CN111766692A - Automatic fluid infusion microsphere super-resolution microscopic imaging system - Google Patents
Automatic fluid infusion microsphere super-resolution microscopic imaging system Download PDFInfo
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- CN111766692A CN111766692A CN202010559039.2A CN202010559039A CN111766692A CN 111766692 A CN111766692 A CN 111766692A CN 202010559039 A CN202010559039 A CN 202010559039A CN 111766692 A CN111766692 A CN 111766692A
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/02—Objectives
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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
- 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
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B27/00—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00
- G02B27/58—Optics for apodization or superresolution; Optical synthetic aperture systems
Abstract
The invention discloses an automatic liquid-supplementing microsphere super-resolution microscopic imaging system which comprises a microscope, a sample stage, a microsphere probe, a first moving mechanism, a capillary tube, a micropump and a second moving mechanism, wherein the microscope is positioned above the sample stage, the microsphere probe comprises a probe and a microsphere lens connected with one end of the probe, the other end of the probe is installed on the first moving mechanism, the capillary tube is installed on the second moving mechanism, the liquid inlet end of the capillary tube is connected with the micropump, and the liquid outlet end of the capillary tube is close to the microsphere lens. The invention can keep the depth of the microsphere lens immersed in the liquid consistent, avoid the change of the contrast and the magnification of the imaging due to the volatilization of the liquid and keep the consistency of the imaging; the microsphere lens achieves the best imaging effect; the diameter of the microsphere lens is in a micron order, the required liquid amount is small, and the liquid can automatically volatilize after observation, so that the sample is prevented from being polluted or damaged.
Description
Technical Field
The invention relates to the technical field of microscopic imaging, in particular to an automatic liquid supplementing microsphere super-resolution microscopic imaging system.
Background
In three hundred years from the first microscope, researchers at home and abroad have proposed various methods for improving the imaging effect and the system resolution, and developed various microscopes based on different mechanisms and principles. For electron microscopes, scanning tunneling microscopes and atomic force microscopes with imaging resolution on the order of nanometers or even atomic scales, although the imaging resolution is very high, there are severe limitations on the imaging conditions and samples, especially the inability to image living biological samples, which limits the application in biomedicine. Because the optical microscope can directly observe a sample in real time and is accompanied by the characteristics of rich functional imaging information (such as color, transparency and the like), the optical microscope still has irreplaceable effect in the field of microscopic imaging such as biomedicine and the like. With the development of modern biotechnology, biological samples that one needs to observe have been extended from biological individuals to the organ, tissue, cell, or even single molecule level. Particularly, with the further development of chemical analysis techniques for nucleic acids and proteins, life sciences have been gradually shifted to the fields of molecular biology, molecular immunology, molecular cytology, molecular genetics, and the like. These technical studies put higher demands on the resolution capability and imaging conditions of the microscope, so that the exploration of an optical super-resolution imaging method, especially a far-field optical microscopic imaging method, which breaks through the diffraction limit and obtains higher resolution capability, has become a very critical technical problem in the biomedical field. In recent decades, with the continuous efforts of researchers, some novel optical super-resolution microscopy methods and techniques have been proposed and made a breakthrough progress, which brings about the observation and research of nanoscale biological samples.
A model for realizing super-resolution microscopic imaging by using transparent medium microspheres under white light is established by a Wangcheng group at the university of Manchester in Britain in 2011, the diameter of the silica medium microspheres capable of realizing super-resolution is required to be 2-9 um, and the microspheres are directly placed on the surface of a sample to be tested, so that super-resolution imaging can be realized under a common optical microscope. The microscope works in a white light mode, and under a transmission mode, transparent medium microspheres with the diameter of 4.74um are adopted to successfully image a fishnet gold-plated anodic aluminum oxide film sample with the diameter of 50nm and spaced holes; under the reflection mode, the 100-nanometer wide lines on the surface of the protective film of the blue-ray DVD can be clearly seen through the silicon dioxide microspheres, and the limit of diffraction limit is successfully broken.
The microsphere lens can realize super-resolution imaging by combining with an optical microscope. In the subsequent research, researchers find that microspheres made of silicon dioxide, barium titanate and polystyrene have super-resolution imaging capability, and that half-immersed silicon dioxide or barium titanate microspheres have better imaging effect in water or alcohol solution. However, during observation, due to evaporation of water or alcohol, the depth of the microsphere lens immersed in liquid is reduced, so that the imaging magnification and the imaging contrast of the microsphere lens are changed, and the imaging consistency of the microsphere lens is influenced; in addition, in the observation of a sample such as a chip, the dispersion of a large amount of liquid on the surface of the sample affects the circuit structure of the chip and the reuse of the chip.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide an automatic liquid supplementing microsphere super-resolution microscopic imaging system.
In order to achieve the above object, an embodiment of the present invention provides the following technical solutions:
the utility model provides an automatic fluid infusion microballon super-resolution microscopic imaging system, includes microscope, sample platform, microballon probe, first moving mechanism, capillary, micropump and second moving mechanism, the microscope is located the top of sample platform, the microballon probe include the probe, with the microballon lens that the one end of probe is connected, the other end of probe is installed on the first moving mechanism, the capillary is installed on the second moving mechanism, the feed liquor end of capillary with the micropump is connected, the play liquid end of capillary is close to microballon lens.
As a further improvement of the present invention, the second moving mechanism is mounted on the first moving mechanism, and the capillary is located below the probe.
As a further improvement of the invention, the distance between the liquid outlet end of the capillary and the microsphere lens is 50-100 μm.
As a further improvement of the invention, the capillary tube is a capillary glass tube.
As a further improvement of the invention, the microsphere lens is made of silicon dioxide or barium titanate.
As a further improvement of the invention, the microsphere lens is adhered to one end of the probe.
As a further improvement of the present invention, the first moving mechanism includes a first horizontal moving platform, a second horizontal moving platform installed on the first horizontal moving platform, a vertical moving platform installed on the second horizontal moving platform, and a first angle adjusting mechanism installed on the vertical moving platform, and the other end of the probe is installed on the first angle adjusting mechanism.
As a further improvement of the present invention, the other end of the probe is connected to an extension bar, and the extension bar is mounted on the first angle adjusting mechanism.
As a further improvement of the present invention, the second moving mechanism includes a three-axis moving platform, and a second angle adjusting mechanism mounted on the three-axis moving platform, and the capillary tube is mounted on the second angle adjusting mechanism.
As a further improvement of the invention, the sample stage is an XY moving platform.
The invention has the beneficial effects that:
(1) the invention can keep the depth of the microsphere lens immersed in the liquid consistent, avoid the contrast and magnification of the imaging changed due to the volatilization of the liquid and keep the consistency of the imaging.
(2) The liquid supplementing device can automatically and quantitatively control the liquid supplementing amount according to the material and the diameter of the microsphere lens, the type of the supplementing liquid, the immersion depth of the microsphere lens in the liquid and the evaporation speed of the liquid, so that the microsphere lens achieves the optimal imaging effect.
(3) During the formation of image of microballon lens super-resolution, need not to fill liquid full whole observation sample surface, solved and need in advance with a large amount of water or alcohol dropwise add at the problem of observing the sample surface when carrying out microballon microscopic observation, reduced the destruction of a large amount of liquid to the sample function, and the diameter of microballon lens is at the micron level, and the liquid volume of needs is few, and the liquid that needs to add is also few, and liquid can volatilize automatically after having observed, avoids causing pollution or damage to the sample.
(4) The microsphere probe and the second moving mechanism on which the capillary is mounted are both mounted on the first moving mechanism, and any region of the sample can be observed by the movement of the first moving mechanism.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments described in the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a schematic structural diagram of a preferred embodiment of the present invention;
FIG. 2 is a front view of the probe mounted on the first movement mechanism and the capillary mounted on the second movement mechanism of the preferred embodiment of the present invention;
FIG. 3 is a schematic structural diagram of a second moving mechanism according to a preferred embodiment of the present invention;
FIG. 4 is an enlarged view of a portion of a microsphere probe and a capillary according to a preferred embodiment of the present invention;
FIG. 5 is a schematic view of an imaging configuration of a preferred embodiment of the present invention;
in the figure: 10. microscope, 12, sample stage, 14, microsphere probe, 16, first moving mechanism, 18, capillary, 20, second moving mechanism, 22, probe, 24, microsphere lens, 26, liquid inlet end, 28, liquid outlet end, 30, first horizontal moving platform, 32, second horizontal moving platform, 34, vertical moving platform, 36, first angle adjusting mechanism, 38, first base, 40, first bearing rod, 42, first angle adjusting knob, 44, extension rod, 46, first connecting rod, 48, three-axis moving platform, 50, second angle adjusting mechanism, 52, second base, 54, second bearing rod, 56, second angle adjusting knob, 57, second connecting rod, 58, sample, 60, liquid, 62, super-resolution image.
Detailed Description
In order to make those skilled in the art better understand the technical solution of the present invention, the technical solution in the embodiment of the present invention will be clearly and completely described below with reference to the drawings in the embodiment of the present invention, and it is obvious that the described embodiment is only a part of the embodiment of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
As shown in fig. 1 and 4, an automatic fluid infusion microsphere super-resolution microscopic imaging system includes a microscope 10, a sample stage 12, a microsphere probe 14, a first moving mechanism 16, a capillary 18, a micropump (not shown in the figure) and a second moving mechanism 20, wherein the microscope 10 is located above the sample stage 12, the microsphere probe 14 includes a probe 22 and a microsphere lens 24 connected to one end of the probe 22, the other end of the probe 22 is mounted on the first moving mechanism 16, the capillary 18 is mounted on the second moving mechanism 20, a fluid inlet end 26 of the capillary 18 is connected to the micropump, and a fluid outlet end 28 of the capillary 18 is close to the microsphere lens 24.
To facilitate scanning of the microsphere lens 24, the present invention preferably has the second movement mechanism 20 mounted on the first movement mechanism 16 with the capillary tube 18 positioned below the probe 22.
In order that the liquid flowing out of the capillary tube 18 does not affect the imaging of the microsphere lens 24, the distance between the liquid outlet end 28 of the capillary tube 18 and the microsphere lens 24 is preferably 50-100 μm. Preferably, the inlet end 26 of the capillary 18 is connected to the micro-pump by a thin hose. Preferably, the capillary tube 18 is not in contact with the sample surface to avoid damage to the sample surface, while the capillary tube 18 does not prevent the microsphere lenses 24 from contacting the sample surface.
According to the invention, the capillary tube 18 is preferably a capillary glass tube, so that the liquid outlet end 28 of the capillary tube 18 can be conveniently formed, and the liquid supplementing accuracy is improved.
In the present invention, it is preferable that the microsphere lenses 24 are made of a silicon dioxide material, but the microsphere lenses 24 are not limited to a silicon dioxide material, and may be made of a barium titanate material.
In order to improve the stability of the connection between the microsphere lens 24 and the probe 22, the present invention preferably attaches the microsphere lens 24 to one end of the probe 22.
In the present invention, it is preferable that the first moving mechanism 16 includes a first horizontal moving platform 30, a second horizontal moving platform 32 mounted on the first horizontal moving platform 30, a vertical moving platform 34 mounted on the second horizontal moving platform 32, and a first angle adjusting mechanism 36 mounted on the vertical moving platform 34, wherein the other end of the probe 22 is mounted on the first angle adjusting mechanism 36, and the probe 22 is tilted by a proper angle by the first angle adjusting mechanism 36, so as to ensure that the bottom of the microsphere lens 24 can contact the surface of the sample. In this embodiment, the first horizontal moving platform 30 realizes movement along the X-axis direction, the second horizontal moving platform 32 realizes movement along the Y-axis direction, the vertical moving platform 34 realizes movement along the Z-axis direction, and the first horizontal moving platform 30, the second horizontal moving platform 32, and the vertical moving platform 34 all adopt general structures in the art, which are not described herein again. As shown in fig. 2, it is preferable that the first angle adjusting mechanism 36 includes a first base 38, a first bearing rod 40 hinged to the first base 38, and a first angle adjusting knob 42 screwed to the first base 38, the first base 38 is mounted on the vertical moving platform 34, a first bearing (not shown) is disposed in the first base 38, a bottom of the first angle adjusting knob 42 is engaged with an inner ring of the first bearing, an outer ring of the first bearing is engaged with the first base 38, and when the first angle adjusting knob 42 is rotated, the first angle adjusting knob 42 is lifted and lowered to allow the first bearing rod 40 to change the inclination angle, so that the probe 22 changes the inclination angle.
The other end of the preferred probe 22 of the present invention is connected to an extension bar 44. the extension bar 44 is mounted to the first angle adjustment mechanism 36. Further, a first connecting rod 46 is provided, an upper portion of the first connecting rod 46 is connected to the first carrier bar 40, a lower portion of the first connecting rod 46 is connected to the extension bar 44, and the extension bar 44 is inclined at a proper angle by rotating the first angle adjusting knob 42, thereby inclining the probe 22 at a proper angle. Of course, it will be appreciated that probe 22 and extension bar 44 may be integrally formed.
As shown in fig. 3, it is preferable that the second moving mechanism 20 of the present invention includes a three-axis moving platform 48, and a second angle adjusting mechanism 50 mounted on the three-axis moving platform 48, the three-axis moving platform 48 being capable of moving in the X-axis direction, the Y-axis direction, and the Z-axis direction, and the capillary tube 18 being mounted on the second angle adjusting mechanism 50. It is further preferable that the second angle adjusting mechanism 50 includes a second base 52, a second bearing rod 54 hinged to the second base 52, and a second angle adjusting knob 56 screwed to the second base 52, the second base 52 is mounted on the three-axis moving platform 48, a second bearing (not shown) is provided in the second base 52, a bottom of the second angle adjusting knob 56 is engaged with an inner ring of the second bearing, an outer ring of the second bearing is engaged with the second base 52, and when the second angle adjusting knob 56 is rotated, the second angle adjusting knob 56 is lifted and lowered to make the second bearing rod 54 change the inclination angle, so that the capillary 18 changes the inclination angle. It is further preferable that the second connecting rod 57 is connected to the second carrier rod 54, and the capillary tube 18 is inserted into the second connecting rod 57.
In the present invention, the sample stage 12 is preferably an XY moving stage, and can move in the X-axis and Y-axis directions.
When the invention is used, the first horizontal moving platform 30 and the second horizontal moving platform 32 are adjusted to enable the microsphere lens 24 to move to the upper part of the sample 58, the first angle adjusting knob 42 is rotated to adjust the position of the microsphere lens 24, then the vertical moving platform 34 is used for adjusting the height of the microsphere lens 24 to enable the microsphere lens 24 to contact the surface of the sample 58, the second moving mechanism 20 is adjusted to enable the liquid outlet end 28 of the capillary tube 18 to be close to the surface of the microsphere lens 24, when in observation, because the microsphere lens 24 needs to be half soaked in liquid to realize super-resolution imaging, when the capillary tube 18 and the microsphere lens 24 are moved to the area needing to be observed, the liquid 60 is conveyed to the surface of the sample 58 through the capillary tube 18, the microsphere lens 24 can realize super-resolution imaging to obtain a super-resolution image 62, as shown in fig. 5, according to the material and diameter of the microsphere lens 24, The type of the supplementary liquid, the depth of the microsphere lens 24 immersed in the liquid and the evaporation speed of the liquid are supplemented, the liquid is observed through a micro-vision system to form feedback to a micro pump, and the micro pump controls the supplement amount of the liquid, so that the microsphere lens 24 achieves the optimal imaging effect. Wherein the liquid may be water or alcohol. Since the second moving mechanism 20 on which the capillary tube 18 is mounted and the micro-ball lens 24 are mounted on the first moving mechanism 16, it is possible to move the first moving mechanism 16 and observe an arbitrary region of the sample 58. The observed trace amount of liquid on the surface of the sample 58 can be rapidly evaporated, thereby avoiding contamination or damage to the sample 58.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (10)
1. The utility model provides an automatic fluid infusion microballon super-resolution microscopic imaging system, its characterized in that, includes microscope, sample platform, microballon probe, first moving mechanism, capillary, micropump and second moving mechanism, the microscope is located the top of sample platform, the microballon probe include the probe, with the microballon lens that the one end of probe is connected, the other end of probe is installed on the first moving mechanism, the capillary is installed on the second moving mechanism, the feed liquor end of capillary with the micropump is connected, the play liquid end of capillary is close to microballon lens.
2. The system of claim 1, wherein the second moving mechanism is mounted on the first moving mechanism, and the capillary is located below the probe.
3. The automatic fluid infusion microsphere super-resolution microscopic imaging system according to claim 1 or 2, wherein the distance between the liquid outlet end of the capillary tube and the microsphere lens is 50-100 μm.
4. The automatic fluid infusion microsphere super-resolution microscopic imaging system according to claim 3, wherein the capillary tube is a capillary glass tube.
5. The automatic fluid infusion microsphere super-resolution microscopic imaging system according to claim 1, wherein the microsphere lens is made of silicon dioxide or barium titanate.
6. The system of claim 1, wherein the microsphere lens is bonded to one end of the probe.
7. The system according to claim 1, wherein the first moving mechanism comprises a first horizontal moving platform, a second horizontal moving platform installed on the first horizontal moving platform, a vertical moving platform installed on the second horizontal moving platform, and a first angle adjusting mechanism installed on the vertical moving platform, and the other end of the probe is installed on the first angle adjusting mechanism.
8. The system of claim 7, wherein an extension bar is connected to the other end of the probe, and the extension bar is mounted on the first angle adjustment mechanism.
9. The system of claim 1, wherein the second moving mechanism comprises a three-axis moving platform, and a second angle adjusting mechanism mounted on the three-axis moving platform, and the capillary tube is mounted on the second angle adjusting mechanism.
10. The automatic fluid infusion microsphere super-resolution microscopic imaging system according to claim 1, wherein the sample stage is an XY moving platform.
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Cited By (2)
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| CN113189357A (en) * | 2021-05-06 | 2021-07-30 | 中国科学院上海微***与信息技术研究所 | Preparation method of dipping type sample applicator and micro-cantilever sensor chip |
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| CN113189357A (en) * | 2021-05-06 | 2021-07-30 | 中国科学院上海微***与信息技术研究所 | Preparation method of dipping type sample applicator and micro-cantilever sensor chip |
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