Frequency-shift super-resolution microscopic imaging chip capable of observing in-situ electrical stimulation in microfluidics and preparation method of frequency-shift super-resolution microscopic imaging chip

Abstract

The invention discloses a frequency shift super-resolution microscopic imaging chip capable of observing in-situ electrical stimulation in microfluidics and a preparation method of the frequency shift super-resolution microscopic imaging chip, the frequency shift super-resolution microscopic imaging chip sequentially comprises a microfluidic structure layer, a microelectrode array layer, a high refractive index waveguide layer and a frequency shift structure layer, and the frequency shift super-resolution microscopic imaging chip presents light transmission; the micro-fluidic structure layer comprises a micro-fluidic structure capable of guiding cell neurons in cell sap to grow along a single-cell channel; the microelectrode array layer comprises a microelectrode array and can perform in-situ electrical stimulation on single cells in the single cell channel; the high-refractive-index waveguide layer is used for supporting the microelectrode array layer and realizing high-refractive-index optical waveguide transmission; the frequency shift structure layer is used for exciting large-wave-vector frequency shift light for incoming illumination light and transmitting the large-wave-vector frequency shift light to the high-refraction waveguide layer; the chip can support frequency shift super-resolution microscopic signal imaging while carrying out in-situ electrical stimulation on single cells in microfluidics.

Description

Technical Field

[0001] The invention relates to the field of super-resolution microscopy and microfluidic chips, and in particular to a frequency-shift super-resolution microscopic imaging chip capable of observing in-situ electrical stimulation in microfluidics and a preparation method thereof. Background Art

[0002] In neurons, electrical activity coordinates many intracellular events, such as calcium signaling, energy homeostasis, and protein distribution. Neuroelectrophysiological research has become the focus of many researchers. Microelectrode arrays prepared by microfabrication technology can be customized with high-density, coded microelectrode arrays, providing a high signal-to-noise ratio and high-throughput means for studying neuroelectrophysiological information. By introducing exogenous electrical stimulation, that is, artificially through a constant voltage, constant current power supply or waveform generator, electrophysiological activity can be generated in vitro cultured neurons for a long time without damage.

[0003] Microfluidic chips, which are also based on this technology, can finely produce tiny fluid structures, making it possible to customize the design of neuronal connections in vitro. In recent years, neural chips that combine microfluidic chips with microelectrode arrays have achieved important results in neuroelectrophysiology research because of their ability to detect and regulate the neuroelectrophysiological information of customized networks. However, the chips currently used to study neuronal electrical activity generally only perform single-dimensional electrophysiological observations, making it difficult to balance spatial and temporal resolution, and unable to record the subcellular dynamic structural changes and calcium ion dynamic changes of living neurons under electric field regulation, which greatly limits the study of neuronal electrical activity.

[0004] Super-resolution microscopy breaks the diffraction limit of traditional optical microscopes and overcomes the limitation of spatial resolution. It has the advantages of wide application range, non-invasiveness, in vivo observation and easy operation. Among them, on-chip frequency-shifting super-resolution microscopy uses the sub-wavelength local field generated by the waveguide surface to illuminate the sample. In this process, the high-frequency information carried by the sample is shifted to low frequency and thus received by the traditional microscope. Then, a high-resolution image is obtained by spectrally reconstructing and splicing a series of low-resolution images received.

[0005] The patent application with application publication number CN111024664A provides an imaging method for a structured light illumination super-resolution microscopy chip. Specifically: based on a block of high-refractive optical waveguide materials, a diffraction structure is prepared on the waveguide surface, two beams of free-space light are coupled into the waveguide material using the diffraction effect, and the interference structured light generated by the evanescent field of the high effective refractive index is used to illuminate the sample, and the high-frequency spatial spectrum information of the object is modulated, and the frequency is shifted to a low frequency to be received by the microscope objective lens, and then the super-resolution micrograph of the sample is restored. This technology focuses on how to provide higher resolution in optics, but it is lacking in optoelectronic integration and multi-functions. If in-situ electrical regulation and super-resolution imaging can be performed on the imaging chip, for biological research, it can start from multiple dimensions, and regulate and detect the dynamic behavior of the object under study with extremely high spatiotemporal resolution, providing a more powerful tool for biological and medical mechanism research, detection and diagnosis, etc. Summary of the invention

[0006] In view of the above, the purpose of the present invention is to optimize the chip and form a biochip with extremely high spatiotemporal resolution to address the technical deficiencies of existing chips for studying neuronal electrical activity, such as limited recording dimensions and single functions.

[0007] To achieve the above-mentioned purpose of the invention, the embodiment provides a frequency-shift super-resolution microscopic imaging chip capable of observing in-situ electrical stimulation in microfluidics, which comprises a microfluidic structure layer, a microelectrode array layer, a high refractive index waveguide layer, and a frequency-shift structure layer in sequence, and exhibits light transmittance;

[0008] The microfluidic structure layer includes a microfluidic structure capable of guiding cell neurons in the cell fluid to grow along a single cell channel;

[0009] The microelectrode array layer includes a microelectrode array, which can realize in situ electrical stimulation of single cells in the single cell channel;

[0010] The high refractive index waveguide layer is used to support the microelectrode array layer and realize high refractive index optical waveguide transmission;

[0011] The frequency-shifting structural layer is used to excite large-wave-vector frequency-shifted light to the incident illumination light, and transmit the large-wave-vector frequency-shifted light to the high-refractive waveguide layer;

[0012] The chip can realize in situ electrical stimulation of single cells in microfluidics while supporting frequency-shift super-resolution microscopic signal imaging.

[0013] Preferably, the microfluidic structure layer includes an inoculation cavity, a connecting channel and a microchannel, and is made of a polymer material (such as PDMS) as a whole, and is designed to guide the growth of neurons. The two ends of the specific multiple groups of microchannels are connected by two connecting channels, and the other two ends of the two connecting channels are respectively connected to the inoculation cavity. The microchannel is a single cell channel, and its width and height only allow a single axon or dendrite in the cell neuron to grow, and the connecting channel and the microchannel are combined. A funnel-shaped structure is used to guide the cell neurons in the connecting channel to be generated along the microchannel. Preferably, the inoculation cavity is set to 4, and the connecting channels are 4, and every 2 inoculation cavities, 2 connecting channels and 1 group of microchannels form a microfluidic structure for cell growth.

[0014] The effective area of ​​the microfluidic structure ensures the culture density and the slow stacking of nutrients and waste, which is conducive to the in vitro culture of neurons. Preferably, the height of the microchannel is 4μm-5μm, the width is 4μm-5μm, and the microchannel spacing is 20-40μm. The microchannel ensures the growth of a single axon structure, avoids cell stacking, and ensures the on-chip super-resolution imaging quality.

[0015] Preferably, the microelectrode array includes a microelectrode array located in the center, a reference electrode array arranged around the microelectrodes, an external contact point array arranged on the periphery, and wires connecting the external contact points with the reference electrodes and the microelectrodes. The microelectrode array corresponds to the single-cell channel of the microfluidic structure layer, thereby achieving in situ electrical stimulation of single cells in the single-cell channel, that is, stimulating and detecting the axonal potential of a single neuron.

[0016] Preferably, the diameter of the microelectrode is in the range of 10 μm-20 μm, and the diameter of the microelectrode is larger than the width of the microchannel, the diameter of the external contact point is 2 mm-2.5 mm, and the size of the reference electrode and the lead wire is reasonably designed to ensure good electrical connection.

[0017] Preferably, an insulating layer is further provided between the microfluidic structure layer and the microelectrode array layer.

[0018] Preferably, the high-refractive waveguide layer is used as a substrate, and its material is selected to be transparent in the selected illumination light band. When the illumination light is visible light, the high-refractive waveguide layer material includes Si 3 N 4 、SiO 2 、Al 2 O 3 、TiO 2 Or Polymer.

[0019] Preferably, the frequency-shifting structural layer includes a multi-turn grating, which is prepared by a micro-nano processing method and is designed to enhance the imaging resolution. A diffraction structure formed by etching multiple multi-turn gratings under the high refractive index waveguide is used to excite large wave vector frequency-shifted light. After being transmitted in the waveguide, the cells above are illuminated by frequency-shifted illumination or structured light for multiple times to different degrees. The frequency-shifted illumination area formed by the multi-turn grating matches the microchannel exactly, and the axonal changes in the microchannel can be dynamically observed under super-resolution.

[0020] The grating design includes gratings of different periods to meet the imaging requirements, and the spacing and angle of the gratings are precisely calculated and designed. Specifically, each circle of gratings is circularly distributed, the centers of multiple groups of gratings coincide, and each circle of gratings contains multiple pairs of gratings symmetrical along the center. The periods of the same circle of gratings are the same, and the periods of different circles of gratings are different; each pair of gratings D m The distance between them satisfies:

[0021]

[0022] Where t represents the thickness of the substrate material, λ represents the wavelength of the illumination light used, n represents the refractive index of the substrate material, and T m Represents the period of the mth grating.

[0023] The embodiment also provides a method for preparing the frequency-shift super-resolution microscopic imaging chip capable of observing in-situ electrical stimulation in microfluidics, comprising the following steps:

[0024] cleaning and drying the high-refractive waveguide to form a high-refractive waveguide layer;

[0025] fabricating a microelectrode array layer pattern on the high-refractive waveguide layer;

[0026] covering the microelectrode array layer with an insulating layer, and preparing the microelectrode array layer on the insulating layer;

[0027] Etching a frequency shifting structure layer on the other side of the high refractive waveguide layer;

[0028] A microfluidic structure layer is prepared, and the microfluidic structure layer is aligned and bonded with the microelectrode array layer.

[0029] The alignment of microfluidics and microelectrodes is determined by multiple sets of precisely designed marks, such as positioning holes. Gratings can be prepared in amplitude or phase mode, and gratings can be mass-produced using nanoimprinting.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The chip of the present invention can guide the growth of single cell size through the microfluidic structure layer, and the microelectrode array layer coordinated with the microfluidic structure layer can realize in situ electrical stimulation observation of single cells in the single cell channel. At the same time, the high refractive index waveguide layer and the frequency shift structure layer are added, which can have super-resolution imaging capabilities, providing new tools and methods for related biomedical research. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0033] Figure 1 It is a side view of a frequency-shift super-resolution microscopic imaging chip capable of observing in-situ electrical stimulation in microfluidics and a front view of each substructure provided in an embodiment;

[0034] Figure 2 is a schematic diagram of a microfluidic structure provided in an embodiment;

[0035] Figure 3 is a schematic diagram of a microelectrode array provided in an embodiment;

[0036] Figure 4 is a schematic diagram of a frequency shift structure provided in an embodiment;

[0037] Figure 5 is a schematic diagram of the chip structures provided in the embodiment after alignment, and the right side shows the one-to-one correspondence between the microchannels and the microelectrodes;

[0038] Figure 6 is a schematic diagram of a spectrum after frequency shift synthesis provided by an embodiment;

[0039] Figure 7 is a flow chart of the preparation method provided in the embodiment;

[0040] Among them: 1 is an external contact point, 2 is a wire, 3 is a reference electrode, 4 is a microelectrode, 5 is an inoculation cavity, 6 is a connecting channel, 7 is a microchannel, 8 is an insulating layer, 9 is a grating with a period of T1, and 10 is a grating with a period of T2. DETAILED DESCRIPTION

[0041] To make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific implementation methods described herein are only used to explain the present invention and do not limit the scope of protection of the present invention.

[0042] The embodiment of the present invention provides a frequency-shifted super-resolution microscopic imaging chip capable of observing in-situ electrical stimulation in microfluidics, which includes a microfluidic structure layer, a microelectrode array layer, a high-refractive-index waveguide layer, and a frequency-shifted structure layer in sequence, and exhibits light transmittance, wherein the microfluidic structure layer includes a microfluidic structure capable of guiding cell neurons in the cell fluid to grow along a single-cell channel; the microelectrode array layer includes a microelectrode array, which can realize in-situ electrical stimulation of single cells in the single-cell channel; the high-refractive waveguide layer is used to support the microelectrode array layer and realize high-refractive-index optical waveguide transmission; the frequency-shifted structure layer is used to excite large-wave-vector frequency-shifted light for incident illumination light, and transmit the large-wave-vector frequency-shifted light to the high-refractive waveguide layer. The chip is mainly used for cell experiments, and the microfluidic structure layer is used to guide and control the cell growth of cells in the cell fluid, and the microelectrode array layer is used to realize in-situ electrical stimulation of single cells in the microfluidics, and the cooperation of the high-refractive waveguide layer and the frequency-shifted structure layer is also used to support frequency-shifted super-resolution microscopic signal imaging.

[0043] In the embodiment, the entire microfluidic structure is made of PDMS. Figure 3 As shown, the microfluidic structure layer includes an inoculation chamber 5, a connecting channel 6 and a microchannel 7. There can be 4 inoculation chambers 5, 2 connecting channels 6 and several microchannels 7, wherein the two ends of multiple groups of microchannels 7 are connected by two connecting channels 6, and the other two ends of the two connecting channels 6 are respectively connected to the inoculation chamber 5. The microchannel 7 is a single cell channel, and its width and height are very small compared to the connecting channel 6, allowing only a single axon or dendrite in the cell neuron to grow, and the funnel-shaped structure is used at the junction of the connecting channel 6 and the microchannel 7 to guide the cell neuron in the connecting channel 6 to grow along the microchannel 7. Among them, the area of ​​the microfluidic structure is 4cm 2 , the effective inoculation area is 1cm 2 , thickness is 200μm, inoculation chamber 5 height is 200μm, inoculation chamber 5 diameter is 2.5mm, connecting channel 6 height is 200μm, connecting channel 6 width is 750μm, microchannel 7 height is 4μm, microchannel 7 width is 4μm, and microchannel 7 length is 600μm.

[0044] In the embodiment, Figure 3 As shown, the microelectrode array includes a microelectrode array 4 located in the center, a reference electrode array 3 arranged around the microelectrodes, an external contact point array 1 arranged on the periphery, and a wire 2 connecting the external contact points with the reference electrodes and the microelectrodes, wherein the microelectrode array 4 corresponds to the single-cell channel (i.e., microchannel 7) of the microfluidic structure layer, thereby realizing in situ electrical stimulation of single cells in the single-cell channel.

[0045] The microelectrode array 4 is composed of Au electrodes combined with an insulating layer SiO 2The microelectrode array 4 has 2 groups, each group has 20-40 electrodes, each microelectrode has a diameter of 10 μm, the wire 2 has a width of 4 μm-80 μm, and the diameter of each external contact point in the external contact point array 1 is 2 mm. The electrode pattern layer of the entire microelectrode array layer is also covered with an insulating layer 8, which can be made of Al 2 O 3 After the microelectrode array layer and the microfluidic structure are aligned using the markers, they are manually bonded with the aid of alcohol or polydimethylsiloxane. Figure 5 As shown, holes of 4 μm to 1800 μm are punched at the microelectrode array 4, the reference electrode array 3 and the external contact point array 1 at the same time. The holes of the microelectrode array 4 are aligned one by one with the microchannels 7 of the upper microfluidic structure, so that the microelectrodes can achieve in-situ electrical stimulation of cells in the microchannels 7.

[0046] In the embodiment, the high-refractive waveguide layer is used as a substrate, and its material is selected to be transparent in the selected illumination light band. When the illumination light is visible light, the high-refractive waveguide layer material includes Si 3 N 4 、SiO 2 、Al 2 O 3 、TiO 2 Or polymer. The imaging resolution depends on the material refractive index and the selected illumination wavelength. The substrate material and illumination wavelength are selected according to the imaging resolution requirements, cost and processing conditions.

[0047] In the embodiment, the frequency shift structure layer includes multiple circles of gratings, each circle of gratings is distributed in a circle, the centers of multiple groups of gratings coincide, each circle of gratings includes multiple pairs of gratings symmetrical along the center, the period of the same circle of gratings is the same, and the period of different circles of gratings is different. Figure 4 As shown, it can specifically include two circles of gratings, which are made under the high refractive index waveguide by micro-nano processing methods such as electron beam direct writing, focused ion beam etching, photolithography or nanoimprinting. The imaging area of ​​the imaging plane is located at the center of the grating circle of the functional surface, and the grating circle is symmetrical about the center of the imaging area. The first circle of gratings is composed of 3 pairs of gratings with a period of T1, and the angle between them is 60°. The second circle of gratings is composed of 6 pairs of gratings with a period of T2 = 600nm, and the angle between them is 30°. The spacing D between the same pair of gratings in each circle m , that is, the radius of the circle where each aperture is located, satisfies:

[0048]

[0049] Where t represents the thickness of the substrate material, λ represents the wavelength of the illumination light used, n represents the refractive index of the substrate material, and T m represents the period of the mth grating. Figure 4Serial No. 9 and 10 correspond to gratings with T1=600nm and T2=300nm, respectively, and their areas are both 7.3mm 2 .

[0050] When the frequency-shift super-resolution microscopic imaging chip is connected to an imaging system, the two apertures in the frequency-shift structure layer can excite the large wave vector frequency-shift light of the incident illumination light, and the collected imaging spectrum is as follows: Figure 6 As shown, k1 and k2 are the frequency shift wave vectors that can be provided by the gratings 9 and 10 respectively, and the spectrum range after synthesis is marked with dotted lines.

[0051] The embodiment also provides a method for preparing the above-mentioned frequency-shift super-resolution microscopic imaging chip, such as Figure 7 As shown, the following steps are included:

[0052] S701, clean and dry the waveguide. Specifically, use acetone, isopropyl ketone, ethanol, and deionized water to clean the Al2O3 layer as the high refractive index waveguide layer in sequence. 2 O 3 The substrate c is then ultrasonically cleaned to obtain a clean Al 2 O 3 Substrate.

[0053] S702, make microelectrodes, reference electrodes, leads and external contacts on the upper surface of the waveguide. Screenshot: Use laser direct writing to pattern the microelectrodes, reference electrodes, leads and external contacts on the upper surface of the waveguide. After development, use an electron beam evaporation coating system to first deposit a layer of Cr seed layer (for example, 30nm) thick, then deposit a layer of Au conductive layer (for example, 200nm) thick, and finally use a stripping process to remove excess film to form microelectrodes, reference electrodes, leads and external contacts to form a microelectrode array layer;

[0054] S703, preparing an insulating layer. Specifically, SiO is deposited on the microelectrode array layer by plasma chemical vapor deposition. 2 / Si 3 N 4 (e.g. 100 nm) insulating layer.

[0055] S704, etching the microelectrode, reference electrode and external contact point on the insulating layer. Specifically, laser direct writing combined with electron beam direct writing is used to expose the microelectrode, reference electrode and external contact point under the insulating layer.

[0056] S705, making culture chambers, connecting channels and microchannels. Specifically, laser direct writing is used to pattern the culture chambers, connecting channels and microchannels on the upper surface of the silicon wafer, and then a layer (for example, 200 μm) thick PDMS is spin-coated on the upper surface. After solidification, the microfluidic structure layer is peeled off.

[0057] S706, etching a grating on the lower surface of the waveguide. Specifically, double-sided alignment lithography combined with electron beam direct writing is used to etch the grating on the Al 2 O 3 A precisely aligned grating is fabricated on the lower surface of the waveguide.

[0058] S707, aligning and fitting the microfluidic structure to the microelectrode array. Specifically, using tweezers and multiple sets of alignment marks, the microfluidic structure is precisely fitted to the microelectrode array under a microscope.

[0059] The preparation method has a simple preparation process and can obtain a high-quality frequency-shift super-resolution microscopic imaging chip.

[0060] The specific implementation methods described above provide a detailed description of the technical solutions and beneficial effects of the present invention. It should be understood that the above is only the most preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, supplements and equivalent substitutions made within the scope of the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A frequency-shifted super-resolution microscopic imaging chip capable of observing in-situ electrical stimulation in microfluidics, characterized in that: It includes a microfluidic structure layer, a microelectrode array layer, a high refractive index waveguide layer, and a frequency shift structure layer in sequence, and exhibits light transmittance; The microfluidic structure layer includes a microfluidic structure capable of guiding cell neurons in the cell fluid to grow along a single cell channel; The microelectrode array layer includes a microelectrode array, which can realize in situ electrical stimulation of single cells in the single cell channel; The high refractive index waveguide layer is used to support the microelectrode array layer and realize high refractive index optical waveguide transmission; The frequency-shifting structural layer is used to excite large-wave-vector frequency-shifted light to the incident illumination light, and transmit the large-wave-vector frequency-shifted light to the high-refractive waveguide layer; The chip can realize in situ electrical stimulation of single cells in microfluidics while supporting frequency-shift super-resolution microscopic signal imaging.

2. The frequency-shifting super-resolution microscopic imaging chip capable of observing in-situ electrical stimulation in microfluidics according to claim 1, characterized in that: The microfluidic structure layer includes an inoculation cavity, a connecting channel and a microchannel. The two ends of multiple groups of microchannels are connected by two connecting channels. The other two ends of the two connecting channels are respectively connected to the inoculation cavity. The microchannel serves as a single cell channel. Its width and height only allow a single axon or dendrite in a cell neuron to grow. At the junction of the connecting channel and the microchannel, a funnel-shaped structure is used to guide the cell neurons in the connecting channel to grow along the microchannel.

3. The frequency-shifting super-resolution microscopic imaging chip capable of observing in-situ electrical stimulation in microfluidics according to claim 2, characterized in that: The microchannel has a height of 4 μm-5 μm, a width of 4 μm-5 μm, and a microchannel interval of 20-40 μm.

4. The frequency-shifting super-resolution microscopic imaging chip capable of observing in-situ electrical stimulation in microfluidics according to claim 1, characterized in that: The microelectrode array includes a microelectrode array located in the center, a reference electrode array arranged around the microelectrodes, an external contact point array arranged on the periphery, and wires connecting the external contact points with the reference electrodes and the microelectrodes. The microelectrode array corresponds to the single cell channel of the microfluidic structure layer to achieve in situ electrical stimulation of single cells in the single cell channel.

5. The frequency-shifting super-resolution microscopic imaging chip capable of observing in-situ electrical stimulation in microfluidics according to claim 4, characterized in that: The diameter of the microelectrode ranges from 10 μm to 20 μm, and the diameter of the outer contact point ranges from 2 mm to 2.5 mm.

6. The frequency-shifting super-resolution microscopic imaging chip capable of observing in-situ electrical stimulation in microfluidics according to claim 1, characterized in that: An insulating layer is also provided between the microfluidic structure layer and the microelectrode array layer.

7. The frequency-shifting super-resolution microscopic imaging chip capable of observing in-situ electrical stimulation in microfluidics according to claim 1, characterized in that: The material of the high-refractive waveguide layer is selected to be transparent in the selected illumination light band. When the illumination light is visible light, the material of the high-refractive waveguide layer includes Si3N4, SiO2, Al2O3, TiO2 or Polymer.

8. The frequency-shifting super-resolution microscopic imaging chip capable of observing in-situ electrical stimulation in microfluidics according to claim 1, characterized in that: The frequency shift structure layer includes multiple circles of gratings, each circle of gratings is distributed in a circle, the centers of multiple groups of gratings coincide, each circle of gratings includes multiple pairs of gratings symmetrical along the center, the periods of the same circle of gratings are the same, and the periods of different circles of gratings are different; Each pair of gratings D m The distance between them satisfies: Where t represents the thickness of the substrate material, λ represents the wavelength of the illumination light used, n represents the refractive index of the substrate material, and T m Represents the period of the mth grating.

9. A method for preparing a frequency-shifting super-resolution microscopic imaging chip capable of observing in-situ electrical stimulation in microfluidics according to any one of claims 1 to 8, comprising the following steps: cleaning and drying the high-refractive waveguide to form a high-refractive waveguide layer; fabricating a microelectrode array layer pattern on the high-refractive waveguide layer; covering the microelectrode array layer with an insulating layer, and preparing the microelectrode array layer on the insulating layer; Etching a frequency shifting structure layer on the other side of the high refractive waveguide layer; A microfluidic structure layer is prepared, and the microfluidic structure layer is aligned and bonded with the microelectrode array layer.

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