CN104198389A - Preparation method of copper-based suspended sample stage used for ultrahigh resolution fluorescence imaging - Google Patents
Preparation method of copper-based suspended sample stage used for ultrahigh resolution fluorescence imaging Download PDFInfo
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- CN104198389A CN104198389A CN201410484928.1A CN201410484928A CN104198389A CN 104198389 A CN104198389 A CN 104198389A CN 201410484928 A CN201410484928 A CN 201410484928A CN 104198389 A CN104198389 A CN 104198389A
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Abstract
The invention relates to a preparation method of copper-based suspended sample stage used for ultrahigh resolution fluorescence imaging, and relates to the technical field of ultrahigh resolution fluorescence imaging, in particular to the manufacturing of a copper foil base graphene sample stage. A micron-sized aperture is etched by utilizing a focused ion beam on a copper foil base where graphene is grown, and a chemical method is combined to protect a suspended graphene layer, so that a fluorescence sample is placed on the graphene layer. When the sample stage is used, a light spot of a smaller local area in a near field can be obtained by using a surface plasma lens to focus and compress an incident laser beam, and the light spot can penetrate through the ultrathin graphene layer to irradiate the sample and stimulate fluorescence. By using the copper-based suspended graphene sample stage and a corresponding surface plasma lens focusing mode, unnecessary background scattering can be reduced, so that the fluorescence quenching of the sample is reduced, the image contrast is improved, and the damage to a living body tissue injury is reduced.
Description
Technical field
The present invention relates to a kind of imaging sample stage preparation method, particularly a kind of unsettled Graphene sample stage of copper base preparation method for ultrahigh resolution fluorescence imaging.
Background technology
The imaging of ultrahigh resolution fluorescent samples is as fluorescence quantum [Appl. Phys. Lett. to fluorescigenic sample, 89,143117 (2006)] or the biomedical samples (as DNA) [Phys.Rev.Lett. 97,260801 (2006)] crossed of fluorescence labeling to carry out resolution be nano level imaging technique.In general, sample and probe that fluorescence needs laser by being inverted object lens, to focus on microslide upper surface excite, as shown in Figure 1 the area schematic between transparent sample and probe.But the spot size minimum that object lens focus on is approximately wavelength half (being hundreds of nanometer).And imaging resolution has reached the level of tens nanometers or several nanometers at present.Resolution depends mainly on the radius-of-curvature of probe tip.Tip is sharper, and radius-of-curvature is less, and resolution is higher.Probe tip minimum profile curvature radius can reach 10 nanometer left and right, even less [ACS Nano, 5 (4), 2570 – 2579 (2011)] at present.Therefore the hot spot, focusing on by object lens is much larger than the size of probe tip.The illumination of this part unnecessary hot spot, can cause fluorescent cancellation, and picture contrast declines, backscatter light, and the infringement of biological tissue.For opaque sample, laser focuses on above sample oblique incidence illumination sample by object lens and probe tip exists the problems referred to above equally, as Fig. 2 laser focuses on oblique incidence by long reach object lens, is radiated at the area schematic between opaque sample and probe.If can find a kind of lighting system, hot spot is focused on only have the zonule of even several nanometers of tens nanometers, and intensity is enough strong, can excited sample fluorescence, just can address the above problem.Although the surface plasma lens that are comprised of precious metal material microstructure can be realized nano-focusing and the enhancing of incident laser, this light field is confined to metal material surface or probe tip near field range.Leave metal surface 100nm and just almost there is no what intensity [Nano Lett., 8 (9), 3041-3045 (2008)].Therefore, cannot see through general microslide is at present irradiated on fluorescent samples.
Graphene (Graphene) is a kind ofly by monolayer carbon atomic arrangement, to be the cancellated carbonaceous novel film material of sexangle, is known thin, the hardest nano material in the world.Single-layer graphene thickness only has 0.3 nanometer.And the Graphene of individual layer has the light transmission rate up to 97.7%.The single crystal graphene size of growing on Copper Foil matrix with chemical vapour deposition technique at present, can reach 5 millimeters or larger [Adv.Funct. Mater. 23,198 – 203 (2013)].
Summary of the invention
The present invention be directed to laser while being positioned at the probe tip of sample on microslide and very close sample by object lens focus illumination (both can be close to and be only had several nanometers by signal feedback control system), hot spot is much larger than needle point size, thereby cause unnecessary laser light scattering background, the fluorescent quenching of sample, the infringement of live body sample, the problem that picture contrast declines, propose a kind of unsettled sample stage of copper base for ultrahigh resolution fluorescence imaging and preparation method thereof, prepared unsettled transparent Graphene micron order aperture on copper matrix as fluorescent material sample stage.On this sample stage, fluorescent material can excite through Graphene with the focus surface plasma wave that is confined near field.
Technical scheme of the present invention is: the unsettled sample stage preparation method of a kind of copper base for ultrahigh resolution fluorescence imaging, in the Copper Foil substrate of the Graphene of having grown, there is a micron order aperture, and micron order aperture only runs through copper foil layer, specifically comprises the steps:
(1) first by the Copper Foil substrate of the Graphene of having grown, copper foil layer 25 micron thickness, purity 99.98%, by focused ion beam, making a call to a degree of depth is 20 microns, radius is the aperture of 2 microns;
(2) on graphene layer surface, drip a small amount of organic glass solution, i.e. polymetylmethacrylate, to being set on graphene layer again;
(3) then the Graphene Copper Foil substrate that scribbles organic glass is put into the liquor ferri trichloridi having prepared and slightly rock, its surface is fully contacted with solution;
After (4) ten minutes, Copper Foil substrate is picked up from liquor ferri trichloridi, put into deionized water and wash, dry, examine under a microscope again in hole and whether the remaining copper base of 5 microns to be dissolved away, repeatedly carry out the process of (3), (4), until the copper base of remaining 5 microns is all dissolved, fall;
(5) copper base is placed on to microscope stage, with microscopical irradiation copper base, printing opacity, illustrates that Kong Shangtong base is dissolved completely;
(6) copper-base graphite alkene is put into acetone soln, the PMMA on Graphene surface is removed, so far complete the preparation of the unsettled Graphene sample stage of ultrahigh resolution fluorescence imaging copper base.
Beneficial effect of the present invention is: the present invention is for the unsettled sample stage preparation method of copper base of ultrahigh resolution fluorescence imaging, adopt and see through the alternative existing microslide of unsettled ultrathin transparent Graphene on copper matrix as fluorescent material sample stage, can use focusing effect better, the surface plasma lens focus that spot size is less and compression light field, thereby fluorescence excitation.Laser facula fluorescence excitation comparison with object lens focus on, can reduce fluorescent quenching, scattering background light, and the infringement of biological tissue sample, improves image comparison figure.
Accompanying drawing explanation
To be laser focus on and see through microslide and be radiated at the area schematic between transparent sample and probe by being inverted high-NA objective Fig. 1;
Fig. 2 is that laser is radiated at the area schematic between opaque sample and probe by long reach object lens focusing oblique incidence;
Fig. 3 is that the present invention is for the unsettled sample stage structural representation of copper base of ultrahigh resolution fluorescence imaging.
Embodiment
For the unsettled sample stage structural representation of copper base of ultrahigh resolution fluorescence imaging, preparation method is as follows as shown in Figure 3:
(1) first by the Copper Foil substrate of the Graphene of having grown (Copper Foil 25 micron thickness, purity 99.98%, Sigma Andric company), by focused ion beam, making a call to a degree of depth is 20 microns, and radius is the aperture of 2 microns;
(2) (polymethylmethacrylate, PMMA), to being set on graphene layer, makes better to protect graphene layer to avoid destroying on graphene layer surface, to drip a small amount of organic glass solution again;
(3) then the Graphene Copper Foil substrate that scribbles organic glass is put into the liquor ferri trichloridi having prepared and slightly rock, its surface is fully contacted with solution;
After (4) ten minutes, Copper Foil substrate is picked up from liquor ferri trichloridi, put into deionized water and wash, dry, examine under a microscope again in hole and whether the remaining copper base of 5 microns to be dissolved away, repeatedly carry out the process of (3), (4), until the copper base of remaining 5 microns is all dissolved, fall;
(5) copper base is placed on to microscope stage, with microscopical irradiation copper base, printing opacity, illustrates that Kong Shangtong base is dissolved completely;
(6) copper-base graphite alkene is put into acetone soln, the PMMA on Graphene surface is removed, so far complete the preparation of the unsettled Graphene sample stage of ultrahigh resolution fluorescence imaging copper base.
According to document [Plasmonics, 8 (2), 931-936 (2013)] in method prepare annular surface plasma lens and be positioned at the nm of gold circular cone at center, with radial polarisation optical illumination annular surface plasma lens, produce surface plasma wave and radially propagate into center, then at golden conical tip near surface, further produce local surface plasma ripple, this local surface plasma ripple can see through the graphene layer of the Copper Foil lower surface of nm of gold circular cone top, by micron order aperture on graphene layer, excite the fluorescent samples being positioned at above graphene layer, thereby send fluorescence.
The present invention proposes to prepare unsettled transparent Graphene micron order aperture on Copper Foil matrix as fluorescent material sample stage.The surface plasma lens that laser consists of precious metal material microstructure can make exciting light focus on and be compressed in than focusing in less region, as shown in Figure 3 with object lens.Due to the ultra-thin characteristic of grapheme material and good transmittancy, thereby this high compression light field that is confined to metal surface near field range can see through unsettled transparent Graphene fluorescence excitation sample on copper matrix.
Claims (1)
1. for the unsettled sample stage preparation method of copper base of ultrahigh resolution fluorescence imaging, it is characterized in that, in the Copper Foil substrate of the Graphene of having grown, have a micron order aperture, micron order aperture only runs through copper foil layer, specifically comprises the steps:
(1) first by the Copper Foil substrate of the Graphene of having grown, copper foil layer 25 micron thickness, purity 99.98%, by focused ion beam, making a call to a degree of depth is 20 microns, radius is the aperture of 2 microns;
(2) on graphene layer surface, drip a small amount of organic glass solution, i.e. polymetylmethacrylate, to being set on graphene layer again;
(3) then the Graphene Copper Foil substrate that scribbles organic glass is put into the liquor ferri trichloridi having prepared and slightly rock, its surface is fully contacted with solution;
After (4) ten minutes, Copper Foil substrate is picked up from liquor ferri trichloridi, put into deionized water and wash, dry, examine under a microscope again in hole and whether the remaining copper base of 5 microns to be dissolved away, repeatedly carry out the process of (3), (4), until the copper base of remaining 5 microns is all dissolved, fall;
(5) copper base is placed on to microscope stage, with microscopical irradiation copper base, printing opacity, illustrates that Kong Shangtong base is dissolved completely;
(6) copper-base graphite alkene is put into acetone soln, the PMMA on Graphene surface is removed, so far complete the preparation of the unsettled Graphene sample stage of ultrahigh resolution fluorescence imaging copper base.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104730048A (en) * | 2015-01-07 | 2015-06-24 | 鲁东大学 | Copper-based graphene carrier table mechanical adjusting system for carrying fluorescent sample |
| WO2020024466A1 (en) * | 2018-08-01 | 2020-02-06 | 广州特种承压设备检测研究院 | Method for preparing graphene optical fibre composite material |
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| CN104730048A (en) * | 2015-01-07 | 2015-06-24 | 鲁东大学 | Copper-based graphene carrier table mechanical adjusting system for carrying fluorescent sample |
| CN104730048B (en) * | 2015-01-07 | 2017-07-07 | 鲁东大学 | A kind of copper-base graphite alkene objective table mechanical control system for carrying fluorescent samples |
| WO2020024466A1 (en) * | 2018-08-01 | 2020-02-06 | 广州特种承压设备检测研究院 | Method for preparing graphene optical fibre composite material |
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