drop lens, scanning of different depths of the sample is realized, no vibration of a mechanical structure exists, and the scanning precision is high;
2. the invention has simple light path, convenient construction, water as the material of the spherical micro-nano liquid drop lens, easy acquisition and low cost.
Drawings
FIG. 1 is a schematic diagram of the structural principle of a focusing controllable super-resolution microscopic device based on a spherical micro-nano liquid drop lens;
wherein: the device comprises a laser 1, a reflecting mirror 2, a first lens group 3, a dichroic mirror 4, a linear polarizer 5, a micro objective lens 6, a packaging box 7, a spherical micro-nano droplet lens 8, a micro flow tube 9, a micro displacement table 10, a sample table 11, a second lens group 12, an imaging device 13 and a computer control system 14.
Fig. 2 is a graph of a fit of the focal length of a spherical micro-nano droplet lens with respect to radius in an embodiment.
Fig. 3 is a graph of fit of half-width versus radius for photon nanojet in an embodiment.
Detailed Description
The present invention will be described in detail below with reference to the drawings of the specification, but the present invention is not limited thereto.
FIG. 1 is a schematic diagram of a focusing controllable super-resolution microscopy device based on a spherical micro-nano droplet lens, comprising: the device comprises a laser 1, a reflecting mirror 2, a first lens group 3, a dichroic mirror 4, a linear polarizer 5, a micro objective lens 6, a packaging box 7, a spherical micro-nano droplet lens 8, a micro flow tube 9, a micro displacement table 10, a sample table 11, a second lens group 12, an imaging device 13 and a computer control system 14.
Wherein, the laser 1 is an all-solid-state laser of LE-LS-589-XXT visible light semiconductor pump produced by Shenzhen European photoelectric technology Co-Ltd, the power is 1-4500 milliwatt, and the working wavelength is 589 nanometers.
The light beam output by the laser 1 is incident on the reflecting mirror 2, the reflecting mirror 2 reflects the incident light beam to the first lens group 3, the light beam collimated by the first lens group 3 enters the dichroic mirror 4, the light beam is transmitted by the dichroic mirror 4 and then is incident on the linear polarizer 5, the vibration direction of the linear polarized light electric field obtained after modulation by the linear polarizer 5 is a horizontal direction, namely, the vibration direction is perpendicular to the light beam transmission direction passing through the linear polarizer 5, and the linear polarized light emitted from the linear polarizer 5 is incident on the back focal plane of the microscope objective 6.
The lower substrate of the packaging box 7 is a glass slide made of silicon dioxide, the side substrate is a spacing bar, a micro-displacement table 10 is placed on the packaging box 7, and a sample table 11 is embedded in the micro-displacement table 10; the parallel light beam emitted after passing through the microscope objective 6 is incident on the spherical micro-nano droplet lens 8 through the lower substrate of the package box 7, and photon nano-jet is generated at the other side of the spherical micro-nano droplet lens 8 opposite to the spherical micro-nano droplet lens 8 irradiated parallel to the light beam.
In this embodiment, spherical micro-nano droplet lenses 8 with radii of 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0 μm are selected respectively, and the focusing characteristics of the spherical micro-nano droplet lenses 8 with different radii are calculated.
And researching photon nano injection of the spherical micro-nano liquid drop lens 8 through a Maxwell Wei Qiu coordinate equation, selecting FDTD-Solution commercial software to conduct accurate numerical simulation of photon nano injection light field distribution, conducting accurate Solution of a Maxwell equation set, and conducting Gaussian fitting through origin software. Fitting curves of the corresponding focal length and half-width with respect to the radius after Gaussian fitting by an origin software are respectively shown in fig. 2 and 3; the larger the radius of the spherical micro-nano liquid drop lens 8 is, the longer the focal length of the corresponding spherical micro-nano liquid drop lens 8 is, the larger the half-width of photon nano jet generated by the corresponding spherical micro-nano liquid drop lens 8 is, but when the radius is between 0.6 micron and 0.7 micron and between 0.8 micron and 0.9 micron, the focal length of the spherical micro-nano liquid drop lens 8 corresponding to the two sections is almost unchanged, and the half-width of photon nano jet is gradually increased; when the radius is in the interval of 0.3 to 0.4 micrometers, the focal length of the spherical micro-nano droplet lens 8 is gradually increased, and the half width is hardly changed.
In this embodiment, the photon nano-jet generated by the spherical micro-nano droplet lens 8 excites the sample fluorescence on the sample stage 11, the fluorescence generated by the sample surface is reversely collected by the micro-objective lens 6 and then is emitted to the linear polarizer 5, the fluorescence is incident on the surface of the dichroic mirror 4 after passing through the linear polarizer 5, and after being reflected by the dichroic mirror 4, the fluorescence is focused by the second lens group 12 and is collected and imaged by the imaging device 13.
The computer control system 14 is connected with the micro-flow tube 9, and is used for programming and driving the micro-flow tube 9 to release or absorb a certain volume of liquid drops in the packaging box 7, so as to control the radius of the spherical micro-nano liquid drop lens 8, and the change of the radius of the spherical micro-nano liquid drop lens 8 can influence the focusing characteristic of the spherical micro-nano liquid drop lens 8, thereby realizing the regulation and control of the focal length of the spherical micro-nano liquid drop lens 8.
In this embodiment, the microscope objective 6 may be a UPLSAP0100XS super apochromatic objective of olympus, the magnification is 100 times, and the numerical aperture is 1.35.
The imaging device 13 in this embodiment may be a DCC1545M type high resolution black and white CMOS camera available from Thorlabs corporation, and the pixels are 1280×1024.
In this embodiment, the sample stage 11 is made of silica, and the sample on the sample stage 11 is calibrated in advance by the fluorescent dye Calcium Crimson, and when the fluorescent dye is irradiated by a light beam with a wavelength of 589 nm, fluorescence with a wavelength of 615 nm is generated.
In the embodiment, the focal length of the spherical micro-nano liquid drop lens 8 is changed by changing the radius of the spherical micro-nano liquid drop lens 8, as shown in fig. 2, the scanning of different depths of a sample can be realized under the condition of no mechanical structure vibration; the half-width of photon nanometer injection corresponding to the radius of the different spherical micro-nano liquid drop lenses 8 is smaller than 250 nanometers as shown in figure 3, namely smaller than half of the wavelength of the light beam emitted by the laser, so as to achieve the super-resolution focusing effect; the micro displacement table 10 is combined to regulate and control the movement of the sample table 11 along the horizontal direction of the plane where the sample table 11 is positioned, so that the multi-dimensional scanning of the observed sample is realized, the observation precision is improved, and the three-dimensional super-resolution microscopic imaging is realized.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solution of the patent and not limiting, and that a person skilled in the art may make several variations and modifications without departing from the principles of the patent, which should also be regarded as the protection scope of the patent.
Claims (4)
1. The focusing controllable super-resolution microscopic device based on the spherical micro-nano droplet lens is characterized by comprising a laser, a reflecting mirror, a first lens group, a dichroic mirror, a linear polarizer, a microscope objective, an encapsulation box, the spherical micro-nano droplet lens, a micro-flow pipe, a micro-displacement table, a sample table, a second lens group, an imaging device and a computer control system;
the light beam output by the laser is incident on a reflecting mirror, the reflecting mirror reflects the incident light beam to a first lens group, the light beam passing through the first lens group enters a dichroic mirror, the light beam passes through the dichroic mirror to be transmitted and then is incident on a linear polarizer, linearly polarized light emitted from the linear polarizer is incident on a rear focal plane of the microscope objective, and forms parallel light beams after passing through the microscope objective to be incident on the spherical micro-nano liquid drop lens, a photon nanometer jet effect is generated on the other side of the spherical micro-nano liquid drop lens, and the jetted nanometer photons excite a sample on the sample stage to generate fluorescence;
fluorescence is reversely collected by the microscope objective, the fluorescence collected by the microscope objective is emitted to the linear polarizer, the fluorescence is incident to the surface of the dichroic mirror after passing through the linear polarizer, and the fluorescence is focused to an imaging device through the second lens group after being reflected by the dichroic mirror, so that imaging is realized;
the computer control system controls the micro-flow pipe to release or absorb a certain volume of liquid drops in the packaging box, so as to regulate and control the radius of the spherical micro-nano liquid drop lens;
the spherical micro-nano liquid drop lens can generate photon nano injection by being irradiated by parallel light beams emitted by a microscope;
the photon nanometer jet refers to a focusing light spot which is generated on the other side of the spherical micro-nano liquid drop lens and has a diameter of a sub-wavelength or a half-sub-wavelength, a small divergence angle, a high focusing light intensity and a focal length of more than 2 lambda or less than 2 lambda when the spherical micro-nano liquid drop lens is irradiated by a parallel light beam.
2. The focusing controllable super-resolution microscopy device based on the spherical micro-nano liquid drop lens according to claim 1, wherein the focusing controllable super-resolution microscopy device is characterized in that: the laser outputs a light beam in the wavelength range of 570 nm to 670 nm.
3. The focusing controllable super-resolution microscopy device based on the spherical micro-nano liquid drop lens according to claim 1, wherein the focusing controllable super-resolution microscopy device is characterized in that: the spherical micro-nano liquid drop lens is made of water.
4. The focusing controllable super-resolution microscopy device based on the spherical micro-nano liquid drop lens according to claim 1, wherein the focusing controllable super-resolution microscopy device is characterized in that: the numerical aperture of the microscope objective is 1.35.
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| CN109387481A (en) * | 2018-11-28 | 2019-02-26 | 同济大学 | A kind of apparatus and method detecting circular dichroism |
| CN111381355B (en) * | 2018-12-29 | 2022-08-02 | 北京雅谱光仪科技有限公司 | Optical imaging apparatus and method |
| CN110646389B (en) * | 2019-09-26 | 2021-07-23 | 中国科学院长春应用化学研究所 | Super-resolution multi-color laser scanning optical fiber probe based on transparent medium microspheres and manufacturing method thereof |
| CN111007065B (en) * | 2019-12-24 | 2022-10-14 | 暨南大学 | Liquid drop microlens mixed solution, liquid drop microlens array preparation method, deformation method, imaging method and signal enhancement method |
| CN112505009A (en) * | 2020-11-12 | 2021-03-16 | 中国科学院长春光学精密机械与物理研究所 | Super surface lens and fluorescence signal collection system formed by same |
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