CN111004722B - Frequency shift super-resolution microscopic chip capable of applying electric field - Google Patents

Frequency shift super-resolution microscopic chip capable of applying electric field Download PDF

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CN111004722B
CN111004722B CN201911148755.5A CN201911148755A CN111004722B CN 111004722 B CN111004722 B CN 111004722B CN 201911148755 A CN201911148755 A CN 201911148755A CN 111004722 B CN111004722 B CN 111004722B
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electric field
resolution
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electrode
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CN111004722A (en
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杨青
汤明炜
庞陈雷
刘伟
陈伟
刘旭
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Zhejiang University ZJU
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Abstract

The invention discloses a frequency shift super-resolution microscopic chip capable of applying an electric field, which comprises: the middle part of the waveguide is an imaging area and is used for introducing exciting light and generating evanescent field interference fringes to excite fluorescent particles positioned in the imaging area to carry out microscopic imaging; and the electrode extends from the edge of the waveguide to the imaging area and is used for introducing an electric field to stimulate the sample in the imaging area. The invention can not only carry out super-resolution microscopy, but also carry out electrical stimulation regulation and control on an observed biological sample, can carry out batch preparation, and has light system and convenient use.

Description

Frequency shift super-resolution microscopic chip capable of applying electric field
Technical Field
The invention relates to the field of biochips, in particular to a frequency shift super-resolution microchip capable of applying an electric field.
Background
The behavior of cells in an organism is regulated by many influencing factors. In addition to chemical gradients and physical forces, biological activity is coordinated by a series of bioelectrical factors (endogenous electrical signals through ion channels and pumps located at the cell membrane) to create and repair structures in the body. The bioelectric field plays an important role in physiological processes such as morphogenesis and growth of organisms, and participates in important pathological processes of organisms, such as wound healing, tissue regeneration, tumor erosion and the like. In order to research the response rule of cells under the action of an electric field, exogenous electric stimulation can be adopted, namely, charges are artificially introduced into the cells through a constant voltage power supply, a constant current power supply or a waveform generator to enable the cells to generate action potential changes. Exogenous electrical stimulation can provide an appropriate physiological environment to mimic endogenous electric fields to study the laws of electric fields that regulate cell transmembrane potential and regulate cell growth, differentiation, and cell function (e.g., morphology, elongation, migration, and gene expression). The observation resolution of the current electric stimulation chip is limited, and the subcellular structure change under the electric field regulation and control state cannot be researched, so that the biological electric field regulation and control research is greatly limited. In order to break through the diffraction limit, the optical super-resolution method is an effective research tool.
The existing classical super-resolution microscopic methods such as SIM, STED, STORM and the like are all based on a large-scale optical system and face the defects of complex operation and expensive and heavy equipment. The optical super-resolution microscopic chip can integrate a precise optical path on the optical waveguide chip through micro-nano processing, so that the reliability and the portability of the super-resolution method are improved, and the cost of super-resolution microscopy can be greatly reduced by mass production of the chips. The frequency shift super-resolution technology is a super-resolution method which can realize large field of view and fast imaging. The frequency shift super-resolution method has great application prospect by combining with the chip technology.
For example, the shift frequency super-resolution microchip provided by publication number CN109374578A utilizes the interference fringes of different directions of evanescent fields of polygonal waveguide to illuminate fluorescence labeled sample. Moire fringes are utilized to transfer high-frequency information of a sample to low-frequency information to be collected by a microscope system, and then a final high-frequency part is transferred to an original position through an algorithm to carry out frequency spectrum splicing, so that the detailed information of the sample is recovered. The super-resolution microscopic chip has the advantages of high integration level and simple and convenient use, is suitable for low-cost batch production, but the super-resolution chip lacks electrical regulation and control capability, and limits the function of the super-resolution microscopic chip in biological cell research.
In the field of biochips, there is still a lack of a chip that can simultaneously realize super-resolution microscopy and electric field regulation of biological cells.
Disclosure of Invention
The invention designs a photoelectric integrated super-resolution microscopic chip based on a frequency shift principle, which can realize electrical stimulation regulation and control on cells, and simultaneously carry out large-field and rapid super-resolution microscopic imaging research on the cells, has important significance for researching cell molecular and biomedicine, and has important and wide application prospect in the aspects of intelligent portable biomedicine research and clinical detection.
The technical scheme adopted by the invention is as follows:
a frequency-shifted super-resolution microchip capable of applying an electric field, comprising:
the middle part of the waveguide is an imaging area and is used for introducing exciting light and generating evanescent field interference fringes to excite fluorescent particles positioned in the imaging area to carry out microscopic imaging;
and the electrode extends from the edge of the waveguide to the imaging area and is used for introducing an electric field to stimulate the sample in the imaging area.
The method comprises the steps of inputting exciting light by using a waveguide, illuminating a fluorescence labeling sample by using interference fringes generating an evanescent field, exciting and collecting fluorescent particles, and carrying out microscopic imaging by frequency shift; meanwhile, the cell electrical stimulation regulation can be observed by combining the electrode.
The waveguide in the application is made of light guide materials with small loss in the visible light range; preferably, the waveguide is made of SiO2, SiN, TiO2, GaP or Al2O 3.
The waveguide can be a rectangle or other regular polygons, and one-dimensional super resolution can be realized by introducing exciting light into two ends of the rectangle waveguide; exciting light is introduced into each side of the regular polygon, and two-dimensional super resolution can be realized.
Preferably, the waveguide is a polygon, an overlapping region of evanescent fields generated by incident excitation light on each side of the waveguide is located in an imaging region in the middle, and each side of the waveguide is provided with an electrode extending to the imaging region.
The electrode is the connecting line of the middle point of the waveguide edge and the vertex of the nearest effective imaging area. The electrodes are connected to a voltage/current controller or a waveform generator through external leads, and different levels and waveforms can be selectively applied to stimulate the cells.
The electrode is selected in consideration of other physical and chemical properties of the material and the processing cost; meanwhile, the material of the electrode needs to have conductivity and biocompatibility, and is conductive polymer, graphene, carbon nanotube, ITO, TiN, platinum or gold, and the like. The conductive polymer includes Ppy, PEDOT, polyaniline, and the like.
Preferably, the electrode is in a strip shape and is perpendicular to the excitation edge of the waveguide.
In the application, the distribution area of the conductive material (i.e. the electrode) is rectangular in a long strip shape, and in order to reduce the influence of the electrode area on the transmission evanescent field, the long edge of the conductive electrode is vertically distributed with the excitation edge of the waveguide.
Preferably, the waveguide further comprises a substrate paved below the waveguide, and the electrode is positioned on the surface of the waveguide or between the waveguide and the substrate.
In the application, the electrodes are distributed in pairs and are symmetrical relative to the center of an imaging area; the horizontal distribution of the electrodes is centrosymmetric, and the distribution in the longitudinal section can be positioned on the surface of the waveguide material or between the waveguide material and the substrate. However, it is necessary to ensure that the upper and lower layers of the light guide layer are low-refractive index materials, and a layer of low-refractive index waveguide material needs to be additionally plated between the light guide layer and the electrode material under necessary conditions. That is, preferably, a low refractive index layer is provided between the waveguide and the substrate, the electrode is located below the low refractive index layer, and the low refractive index layer has a lower refractive index than the waveguide.
Preferably, the substrate is a Si substrate.
The invention has the beneficial effects that: the device can be used for performing super-resolution microscopy, performing electrical stimulation regulation and control on an observed biological sample, performing batch preparation, and being light in system and convenient to use.
Drawings
FIG. 1 is a schematic diagram of an electrode distribution design;
FIG. 2 is a top view of a one-dimensional waveguide;
FIG. 3 is a cross-sectional design of a one-dimensional chip with electrodes distributed on the top surface of the optical waveguide;
FIG. 4 is a cross-sectional design of another one-dimensional chip with electrodes distributed under the waveguides and a layer of low index optical waveguide between the optical waveguide transmission layer and the electrodes;
FIG. 5 is a simulation diagram of the electric field distribution of one-dimensional electrodes;
FIG. 6 is a schematic diagram of the practical use of the chip, with a cell sample placed in the middle imaging area.
Wherein 101 is an electrode, 102 is an optical waveguide, and 103 is an imaging region; 201 is excitation light, 202 is an electrode, 203 is an imaging region, 204 is fluorescent particles, 205 is a high refractive light guide material, 206 is a substrate material, and 207 is a low refractive light guide material.
Detailed Description
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, however, the present invention may be practiced in other ways than those specifically described herein, and thus the present invention is not limited to the specific embodiments disclosed below.
The two-dimensional frequency shift imaging super-resolution microchip shown in fig. 1 is an example of a decagonal waveguide, and comprises an electrode 101 and a polygonal optical waveguide 102, wherein a white area in the middle of the optical waveguide 102 is an effective imaging area 103, namely an illumination light overlapping area of the decagonal waveguide. The electrode is a black strip shape and is a connecting line of the midpoint of the waveguide side and the vertex of the nearest effective imaging area. The electrodes are connected to a voltage/current controller or a waveform generator through external leads, and different levels and waveforms can be selectively applied to stimulate the cells.
Fig. 2 illustrates a top view of a one-dimensional super-resolution waveguide and electrode design, which uses rectangular optical waveguides. The imaging region 203 is located in the middle region of the waveguide, and the electrodes 202 are symmetrically distributed at two ends of the waveguide outside the imaging region. Excitation light 201 enters the waveguide through the coupling of the two ends of the waveguide, evanescent field interference fringes are generated on the surface of the waveguide, fluorescent particles 204 on the surface of the waveguide imaging area are excited, and finally a one-dimensional super-resolution image is restored through a frequency shift effect.
Fig. 3 and 4 illustrate side views of one-dimensional super-resolution waveguide and electrode designs, with substrate material 206 disposed, using rectangular waveguides. The conductive material (electrode 202) can be located on the surface of the imaging waveguide, as shown in fig. 3, or at the bottom of the imaging waveguide, as shown in fig. 4, where the low refractive index waveguide material 207 refers to a material with an optical refractive index lower than that of the evanescent field transmission material (high refractive index optical waveguide material 205, i.e., waveguide), and optionally SiO2 or its low refractive index doped material. In the design shown in fig. 3, the metal (electrode 202) is located on the surface of the waveguide, which has an effect on the transmission of the evanescent field, so that in order to reduce the effect as much as possible, it is necessary to reduce the width of the electrode as much as possible, and at the same time, to ensure that the conductivity, i.e., the resistance, is not too high. The design of fig. 4 avoids the influence of metal on the transmission evanescent field, so that the width of the electrode is not limited, but the processing difficulty is increased because the conductive material needs to be additionally plated and etched first and then the subsequent plating is carried out. In practical situations, the cost and the implementation possibility need to be considered, and a proper scheme is selected according to specific use conditions.
Fig. 5 illustrates a schematic diagram of an electric field generated by applying a voltage to one-dimensional electrodes.
FIG. 6 illustrates the method of use of the chip and the distribution of the cell samples. The actual imaging sample needs to be attached to the surface of the chip within one hundred nanometers, and cells can be cultured to grow in an attached manner in the middle imaging area for cell research.
Assuming gold is used as the electrode material, the preparation process of the chip is as follows:
preparing a polygonal area of the optical waveguide;
plating a layer of photoresist, exposing a mask plate, and developing;
a gold plating film having a thickness less than that of the photoresist;
and cleaning the residual photoresist.
The above description is only exemplary of the preferred embodiments of the present invention, and is not intended to limit the present invention, and any modifications, equivalents, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (6)

1. A frequency-shift super-resolution microchip capable of applying an electric field, comprising:
the middle part of the waveguide is an imaging area and is used for introducing exciting light and generating evanescent field interference fringes to excite fluorescent particles positioned in the imaging area to carry out microscopic imaging;
an electrode extending from the waveguide edge to the imaging region for introducing an electric field to stimulate the sample within the imaging region;
the waveguide is polygonal, an evanescent field overlapping region generated by incident exciting light on each side of the waveguide is positioned in an imaging region in the middle, and each side is provided with an electrode extending to the imaging region;
the electrode is positioned on the surface of the waveguide or positioned between the waveguide and the substrate; the electrodes are strip-shaped and are vertical to the excitation edge of the waveguide; a low refractive index layer is arranged between the waveguide and the substrate, and the electrode is positioned below the low refractive index layer; the low refractive index layer has a lower refractive index than the waveguide.
2. The frequency-shift super-resolution microchip with electric field application according to claim 1, wherein the material of the waveguide is SiO2、SiN、TiO2GaP or Al2O3。
3. The frequency-shift super-resolution microchip with electric field application according to claim 1, wherein the electrode is made of conductive polymer with conductivity and biocompatibility.
4. The frequency-shift super-resolution microchip with electric field application according to claim 1, wherein the material of the electrodes is graphene, carbon nanotubes, ITO, TiN, platinum or gold.
5. The frequency-shift super-resolution microchip with an applied electric field according to claim 4, wherein the electrodes are arranged in pairs and are symmetrical with respect to the center of the imaging area.
6. The frequency-shift super-resolution microchip with electric field application according to claim 1, wherein the substrate is a Si substrate.
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