CN109269979B - Sample placing system and method for obtaining single-particle fluorescence-micro morphology - Google Patents
Sample placing system and method for obtaining single-particle fluorescence-micro morphology Download PDFInfo
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- CN109269979B CN109269979B CN201811074855.3A CN201811074855A CN109269979B CN 109269979 B CN109269979 B CN 109269979B CN 201811074855 A CN201811074855 A CN 201811074855A CN 109269979 B CN109269979 B CN 109269979B
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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
- G01N21/62—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
Abstract
The invention discloses a sample placing system and a sample placing method for obtaining single-particle fluorescence-microscopic morphology. The system comprises: the device comprises a first sealing ring, a slide glass, a second sealing ring, a cover plate, a fixing ring, a film window for a transmission electron microscope and a gasket; the single particle sample is loaded on the lower surface of the film window; when the system is used for single-particle fluorescence measurement, the whole set of sample placement system is arranged above an objective lens of a copolymerization light fluorescence microscope, and a fluorescence spectrum and a fluorescence image can be obtained; when the micro-morphology analysis is carried out, the film window in the system is taken down, the film window is placed in a transmission electron microscope sample rod in a reversed mode, and a transmission electron microscope is used for representing, so that a micro-morphology image of a corresponding area can be obtained; the invention can realize the single-particle fluorescence and microscopic morphology analysis of the same nano-particle, and has the characteristics of clear single-particle fluorescence signal and high microscopic morphology resolution.
Description
Technical Field
The invention relates to the field of single-particle fluorescence spectrum measurement and micro-morphology analysis research, in particular to a sample placing system for obtaining single-particle fluorescence-micro-morphology and a using method thereof.
Background
The single-particle fluorescence spectrum technology is based on a laser scanning confocal microscope, can reveal an energy relaxation mechanism of a single nano particle on the scale of the single nano particle and monitor a photochemical reaction process generated on the surface of the single nano particle in real time, and becomes an effective means for researching a charge transfer mechanism and a catalytic reaction mechanism. The traditional single-particle fluorescence spectrum technology cannot provide high-resolution micro-morphology information at the same time, and the research of the technology on a charge transfer mechanism and a photochemical reaction process in a fine nano structure is limited. The difficulty lies in that:
(1) the acquisition of high-resolution micro-morphology information needs the assistance of a Transmission Electron Microscope (TEM), while in the traditional single-particle fluorescence test, a sample is polydispersed on a quartz glass slide, and after the single-particle fluorescence measurement, the analysis of the micro-morphology can only be carried out by means of a Scanning Electron Microscope (SEM), so that the resolution of the analysis of the micro-morphology is low; if a transmission electron microscope is used for carrying out appearance analysis on the sample, an optimal design needs to be carried out on a sample setting system;
(2) the fluorescence of a single nanoparticle is very weak, stronger laser is needed to be used as a light source for excitation, the quartz glass slide has the advantages of low fluorescence background noise and the like, the acquisition of single-particle fluorescence signals is facilitated, and if a new sample placing system capable of carrying out transmission electron microscope analysis is used, the problems of fluorescence background noise and the like under strong laser are required to be overcome;
(3) the conventional quartz glass slide has sufficient mechanical strength, and the copper mesh/carbon film for the conventional transmission electron microscope cannot be subjected to high-speed spin coating sample preparation, so that a sample with dispersed single particles cannot be obtained.
Therefore, new research designs for sample placement systems for obtaining single particle fluorescence-microtopography are needed.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a sample placement system for obtaining single-particle fluorescence-micro morphology, so that the fluorescence characteristics and high-resolution micro morphology of a single nano particle are measured and analyzed, the micro morphology resolution of a single-particle spectrum technology is greatly improved, and a new research means is provided for deeply researching a photon-generated carrier transfer mechanism and a single-particle surface catalytic reaction process.
The specific scheme of the sample placement system for obtaining single-particle fluorescence-micro morphology is as follows:
a sample placement system for obtaining single-particle fluorescence-microscopic morphology comprises a slide glass and a cover plate arranged at a set distance from the slide glass, wherein a thin film window is supported on the surface of the slide glass through a gasket, a fixing ring is arranged on one side of the cover plate in the circumferential direction of the thin film window to limit the thin film window, a space is reserved between the thin film window and the slide glass, and a sample is arranged on the surface of the thin film window.
The sample placing system can effectively place single-particle dispersed samples, is particularly arranged on the lower surface of the film window, the samples only contact with the silicon oxide film window and do not directly contact with a quartz slide glass below, the sample placing component is convenient to set, and the stability and the reliability of sample setting can be guaranteed.
Further, the fixing ring is connected or contacted with the cover plate, the inner diameter of the fixing ring is larger than the outer diameter of the silicon oxide film window by 0.01-0.8 mm, the fixing effect is achieved, and the inner diameter of the stainless steel gasket is slightly smaller than the size of the silicon oxide film window for the transmission electron microscope.
The fixing ring and the gasket are made of metal materials, preferably stainless steel materials, and have good chemical and physical stability, and the cover plate and the slide glass can be made of silicon oxide, aluminum oxide, silicon carbide or glass which are transparent to light, so that the good light transmittance of the cover plate and the slide glass is ensured.
Furthermore, the film window is a silicon oxide film window, the film material on the surface of the film window is silicon oxide or aluminum oxide or silicon nitride, and the window is transparent to the projection electron beam, so that the window is ensured to have good light transmittance.
Further, the cover plate is clamped through the first sealing ring, the slide glass is supported through the second sealing ring, the second sealing ring is connected with the first sealing ring, the two sealing rings are made of metal materials or plastic materials, and the two sealing rings are connected with each other through a threaded structure or a magnetic structure.
Further, the film window is in a grid shape and is single-hole or multi-hole.
Further, the gasket is an annular gasket.
In order to overcome the defects of the prior art, the invention provides an installation method of a sample placement system for acquiring single-particle fluorescence-micro morphology, which comprises the following specific steps:
1) carrying out ozone cleaning treatment and aluminum oxide atomic layer deposition pretreatment on the film window;
2) cleaning the fixing ring, the gasket, the cover plate and the slide glass;
3) arranging a slide glass in the second sealing ring, and arranging a gasket in the center of the slide glass;
4) installing a film window with a sample on a gasket, and sleeving the film window by using a fixing ring for fixing;
5) the cover plate is arranged above the fixing ring, and the first sealing ring is sleeved on the circumference of the cover plate to be compressed and sealed.
The method combines the aluminum oxide atomic layer deposition and the ozone cleaning treatment mode, overcomes the difficulties of fluorescence background noise and the like, greatly improves the resolution of the single-particle fluorescence spectrum technology, and provides a new research means for deeply researching a photon-generated carrier transfer mechanism and a single-particle surface catalytic reaction process.
The thickness of the deposited aluminum oxide atomic layer of the film window in the step 1) is 2-20 nanometers, the thickness of the film window is 10-100 nanometers, and the deposition temperature of the aluminum oxide atomic layer is 120-200 ℃;
the setting process of the film window with the sample in the step 4) is as follows: carrying out high-speed centrifugal cleaning on the suspension of a set sample for a set number of times, ultrasonically dispersing the suspension in deionized water at a set dilution multiple, fixing a film window in the center of a spin coater, coating the suspension with a set volume on the surface of the film window, carrying out spin coating at a set rotating speed and drying at normal temperature to obtain a single dispersed sample.
The invention also provides a use method of the sample placement system for obtaining the single-particle fluorescence-micro morphology,
when the system is used for single-particle fluorescence measurement, the whole set of sample placement system is arranged above an objective lens of a copolymerization light fluorescence microscope, a slide glass is directly contacted with the objective lens through oil drops with set refractive index, a fluorescence image of single dispersed nano particles can be obtained after laser excitation, and a corresponding fluorescence spectrum can be obtained at the same time;
when the microscopic morphology is analyzed, the film window is taken out, placed in a transmission electron microscope sample rod in a reversed mode, and characterized by a transmission electron microscope, so that a transmission electron microscope image of a region corresponding to a fluorescence image can be obtained, and finally the fluorescence characteristics (fluorescence spectrum and fluorescence image) of a single nanoparticle and the transmission electron microscope microscopic morphology corresponding to the fluorescence characteristics can be obtained.
Compared with the prior art, the invention has the beneficial effects that:
1) the invention can be used for fluorescence image and fluorescence spectrum analysis of a confocal fluorescence microscope and can also be used for microscopic morphology analysis of a high-resolution transmission electron microscope by arranging the film window for the transmission electron microscope; when the traditional single-particle fluorescence analysis is carried out, the sample is polydispersed on a quartz glass slide, and after the fluorescence characteristic measurement is carried out, the analysis of the micro-morphology can only be carried out by means of a scanning electron microscope, so that the resolution of the micro-morphology analysis is lower, and therefore, the high-resolution transmission electron microscope greatly improves the resolution of the micro-morphology of the sample in the single-particle fluorescence analysis process.
2) According to the invention, the single-particle sample is separated from the quartz slide by the stainless steel gasket, so that adverse effects of the quartz slide on the single-particle sample, such as mechanical displacement of single nanoparticles caused by pollution or external force, are effectively avoided.
3) The silicon oxide film window is subjected to ozone cleaning treatment and aluminum oxide atomic layer deposition pretreatment, so that the fluorescence background noise under strong laser excitation is effectively reduced, and the signal resolution of single-particle fluorescence is greatly improved.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the application and, together with the description, serve to explain the application and are not intended to limit the application.
FIG. 1 is a schematic diagram of a sample placement system according to the present invention;
FIG. 2(a) is a top view of a coverslip/slide of the present invention;
FIG. 2(b) is a top view of the retaining ring of the present invention;
FIG. 2(c) is a top view of a gasket of the present invention;
FIG. 3(a) is a side view of a single aperture film window of the present invention;
FIG. 3(b) is a top view of a single aperture film window of the present invention;
FIG. 3(c) is a side view of a window of a porous membrane of the present invention;
FIG. 3(d) is a top view of a porous membrane window of the present invention;
FIG. 4(a) is a fluorescence image of a single gold nanoparticle obtained in example of the present invention (in the figure, the flower circle portion is weakly colored to indicate fluorescence, and is converted to black and white);
FIG. 4(b) is a transmission electron micrograph corresponding to a fluorescence image of a single gold nanoparticle obtained in an example of the present invention;
FIG. 4(c) is a diagram of the embodiment of the present invention shown in FIG. 4(a) -3 in FIG. 4(b)#Fluorescence spectrum image of gold nanoparticles.
The device comprises a base, a first sealing ring, a second sealing ring, a slide glass, a second sealing ring, a cover plate, a first fixing ring, a second fixing ring, a film window, a second fixing ring, a first sealing ring, a second sealing ring.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
As described in the background, the prior art is deficient in that the present application provides a sample placement system for obtaining single particle fluorescence-microtopography.
In a typical embodiment of the present application, as shown in fig. 1, a sample placement system for obtaining single-particle fluorescence-microscopic morphology includes a slide 2 and a cover plate 4 disposed at a set distance from the slide, a gasket 7 is disposed on the surface of the slide 2 to support a thin film window 6, the thin film window is disposed on the upper surface of the gasket, a fixing ring 5 is disposed between the thin film window 6 and the cover plate to limit the thin film window 6, a space is left between the thin film window 6 and the slide 2, a sample is disposed on the lower surface of the thin film window, and the gasket 7 is an annular gasket, as shown in fig. 2 (c).
The sample placing component can effectively place single-particle dispersed samples, is particularly arranged on the lower surface of the film window 6, the samples only contact with the silicon oxide film window 6 and do not directly contact with the quartz slide 2 below, the sample placing component is convenient to set, and the stability and the reliability of sample setting can be guaranteed.
The fixing ring 5 is connected or contacted with the cover plate 4, the inner diameter of the fixing ring 5 is larger than the peripheral size of the silicon oxide film window protruding block and smaller than the bottom size of the film window, the fixing ring plays a role in fixing the clamping position of the film window, the inner diameter of the stainless steel gasket is slightly smaller than the size of the silicon oxide film window for the transmission electron microscope, and the fixing ring and the gasket are spaced at a set distance.
The fixing ring 5 and the gasket 7 are made of metal materials, preferably stainless steel materials, and have good chemical and physical stability, and the cover plate and the slide glass can be made of silicon oxide, aluminum oxide, silicon carbide or glass which are transparent to light, so that the good light transmittance is ensured.
The thin film window 6 is a silicon oxide thin film window, and the thin film window 6 is composed of silicon oxide, aluminum oxide, or silicon nitride transparent to the transmitted electron beam.
Cover plate 4 is through 1 block of first sealing washer, slide glass 2 supports through second sealing washer 3, and second sealing washer 3 is connected with first sealing washer 1, and two sealing washer materials are metal material or plastic material, and two sealing washers pass through helicitic texture or magnetic force structure interconnect. The film window 6 is in a grid shape, and is single-hole or multi-hole, as shown in fig. 3(a) -3 (b).
The invention provides an installation method of a sample placement system for obtaining single-particle fluorescence-microscopic morphology, which comprises the following specific steps:
1) carrying out ozone cleaning treatment and aluminum oxide atomic layer deposition pretreatment on the thin film window 6;
2) cleaning the fixing ring 5, the gasket 7, the cover plate 4 and the slide 2;
3) arranging a slide glass 2 in the second sealing ring 3, and arranging a gasket 7 in the center of the slide glass 2;
4) installing a film window 6 with a sample on a gasket 7, and fixing the film window 6 by sleeving a fixing ring 5;
5) the cover plate 4 is arranged above the fixing ring 5, and the first sealing ring 1 is sleeved on the circumference of the cover plate 4 for compression sealing.
The method combines the aluminum oxide atomic layer deposition and the ozone cleaning treatment mode, overcomes the difficulties of fluorescence background noise and the like, and greatly improves the signal resolution of single-particle fluorescence. Provides a new research means for deeply researching a photon-generated carrier transfer mechanism and a single-particle surface catalytic reaction process.
The thickness of the deposited aluminum oxide atomic layer of the film window in the step 1) is 2-10 nanometers, the thickness of the film window is 10-100 nanometers, and the deposition temperature of the aluminum oxide atomic layer is 120-200 ℃;
taking single particle fluorescence analysis of gold nanorods as an example: when a sample is prepared, the suspension of the gold nanorods is subjected to high-speed centrifugal cleaning twice, and finally diluted ten times of ultrasonic dispersion is carried out in deionized water, a silicon oxide film window is fixed in the center of a spin coater, 10 microliters of diluted gold nanorod suspension is dropped on the surface of the silicon oxide film window for a transmission electron microscope, spin coating is carried out at the rotating speed of 2000rpm, and drying is carried out at normal temperature, so that a single dispersed sample is obtained.
The invention also provides a use method of the sample placement system for obtaining the single-particle fluorescence-micro morphology,
when the system is used for single-particle fluorescence measurement, the whole set of sample placement system is arranged above an objective lens of a copolymerization light fluorescence microscope, a slide glass is directly contacted with the objective lens through oil drops with specific refractive indexes, and a fluorescence image of gold nanoparticles can be obtained after laser excitation, as shown in fig. 4 (a); corresponding fluorescence spectra of the gold nanoparticles can be obtained at the same time, FIG. 4(c) is 3 in FIG. 4(a-b)#Fluorescence spectra of gold nanoparticles.
When the microscopic morphology analysis is carried out, the film window is taken out, placed in a transmission electron microscope sample rod in a reversed mode, and characterized by a transmission electron microscope, so that a transmission electron microscope image of the gold nanoparticles in the corresponding area of the graph 4(a) can be obtained and is shown in the graph 4 (b); wherein the magnified transmission electron microscopy topography is placed as an inset next to the corresponding fluorescence spot of FIG. 4 (a).
Therefore, the fluorescence characteristics (fluorescence spectrum and fluorescence image) of a single specific nanoparticle and the corresponding transmission electron microscope microscopic morphology thereof can be finally obtained.
The embodiment result shows that the fluorescence image and the spectrum of a single gold nanorod and the transmission electron microscope image can be obtained simultaneously, the fluorescence background noise is low, the fluorescence signal and the microscopic morphology resolution of the single-particle spectrum technology are greatly improved, and an effective research means is provided for deeply researching the fluorescence characteristic and the surface characteristic of a single particle.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims (8)
1. The sample placement system is characterized by comprising a slide glass and a cover plate arranged at a set distance from the slide glass, wherein the surface of the slide glass supports a thin film window through a gasket, one side of the cover plate is provided with a fixing ring in the circumferential direction of the thin film window so as to limit the thin film window, a space is reserved between the thin film window and the slide glass, and a sample is arranged on the surface of the thin film window; the film material on the surface of the film window is silicon oxide or aluminum oxide or silicon nitride which is transparent to light rays, and the film window is transparent to transmitted electron beams; the cover and slide are transparent to light.
2. The sample placement system for obtaining single particle fluorescence-microtopography of claim 1, wherein the fixing ring is connected or in contact with the cover plate.
3. The sample placement system for obtaining single-particle fluorescence-microtopography of claim 1, wherein the cover plate is clamped by a first sealing ring, the slide is supported by a second sealing ring, and the second sealing ring is connected with the first sealing ring.
4. The sample placement system for obtaining single particle fluorescence-microtopography of claim 1, wherein the thin film window is grid-shaped, single-hole or multi-hole.
5. The sample placement system for obtaining single-particle fluorescence-microtopography of claim 1, wherein the gasket is an annular gasket.
6. The method for installing a sample placement system for obtaining single-particle fluorescence-microtopography according to any one of claims 1 to 5, characterized by comprising the following steps:
1) carrying out ozone cleaning treatment and aluminum oxide atomic layer deposition pretreatment on the film window;
2) cleaning the fixing ring, the gasket, the cover plate and the slide glass;
3) arranging a slide glass in the second sealing ring, and arranging a gasket in the center of the slide glass;
4) installing a film window with a sample on a gasket, and sleeving the film window by using a fixing ring for fixing;
5) the cover plate is arranged above the fixing ring, and the first sealing ring is sleeved on the circumference of the cover plate to be compressed and sealed.
7. The installation method of the sample placement system for obtaining single-particle fluorescence-microtopography according to claim 6, wherein the thickness of the thin-film window aluminum oxide atomic layer deposition in the step 1) is 2-20 nm, and the deposition temperature of the aluminum oxide atomic layer is 120-200 ℃;
the setting process of the film window with the sample in the step 4) is as follows: carrying out high-speed centrifugal cleaning on the suspension of a set sample for a set number of times, ultrasonically dispersing the suspension in deionized water at a set dilution multiple, fixing a film window in the center of a spin coater, coating the suspension with a set volume on the surface of the film window, carrying out spin coating at a set rotating speed and drying at normal temperature to obtain a single dispersed sample.
8. Use of a sample placement system for obtaining single particle fluorescence-microtopography according to any of claims 1 to 5,
when the system is used for single-particle fluorescence measurement, the whole set of sample placement system is arranged above an objective lens of a copolymerization light fluorescence microscope, a slide glass is directly contacted with the objective lens through oil drops with set refractive index, a fluorescence image of single dispersed nano particles can be obtained after laser excitation, and a corresponding fluorescence spectrum can be obtained at the same time;
when the microscopic morphology is analyzed, the film window is taken out, placed in a transmission electron microscope sample rod in a reversed mode, and represented by a transmission electron microscope, so that a transmission electron microscope image of a region corresponding to a fluorescence image can be obtained, and finally the fluorescence characteristic of a single nanoparticle and the transmission electron microscope microscopic morphology corresponding to the fluorescence characteristic can be obtained.
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