TiO2 solid immersion lens and preparation method and application thereof

Abstract

The application belongs to the technical field of optical functional materials, and relates to a TiO2 solid immersion lens, a preparation method thereof and an application of the solid immersion lens in super-resolution optical microscopic imaging. The preparation method comprises the following steps: (1) adding a water dispersion liquid containing anatase TiO2 nanoparticles with a refractive index of 2.55 into a container containing silicone oil and having a hydrophobic base material sheet placed at the bottom, to form a hemispherical TiO2 nanoparticle water dispersion liquid drop; and (2) performing two-step dehydration on the hemispherical TiO2 nanoparticle water dispersion liquid drop, to obtain a TiO2 solid immersion lens formed by closely packed TiO2 nanoparticles. The prepared solid immersion lens has the advantages of high refractive index, high transparency, high mechanical strength, controllable morphology, simple preparation, low cost, convenient use and the like, and can be used for super-resolution imaging of samples such as nanomaterials, cancer cell pathological tissues, living cells or bacteria in a large field of view, in real time and with high contrast.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of optical functional materials, and particularly relates to a TiO2 solid immersion lens, a preparation method thereof and an application of the TiO2 solid immersion lens in super-resolution optical microscopic imaging. BACKGROUND

[0002] An optical microscope is an optical device for realizing magnified imaging of a micro-object by using an optical lens group and visible light, and has important and wide applications in modern scientific research and industrial production. However, due to the diffraction characteristics of light, the highest imaging resolution of a conventional optical microscope can only reach about 200 nm, which cannot meet the demand of people for more fine microscopic observation. Breaking through the limit resolution of an optical microscope to realize super-resolution optical microscopic imaging has important significance for promoting the progress of modern biomedicine, nanomaterials, micro-nano manufacturing and information technology and other high-techs.

[0003] A solid immersion lens is used to fill the air gap between an objective lens of an optical microscope and a sample surface by using a dielectric material with a high refractive index (usually greater than 2), high transparency and a semi-spherical morphology, such as high-refractive glass or diamond, so that the numerical aperture (NA), light collection efficiency and optical imaging resolution of the objective lens can be improved by the optical focusing magnified imaging effect of the solid immersion lens. In addition, the solid immersion lens also has important applications in improving the fluorescence collection efficiency of a fluorescence microscope, high-density optical data storage, nanolithography and photonic devices. However, the manufacturing of the solid immersion lens at present mainly depends on expensive and complex top-down microfabrication technologies, such as electron beam lithography and focused ion beam milling, which have the application bottlenecks of limited selection of high-refractive optical materials, high processing difficulty, high manufacturing cost and low efficiency, which to some extent hinders the promotion and wide application of the solid immersion lens. Although the manufacturing difficulty and cost of a polymer solid immersion lens are relatively low, the relatively low refractive index (usually less than 1.7) of the polymer material will limit the optical and imaging performance of the solid immersion lens. SUMMARY

[0004] In view of the above defects or improvement needs of the prior art, the present application aims to provide a self-developed bottom-up two-step dehydration self-assembly method for silicon oil, which can self-assemble dielectric TiO2 nanoparticle material with high refractive index, low optical absorption loss and deep sub-wavelength scale into a solid immersion lens with a controllable width of several microns to centimeters. The TiO2 solid immersion lens assembled by the close packing of TiO2 nanoparticles can be simply, low-cost and batch-prepared, and has the advantages of high refractive index, high transparency, high mechanical strength, convenient operation, and high resolution and high contrast of imaging, etc. The TiO2 solid immersion lens with super-resolution optical microscopic magnification imaging capability is expected to have diversified and practical applications in the fields of observation of surface structure of nanomaterials, precise imaging diagnosis of pathological tissue such as cancer cell staining sections, and real-time identification and detection of microstructure of living cells or bacteria, etc.

[0005] To achieve the above-mentioned purpose, according to one aspect of the present application, a preparation method of a TiO2 solid immersion lens is provided, comprising the following steps:

[0006] (1) adding a water dispersion liquid containing anatase TiO2 nanoparticles with a refractive index of 2.55 into a container containing silicon oil and placing a hydrophobic substrate sheet on the bottom surface of the container to form a hemispherical TiO2 nanoparticle water dispersion liquid drop;

[0007] (2) performing two-step dehydration on the hemispherical TiO2 nanoparticle water dispersion liquid drop to obtain a TiO2 solid immersion lens assembled by close packing of TiO2 nanoparticles.

[0008] Further, the particle size of the TiO2 nanoparticles in step (1) ranges from 1 nm to 100 nm.

[0009] Further, before the TiO2 nanoparticle water dispersion liquid drop is added into the container in step (1), it further comprises: purifying the TiO2 nanoparticle water dispersion liquid by centrifugation to remove TiO2 nanoparticle agglomerates therein.

[0010] Further, the two-step dehydration of the hemispherical TiO2 nanoparticle water dispersion liquid drop in step (2) comprises:

[0011] First step dehydration: heating the container in an oven to a temperature at which the hemispherical TiO2 water dispersion liquid drop changes from a flowable liquid state to a plastic gel-like semi-solid state;

[0012] Second step dehydration: pouring out the silicon oil in the container, and drying the container at room temperature until the remaining water in the semi-solid drop is completely evaporated, and the TiO2 nanoparticles are assembled into a solid hemispherical lens by close packing.

[0013] Further, step (2) also includes: after the two-step dehydration is completed, using water absorption paper to absorb or using organic solvent to wash the residual silicone oil on the surface of the lens.

[0014] According to another aspect of the present application, there is provided a TiO2 solid immersion lens prepared according to the preparation method of the TiO2 solid immersion lens as described above.

[0015] Further, the width of the TiO2 solid immersion lens ranges from 1 μm to 1 cm.

[0016] According to a third aspect of the present application, there is provided an application of the TiO2 solid immersion lens to super-resolution optical microscopic imaging, which comprises: transferring the solid immersion lens to the surface of a solid sample to be observed or the surface of a liquid sample covered by an ultrathin cover glass, and observing the super-resolution detail information of the sample by focusing magnification imaging of the solid immersion lens under the observation of an optical microscope objective.

[0017] Further, the ultrathin cover glass is a transparent film with a thickness less than 50 μm.

[0018] Compared with the prior art, the present application can achieve the following beneficial effects:

[0019] 1. The solid immersion lens of the present application is formed by closely and uniformly stacking TiO2 nanoparticles with high refractive index, low optical absorption loss and deep sub-wavelength scale, and thus has high refractive index, transparency, mechanical strength and optical imaging performance.

[0020] 2. The width and aspect ratio of the solid immersion lens of the present application can be finely controlled by adjusting the concentration and volume of the TiO2 nanoparticle aqueous dispersion, and the hydrophilic or hydrophobic properties of the hydrophobic substrate placed in the container during dehydration.

[0021] 3. The solid immersion lens of the present application can be prepared with a width ranging from 1 μm to 1 cm, and the solid immersion lens with a width of 200 μm to 600 μm is most suitable for super-resolution optical microscopic imaging. This size range is convenient for picking up and transferring the lens with tweezers, and can also satisfy the condition that the height of the lens is less than the working distance of the high-power objective, so that the lens can be placed in the gap between the sample to be observed and the objective.

[0022] 4. The thickness of a conventional glass cover glass is 130 to 170 μm, and the use of a transparent film with a thickness less than 50 μm as an ultrathin cover glass can make the focusing distance between the lens and the sample closer, so that the lens can perform real-time focusing imaging observation on the sample such as cells packaged under the cover glass.

[0023] 5、The preparation method of the present application only needs common and easily available TiO2 nanoparticle water dispersion and silicone oil as raw materials, and the silicone oil can be reused multiple times; only an ordinary oven is needed as equipment. Therefore, the present application has the advantages of simple preparation process, low cost, high efficiency, high yield, environmental friendliness, etc., and can meet the preparation of high refractive index TiO2 solid immersion lens in small batches in the laboratory and in large batches in industrial production, thereby helping to promote and apply the solid immersion lens technology in the future. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The flowchart of the two-step dehydration method for preparing the TiO2 solid immersion lens of the silicone oil embodiment of the present application is shown in the figure.

[0025] Figure 2 The physical photo of the TiO2 solid immersion lens of the embodiment of the present application is shown in the figure.

[0026] Figure 3 The schematic diagram of the TiO2 solid immersion lens of the embodiment of the present application for super-resolution optical microscopic imaging is shown in the figure.

[0027] Figure 4 The super-resolution optical microscopic imaging photo of the TiO2 solid immersion lens of the embodiment of the present application on the nanosphere array is shown in the figure.

[0028] Figure 5 The super-resolution optical microscopic imaging photo of the TiO2 solid immersion lens of the embodiment of the present application on cancer cells is shown in the figure.

[0029] Figure 6 The super-resolution optical microscopic imaging photo of the TiO2 solid immersion lens of the embodiment of the present application on living red blood cells is shown in the figure.

[0030] In all the figures, the same reference signs represent the same technical features, specifically: 1. culture dish; 2. silicone oil; 3. hydrophobic substrate sheet; 4. water dispersion droplet containing TiO2 nanoparticles; 5. semi-solid TiO2 nanoparticle water dispersion droplet after the first dehydration step; 6. silicone oil layer remaining on the surface of the semi-solid droplet; 7. solid immersion lens assembled by the close packing of TiO2 nanoparticles after the second dehydration step; 8. objective lens of an optical microscope; 9. TiO2 solid immersion lens; 10. solid sample to be observed; 11. ultrathin cover glass; 12. cells and other samples encapsulated in gum or water; 13. glass slide. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical scheme and advantages of the present application clearer and more apparent, the present application will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and do not limit the present application.

[0032] The present application prepares TiO2 solid immersion lens by two-step dehydration of silicone oil, and the steps are as follows:

[0033] 1) Treatment and purification of raw materials:

[0034] In the self-assembly process of high refractive nanoparticle materials, the agglomeration between nanoparticles often produces structural defects such as voids. These microstructure defects will produce strong optical scattering loss, thereby seriously affecting the optical transparency and final optical performance of the prepared optical device. Therefore, in order to remove the possible nanoparticle agglomerates in the TiO2 nanoparticle aqueous dispersion, 30 mL of 15% aqueous dispersion containing anatase TiO2 nanoparticles with a refractive index of 2.55 was loaded into a 50 mL centrifuge tube, and high-speed centrifugation was performed for 30 minutes. The agglomerated TiO2 nanoparticles will be precipitated at the bottom of the centrifuge tube, and the unprecipitated dispersion is used. The particle size of the anatase TiO2 nanoparticles ranges from 1 nm to 100 nm, and in this embodiment, 15 nm is taken.

[0035] 2) First step dehydration:

[0036] As Figure 1 As shown in the left schematic diagram, a certain volume (0.5 μL to 1.2 μL in this embodiment) of aqueous dispersion containing anatase TiO2 nanoparticles is added dropwise into a culture dish containing low-viscosity silicone oil (the viscosity of the silicone oil in this embodiment is 50 mm 2 / s) and a hydrophobic plastic sheet (polycarbonate sheet in this embodiment) is placed at the bottom. The TiO2 nanoparticle aqueous dispersion droplet will form a hemispherical droplet morphology on the plastic sheet substrate under the combined action of the oil-water interfacial tension and gravity. Then the culture dish is heated in a 70°C oven for 2-4 hours, and the water in the hemispherical aqueous dispersion droplet will partially evaporate and produce a corresponding volume shrinkage. The volume shrinkage of the aqueous dispersion droplet will drive the reduction of the distance between the TiO2 nanoparticles therein. Further, in the limited space, the mutual approach of the electrostatic repulsion layers of adjacent TiO2 nanoparticles will produce a blocking phase transition effect, so that the TiO2 aqueous dispersion droplet is transformed from a flowable liquid state to a plastic gel-like semi-solid state. Through the first step of dehydration, the shape of the hemispherical lens to be prepared can be fixed.

[0037] 3) Second step dehydration:

[0038] As Figure 1As shown in the middle schematic diagram, the silicon oil in the petri dish was poured out and the petri dish was placed at room temperature (25-30°C in this example) for further drying for about 12 hours until the residual water in the semi-solid droplet was evaporated. Due to the low surface tension of the silicon oil, a thin layer of silicon oil remained on the surface of the semi-solid droplet. This thin layer of silicon oil served as a protective layer, allowing the residual water in the gap between the Ti02nanoparticles in the droplet to gradually evaporate, and the nanoparticles to self-assemble into a solid semi-spherical lens under the action of capillary force and van der Waals force. As shown in the left schematic diagram, the Ti02nanoparticles self-assembled into a semi-spherical lens. Figure 1 As shown in the right schematic diagram, the Ti02nanoparticles self-assembled into a solid immersion lens.

[0039] Finally, the residual silicon oil on the surface of the lens was removed by using a water-absorbing paper or washing with an organic solvent (n-hexane in this example).

[0040] The width of the Ti02solid immersion lens prepared by the present application ranged from 1 μm to 1 cm, and a photograph of the actual lens is shown in Figure 2 As shown in the photograph, the left lens had a width of 600 μm, and the right lens had a width of nearly 1 cm. Figure 2

[0041] In the two-step dehydration method of silicon oil, the volume of the Ti02nanoparticle aqueous dispersion added in step 2) above could be adjusted to control the width of the lens prepared. For example, to prepare a small-sized lens with a width of several microns to tens of microns, a spray bottle could be used to spray the Ti02nanoparticle aqueous dispersion into the silicon oil; to prepare a larger-sized lens, a pipette could be used to add a larger volume of Ti02nanoparticle aqueous dispersion droplets into the silicon oil.

[0042] In addition, by adjusting the surface hydrophilic or hydrophobic properties of the substrate sheet at the bottom of the container in step 2), the aspect ratio of the lens prepared could be controlled. For example, a substrate sheet with a more hydrophobic surface was beneficial for preparing a lens with a higher aspect ratio. Generally, a lens with a higher aspect ratio had a larger imaging magnification, but the contrast of the lens imaging would decrease as the aspect ratio increased. The commonly used aspect ratio of the Ti02solid immersion lens was between 0.5 and 0.7.

[0043] The Ti02solid immersion lens was used for super-resolution optical microscopic imaging observation:

[0044] The prepared Ti02solid immersion lens was transferred from the plastic sheet to the surface of the sample to be observed using a sharp-tipped tweezers. Under the focusing of the transmission or reflection type illumination optical microscope, the super-resolution detail information of the sample was observed through the magnifying imaging of the lens. For different samples, different lens observation methods could be selected.

[0045] ​Figure 3 Schematic diagram of TiO2 solid immersion lens for super-resolution optical microscopy imaging. Wherein, Figure 3 The left schematic diagram is for observing the solid sample, the lens can be directly placed on the surface of the sample, and then the microstructure details of the sample surface are observed by super-resolution imaging through the focusing magnification imaging of the lens under the observation of the transmitted or reflected illumination optical microscope objective. Figure 3 The right schematic diagram is for observing the sample such as cells which need to be preserved in gum or water, a transparent plastic film with a thickness less than 50 μm can be used as an ultrathin cover glass to cover and package the sample, and then the lens is placed on the surface of the ultrathin cover glass to observe the magnified imaging of the sample by the lens under the optical microscope objective.

[0046] As Figure 4 shown, the nanosphere array is observed by super-resolution optical microscopy imaging. Figure 4 The left photo is a photo of a monolayer array of monodisperse polystyrene nanospheres with a particle size of 300 nm closely arranged, taken by SEM. Figure 4 The middle photo is an optical microscopy photo of the nanosphere array taken under the direct observation of the 100x objective (model Nikon LU Plan Fluor, NA = 0.9) of the reflected illumination optical microscope (model Nikon Eclipse LV-100). Figure 4 The right photo is a photo of the optical microscopy imaging of the nanosphere array by the lens taken by the optical microscope after the TiO2 solid immersion lens with a width of 223 μm and an aspect ratio of 0.61 is placed on the surface of the nanosphere array. It can be seen that due to the limitation of the optical diffraction limit, the direct observation of the optical microscope cannot distinguish the periodic arrangement structure in the nanosphere array; and the regular hexagonal lattice arrangement structure in the nanosphere array can be clearly observed by the 2.5 times magnification imaging of the TiO2 solid immersion lens, thereby realizing super-resolution optical microscopy imaging. For comparison, the photo of the direct observation of the optical microscope in the middle is magnified to the same magnification as the photo of the lens observation in the right, and the same applies below.

[0047] As Figure 5 shown, the cancer cells are observed by super-resolution optical microscopy imaging. In Figure 5In the middle, first Hela cells (human cervical cancer cells) attached to the glass slide were stained with hematoxylin-eosin, then neutral gum was added on the cell surface, and a fluorinated ethylene propylene (FEP) film with a thickness of 25 μm was used as an ultrathin cover glass to cover and encapsulate the cells. Among them, the left photo is the optical micrograph of Hela cells taken under the direct observation of the 40x objective lens (model Nikon Plan Fluor, NA=0.75) of the transmission illumination optical microscope (model Nikon Eclipse Ci-L); the right photo is the photo of the optical microscopic imaging of the lens on the Hela cells taken by the optical microscope after placing a TiO2 solid immersion lens with a width of 578 μm and an aspect ratio of 0.56 on the surface of the ultrathin cover glass. It can be seen that through the 2.6 times magnification imaging of the lens, the chromatin condensation in the cancer cell nucleus has a clearer profile (as shown in the upper row of boxes in the left and right two figures and the corresponding enlarged images); in addition, the filamentous structure oriented and arranged between two adjacent cell nuclei in the cytoplasm is also more clearly visible (as shown in the lower row of boxes in the left and right two figures and the corresponding enlarged images).

[0048] As shown in Figure 6 , the living red blood cells were observed by super-resolution optical microscopic imaging. In Figure 6 , first the mouse tail vein blood was added on the glass slide, and the FEP ultrathin cover glass with a thickness of 25 μm was used to cover and encapsulate. The left photo is the optical micrograph of mouse red blood cells taken under the direct observation of the 40x objective lens (model Nikon Plan Fluor, NA=0.75) of the transmission illumination optical microscope (model Nikon Eclipse Ci-L); the right photo is the photo of the optical microscopic imaging of the lens on the mouse red blood cells taken by the optical microscope after placing a TiO2 solid immersion lens with a width of 575 μm and an aspect ratio of 0.67 on the surface of the ultrathin cover glass. It can be seen that through the 3.2 times magnification imaging of the lens, the uniformly distributed spike-like structures on the mouse red blood cell membrane can be more clearly observed, and the imaging of the lens can present a certain three-dimensional relief-like stereoscopic effect, as shown in the boxes in the left and right two figures and the corresponding enlarged images, which will be beneficial to the fine observation and identification of the subcellular structures.

[0049] The above-described embodiments only express the implementation of the present application, which is described in a more specific and detailed manner, but it cannot be understood as a limitation on the scope of the patent of the present application. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are within the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. A super-resolution optical microscopy imaging application using a TiO2 solid immersion lens, characterized in that, include: The solid immersion lens is transferred to the surface of the liquid sample covered by the ultrathin coverslip to be observed. Under the observation of the objective lens of an optical microscope, the super-resolution details of the sample are observed by focusing and magnifying the imaging through the solid immersion lens. The sample was HeLa cells or mouse erythrocytes; The method for preparing the TiO2 solid immersion lens includes the following steps: (1) A water dispersion containing anatase TiO2 nanoparticles with a refractive index of 2.55 is dropped into a container filled with silicone oil and a hydrophobic substrate sheet is placed on the bottom to form a hemispherical water dispersion of TiO2 nanoparticles. (2) The water-dispersed droplets of hemispherical TiO2 nanoparticles were dehydrated in two steps to obtain a TiO2 solid immersion lens made of tightly packed TiO2 nanoparticles. The particle size range of the TiO2 nanoparticles mentioned in step (1) is 15 nm; Before adding the TiO2 nanoparticle aqueous dispersion to the container in step (1), the process further includes: purifying the TiO2 nanoparticle aqueous dispersion by centrifugation to remove TiO2 nanoparticle aggregates. Step (2) involves a two-step dehydration process for the hemispherical TiO2 nanoparticle aqueous dispersion droplets, including: Step 1: Dehydration: Heat the container in an oven at 70°C for 2-4 hours until the hemispherical TiO2 aqueous dispersion droplets change from a flowable liquid state to a plastic gel-like semi-solid state. The second step is dehydration: the silicone oil in the container is poured out and the container is dried at room temperature until the remaining water in the semi-solid droplets is completely evaporated, and the TiO2 nanoparticles are tightly packed and assembled into a solid hemispherical lens. The TiO2 solid immersion lens has a width of 578 μm and an aspect ratio of 0.56; Alternatively, the TiO2 solid immersion lens has a width of 575 μm and an aspect ratio of 0.

67.

2. The application of the TiO2 solid immersion lens in super-resolution optical microscopy according to claim 1, characterized in that, The ultrathin cover glass is a transparent film with a thickness of less than 50 μm.

3. The application of the TiO2 solid immersion lens in super-resolution optical microscopy according to claim 1, characterized in that, Step (2) also includes: after the two-step dehydration is completed, the residual silicone oil on the lens surface is removed by absorbing with absorbent paper or washing with organic solvent.

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