Parallelized ring array platform for high speed cell imaging

Abstract

An ultra-high flux quantitative phase imaging (QPI) system incorporates a real-time rotating cell detection based platform with concentric annular cell chambers (20). Each chamber (20) has a detection region (22) and a pair of access channels (24) with openings (25) between the ends. The access channel (24) serves to deliver a fluid containing the cell sample introduced into the opening (25) to the detection region (22). The opening (25) faces an inner radial position of the rotating platform compared to the detection area (22) so as to prevent liquid within the detection area (22) from being squeezed out by centrifugal force when the platform rotates, while communicating with the external environment before, during and after rotation.

Description

Cross - reference to related patent applications

[0001] This application claims the priority of U.S. Application No. 63 / 410,363, filed on September 27, 2022, the entire content of which is incorporated herein by reference. Technical Field

[0002] The present invention relates to the field of various biological detection chambers, and particularly to a biological detection chamber with a rotating imaging cell detection platform. Background Art

[0003] Cell morphology is widely regarded as the basis for determining complex cell types, states, and functions. Therefore, image - based screening assays are becoming increasingly popular in drug development. Most current strategies involve fluorescence labeling, but the operation becomes cumbersome when multiple fluorescence labels are required. Cell - based assays are currently replacing traditional population - based assays because of their advantages in single - cell analysis. Optical microscopy imaging is one of the effective means to achieve this goal. However, in order to discover rare cell subpopulations or obtain representative cell population results (usually greater than 10 6 cells), a large number of cells must be analyzed. Another requirement for these assays is the ability to analyze multiple conditions simultaneously.

[0004] To meet these criteria, a high - throughput, parallelized imaging system is needed. One technique to increase the imaging speed of assays is to use a high - speed rotating disk cytometer platform. By utilizing rotational motion for high - speed scanning, a class of high - throughput imaging techniques has been developed for parallelized image - based assays. See Tang, A.H. et al., "DVD - based time - stretched microscopy for high - throughput imaging cytometry," published in Biomedical Optics Express, Vol. 8, No. 2, pp. 640 - 652 (2017). Similarly, U.S. Application Publication No. 2021 - 0381979A1 describes a method for cell / tissue imaging assays using high - speed rotational motion. In this method, cells are irreversibly sealed with ultraviolet glue to prevent leakage of the culture medium during rotation. With the ability of high - throughput imaging, these techniques have the potential to be applied to many cell - based assays, including but not limited to drug development, CRISPR screening, and cell biophysical analysis. However, some practical problems have hindered the mainstream application of these techniques.

[0005] U.S. Patent No. 8,420,026 discloses a similar rotary platform system. It has a microfluidic device based on centrifugal force, including a rotary operation unit for rotating a rotor, and an external energy source for irradiating a sample with electromagnetic waves. The rotor contains a microfluidic structure disposed therein, and the microfluidic structure includes a plurality of chambers, channels connecting the chambers, and valves disposed in the channels for controlling fluid flow. The microfluidic structure uses the centrifugal force generated by the rotation of the rotor to transport fluid; magnetic beads in the chambers collect target materials from the biological material samples flowing into the chambers. As a result, the microfluidic structure cleans the magnetic beads collecting the target materials and separates nucleic acids by electromagnetic wave irradiation. However, this design does not consider microscope imaging.

[0006] U.S. Patent No. 10,162,162 discloses a different technique that uses a microfluidic device to generate microvortices to rotate individual cells or cell clusters around their centers for tomography. The purpose is to rotate the cells so as to generate three-dimensional images of each cell or cell cluster one by one. Since a rotary platform is not used, the throughput is limited to individual cells or cell clusters. In addition, the chamber has a trapezoidal cross-sectional shape below the flow channel for generating microvortices.

[0007] U.S. Application Publication No. 2022-0195486 also discloses a multi-microfluidic culture chamber that can be used for imaging the growth of a single-cell monolayer. The cell chamber is separated into upper and lower compartments by a thin polydimethylsiloxane (PDMS) membrane. The lower compartment is used for culturing biological samples. The membrane is also used to "press" the sample and fix it in place because the sample may not necessarily adhere to the bottom of the chamber. The movement of the membrane is controlled by the air pressure in the upper compartment. This design is specifically for automatically generating concentration gradients of different chemicals. This design also relies on a flow controller to precisely control the flow rate and the movement of the membrane.

[0008] Living cells must always be cultured in a culture medium. Therefore, for rotary imaging techniques, the chamber must be sealed to avoid leakage of the liquid culture medium. These liquid-tight seals are usually irreversible. Therefore, the cells cannot be accessed during or after the detection. It may be very important to obtain the cells for other genetic and biological tests after the imaging detection. In addition, for more complex experiments that require adding chemicals during the detection, using a sealed chamber is not feasible. Finally, equally importantly, the isolation of the chamber from the external environment due to the seal also makes long-term detection impossible because it will suffocate the cells.

[0009] Another problem is the difference in image quality between individual chambers on a plastic-based substrate, which brings difficulties to further analysis. To achieve quantitative analysis, the chambers must have certain tolerances to minimize aberration and focus changes, which lead to inconsistent image quality between individual chambers.

[0010] U.S. Patent No. 9,612,199 B2 discloses a system for imaging captured cells, which uses an autofocus module to maintain consistent image quality among the respective wells containing cells. These images are snapshots taken of each well while the well is stationary. Therefore, the process is very slow. U.S. Patent No. 7,709,248 B2 describes a circular array substrate for fluorescence-based biological detection. Its substrate is used to detect fluorescently labeled substances such as proteins, DNA, lipid molecules, and does not involve imaging technology.

[0011] International Patent Application WO2018 - 045978 A1 describes a method of constructing a substrate using a polycarbonate sheet and spacers. The chamber is sealed with a fully ultraviolet-cured adhesive to prevent leakage. However, this patent does not specifically describe the shape of the chamber. SUMMARY OF THE INVENTION

[0012] The present invention relates to a large-scale single-cell intrinsic morphology analysis strategy that employs ultra-high-throughput scanning field of view (FOV) imaging technology and combines a novel real-time rotating cell detection platform. This integrated system demonstrates unprecedented functional detection capabilities, not only improving the detection throughput but also endowing new cell analysis capabilities, enabling multiplex live-cell drug screening (e.g., 96 conditions can be detected in a single run) and gene perturbation detection (through CRISPR technology). This platform can generate a large amount of cell quantitative phase imaging (QPI) data sets (e.g., 4.85 TB), which can provide a cost-effective label-free solution for identifying disease- or gene-related cell morphological phenotypes in therapeutic screening.

[0013] The present invention aims to set up a plurality of biological detection chambers on a rotating platform to achieve parallel detection similar to commercially available microplates (96 wells or more), but with improved characteristics that enable it to be uniquely suitable for high-resolution and high-throughput rotating imaging detection. These chambers are arranged in a circular array (or ring) for rapid rotation and imaging. The detection samples can be two-dimensional monolayer adherent cells, three-dimensional cell cultures, or three-dimensional tissues / organs.

[0014] Although there are multiple embodiments of the present invention, one embodiment features fluidic adherent cell chambers designed for rotating imaging technology, and these chambers are concentric and arranged in a circular pattern. The chambers of this platform are composed of three pieces of specially patterned fused silica plates bonded with ultraviolet-cured adhesives, ensuring strict tolerances for the flatness of the substrate and being suitable for fast, large-field-of-view, high-resolution optical imaging.

[0015] The chamber is designed to be in direct contact with the external environment, which is beneficial for the growth of cells at any time, ensuring the long-term health of cells and enabling continuous contact with the internal culture medium and cells, while maintaining no leakage during high-speed rotational motion (no upper limit on rotational speed). This design does not require complete sealing during rotation to prevent leakage, thus ensuring the recyclability, accessibility, and viability of cells.

[0016] Specifically, the platform includes a specially designed chamber with a shape having two inlet and outlet channels that lead to an opening located at the inner radius of the platform. This design enables the liquid culture medium in the chamber not to overflow during high-speed rotation even if the opening is not sealed. Therefore, the liquid culture medium in the chamber can remain in contact with the external environment. In particular, the gas components (such as oxygen and carbon dioxide) in the culture medium can maintain balance indefinitely. This ensures the health status of cells during the detection process and supports long-term cell time-course studies. In addition, the cells can also be recovered after detection.

[0017] As mentioned above, this design uses three fused silica substrates or wafers to define the bottom, top, and sides of the chamber structure. These substrates are bonded together using an optically curable ultraviolet adhesive (NOA61). This manufacturing process enables more stringent tolerance control, thus minimizing the difference in image quality between individual chambers. Consistent image quality is a key element for quantitative cell analysis, which can ensure that the changes in cell images in each chamber are based on the biophysical phenotypes of the cells themselves (such as morphology and dry weight), rather than differences in the manufacturing process.

[0018] When using fused silica and ultraviolet curable adhesive in manufacturing the substrates, the thickness variation can be controlled to a minimum (less than 10 μm), and the entire platform can be made as flat as possible (with a thickness variation of less than 100 μm on a platform with a diameter of 120 mm). Compared with plastic-based substrates, these tolerances minimize the aberrations and focus variations that would cause inconsistent image quality between individual chambers.

[0019] The present invention has the following features: - During or after the rotational imaging detection process, the cell samples and culture medium in the chamber can be accessed. This design allows the culture medium in the chamber to be in contact with air at any time, including during rotation, to ensure cell health. It also supports long-term cell time-course studies. The detection samples can range from two-dimensional monolayer adherent cells to three-dimensional cell cultures and three-dimensional tissues / organs. - The geometric shape of the chamber and the shape of the channels are designed to ensure that the liquid culture medium for the samples can completely fill the well plate without generating bubbles, which is crucial for stable rotational imaging. - Depending on the manufacturing technology, the number of chambers can be highly scalable, up to 384 wells, 1536 wells or even more. These well plates can be single-layer two-dimensional patterns or multi-layer three-dimensional patterns. - This new platform uses three laser-patterned fused silica wafers as substrates. This ensures that the platform has strict tolerance control in terms of thickness and flatness, i.e., the tolerance is within about 100 μm. This fundamentally reduces the variation range of the imaging focal plane between individual chambers, shortens the focusing adjustment distance, and thus enables a faster imaging workflow.

[0020] The overall design is tailored for high-throughput, parallelized, long-term image-based cell assays, which are very useful (but previously scarce) in chemical (drug) screening, gene (such as through CRISPR technology) screening, and cell analysis applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] This patent or application document contains at least one color drawing. If requested and the necessary fees are paid, the Patent Office will provide a copy of this patent or patent application publication document with color drawings.

[0022] In conjunction with the following detailed description and the accompanying drawings, the above and other objects and advantages of the present invention will become more apparent. In the drawings, the same reference numerals denote the same elements, wherein:

[0023] Figure 1A is a schematic diagram of a high-speed rotating imaging substrate with sample wells or chambers; Figure 1B is an enlarged view of a specific area of the substrate for detailed display of the size of a single chamber; Figure 1C is an exploded view with a chamber area, detailing the patterns on each fused silica wafer; Figure 1D is Figure 1C an assembly schematic diagram of the exploded view.

[0024] Figure 2A is Figure 1A an enlarged example of the design with more wells or chambers, a larger substrate diameter, and two layers of chambers; Figure 2B is Figure 2A an enlarged view of a specific area marked with a dashed line on the substrate in Figure 2C is Figure 2B for detailed display of the size of the chambers in this figure; Figure 2D is Figure 2A a cross-sectional side view at the designated line in

[0025] Figure 3 is a prospective schematic diagram of a high-speed rotating imaging system platform according to the present invention, including part of the imaging system.

[0026] Figure 4 is a flowchart of the overall workflow for manufacturing the high-speed rotating imaging platform of the present invention.

[0027] Figure 5A is a prospective schematic diagram of the experimental configuration of the present invention, including an imaging system; Figure 5B shows an image of the rotating platform; Figure 5C shows the enlarged part of the chamber, and living cells are shown by quantitative phase imaging contrast; Figure 5D shows Figure 5C a further enlarged area of the chamber where the specified part contains cell clusters in

[0028] Figure 6A shows the experimental conditions of the outermost ring, the innermost ring of the rotating disk, and the stationary disk; Figure 6B shows under Figure 6A the shown conditions the viability of living cells. Detailed implementation manners

[0029] In the present invention, the cell detection platform is characterized in that, as shown in Figure 1A and Figure 2A , the fluidically designed cell chambers 20 are concentrically and circularly arranged on the substrate 10. For example, Figure 1A the rotating substrate design in Figure 3 has a diameter of 120 mm and 96 holes or chambers. These cell chambers are used for rotating imaging techniques, such as the quantitative phase imaging (QPI) technique shown in

[0030] The main novel elements for realizing the above functions lie in the new chamber shape, the design of the inlet and outlet channels, and the position of the openings. In the prior art, the chambers adopt a completely sealed strategy (usually irreversible) to avoid leakage during high-speed rotation. In the present invention, the chambers 20 can be arranged concentrically in a circular pattern (as shown in Figure 1A , with four rows 11, 12, 13, 14). Each chamber 20 is divided into three main parts: a detection area 22, a pair of inlet and outlet channels 24, and an opening 25 (see Figure 1B)。A cell sample is attached to the bottom of the detection area 22 of the chamber 20, i.e., the cells have the inherent ability to attach to the bottom surface. To assist this process, the bottom surface is coated with a layer of protein (such as fibronectin). The inlet and outlet channels 24 deliver the fluid medium to the detection area 22.

[0031] The arms forming channels with the detection area 22 can be uniform, and in Figure 1B these arms have a width of 1 mm. The opening 25 allows a micropipette to pump liquid through the channel 24, thereby delivering the fluid to the detection area 22. The opening is designed in such a direction that, compared to the detection area, it is towards the inner radial position of the entire platform. This ensures that during the rotation of the substrate 10, the liquid in the detection area will not be squeezed out by the centrifugal force, while remaining in communication with the external environment before, during, and after rotation. The diameter of the opening (1 mm and 1.5 mm in the Figure 1B embodiment) is designed according to the diameter of the pipette tip to enable effective pumping of the liquid through the channel to the detection area. Figure 1B This is shown where one hole is designed to accommodate medium tips (100 - 200 μl) and large tips (1000 μl), so that both types of tips can easily enter the chamber.

[0032] To ensure that the entire chamber is filled with the required liquid medium during pipetting and to prevent the formation of air bubbles, the design of the chamber walls avoids sharp corners and sudden changes in channel width. When pipetting into the chamber, the liquid front will advance in a controlled manner and fill the chamber without generating air bubbles.

[0033] These chambers are composed of three fused silica plates 31, 33, 35 bonded by ultraviolet - cured adhesive and laser - patterned to ensure extremely small tolerances in substrate flatness, suitable for fast, large - field - of - view, and high - resolution optical imaging (see Figure 1C ). This structure minimizes thickness variations (less than 10 μm) and makes the entire platform as flat as possible (thickness variation less than 100 μm on a 120 - mm - diameter platform). Figure 1C is an exploded view of a chamber - containing area on the substrate, showing in detail the patterns on each fused silica wafer. In this embodiment, the top layer 31 has a thickness of 0.5 mm and has two holes. The middle layer 33 has a thickness of 1 mm and is engraved with the shape of the detection area 22 and the channels 24. The bottom layer has a thickness of 0.5 mm and is flat without any incisions. The assembled chamber of this embodiment is as shown in Figure 1D with a total thickness of 2 mm.

[0034] Figure 2A is Figure 1A an enlarged example of the platform design with 384 holes and a double - layer design, with light - colored chambers on the lower layer and dark - colored chambers on the upper layer (see Figure 2C) Its platform diameter is 158 mm. Depending on the manufacturing technology, it can be further extended to 1536 wells or even more. Figure 2B is Figure 2A An enlarged view of a specific area on the platform for detailing the dimensions of the chamber. In this embodiment, the detection area is 5 mm and the arm width forming the channel is 0.5 mm. Figure 2C is Figure 2B A cross-sectional side view at the specified line in, showing a double-layer design. In this embodiment, the thickness of both the upper and lower layer chambers is 1 mm and they are separated by a 0.5 mm space in the vertical direction. Figure 2D is Figure 2A The three-dimensional enlarged view of the specified area in (see Figure 2B ). Note that the upper and lower layer chambers are staggered so that each detection area can be illuminated as Figure 3 shown.

[0035] When performing fast, large field-of-view, high-resolution optical imaging, a general imaging system configuration 40 is adopted (see Figure 3 ). The optical system includes an objective lens 42 for focusing the light emitted by the light source onto the cells, and another objective lens 44 for transmitting the light to the image receiver. When the light irradiates the chamber 20, it passes through the chambers on the substrate 10 (the substrate consists of 3 pieces of fused quartz chambers bonded by ultraviolet-cured adhesive and specially patterned) and the sample. Finally, an image of the sample is obtained, which can be used for single-cell intrinsic morphology analysis research. In Figure 3 , the imaging system only images the outer ring 11 of the chamber. However, it can move to all other rings 11 - 14 for imaging. Due to the extremely small flatness tolerance of the substrate, almost no autofocus is required when performing fast, large field-of-view, high-resolution optical imaging.

[0036] Figure 4 Shows a more detailed manufacturing process or flow chart. In step 502, the design pattern is drawn using CAD software. Then in step 503, the pattern is cut on the fused quartz wafer using a laser, as Figure 1C shown. Next, in step 504, the bottom wafer 35 and the middle wafer 33 (see Figure 1C ) are bonded together using the bonding steps 602 - 607.

[0037] In the bonding step 602, the wafer is cleaned successively with mild detergent, acetone, and isopropyl alcohol, and then assembled. Then in step 603, the wafer is thoroughly dried and gently wiped with a Kimwipe. In step 604, an ultraviolet-curable adhesive is applied to the bonding surface of the middle wafer 33. Subsequently in step 605, the wafers are aligned and the middle wafer 33 is bonded to the bottom wafer 35. In step 606, the bonded wafers are clamped between two glass plates, and a uniform compressive stress is applied to the assembly (e.g., using a jig) to hold the wafers together. Then in step 607, the assembly is irradiated with a 365-nanometer light source to pre-cure the adhesive used in the structure. The pre-curing, final curing, and aging times depend on the ultraviolet-curable adhesive used.

[0038] After the bonding of the bottom wafer 35 and the middle wafer 33 is completed, in step 505, the bonding steps 602 - 607 are repeated to bond the top wafer 31 to the middle-bottom assembly. In step 506, the top-middle-bottom assembly is wiped with a Kimwipe soaked in acetone to remove excess glue. In step 507, the assembly is irradiated with a 365-nanometer light source to fully cure the adhesive. As in step 607, the pre-curing, final curing, and aging times depend on the ultraviolet-curable adhesive used. Finally, in step 508, the assembly is stored to age the adhesive, thereby forming a stronger bond and chemical resistance.

[0039] None of the existing rotational imaging detection techniques can achieve both leak-free and seal-free simultaneously. The present invention employs a chamber design that includes a main detection area and a pair of channels leading to openings at radial positions within the platform. This structural difference makes the present invention stand out among the existing platforms of the same kind.

[0040] Using the present invention and quantitative phase imaging (QPI) technology, two large-scale functional image-based assays (with a data volume of 4.85 TB) have been completed, and the QPI technology has not been fully utilized in the past. First, drug assays were performed on two lung cancer cell lines (NCI-H1975 and NCI-H2170). This QPI-based cell assay showed high specificity / sensitivity to four drugs with different mechanisms of action, including a microtubule depolymerizing agent (docetaxel), DNA interfering agents (cisplatin and gemcitabine), and an epidermal growth factor receptor (EGFR) targeting drug (erlotinib). Second, a CRISPR-based cell-cell fusion assay was developed by co-culturing human cells expressing the receptor ACE2 (gene knockout) with cells expressing the SARS-CoV-2 spike protein. The large amount of QPI data sets generated helped to more deeply evaluate the association and impact between the potential mechanism of virus entry into cells and the inherent morphology of cells. This analysis strategy has new potential in identifying novel morphological phenotypes related to disease-genes, which can serve as cost-effective indicators for developing therapeutic methods.

[0041] Figures 5A - 5C The experimental setup using the optical system of the present invention and the obtained images are shown. These images are live cell images under quantitative phase imaging (QPI) contrast, which is commonly used to display cell characteristics. Figure 5A is a prospective schematic diagram of an experimental configuration similar to Figure 3 but shows the entire platform. In Figure 5A , the platform with a diameter of 120 mm rotates at a speed of 1300 revolutions per minute. Figure 5B The image of the rotating platform is shown, illustrating that the present invention can achieve high-resolution imaging of a large area. Figure 5C The enlarged part of the chamber is shown, with live cells displayed under QPI contrast, Figure 5D and Figure 5C shows a further enlarged area of a specified part of the chamber in Figure 5D . QPI is a microscopy method used to quantify the phase shift that occurs when light waves pass through an optically denser object such as a translucent living human cell. As shown in

[0042] Figure 6A and 6B show the viability of live cells under different conditions of the rotating platform of the present invention. These conditions are placing for one hour at the outermost ring of the rotating platform, placing for one hour at the innermost ring, and placing for one hour on the stationary disk. Figure 6B The bar chart in

[0043] Although the present invention is described in connection with specific embodiments, it should be understood that those skilled in the art will appreciate various modifications thereto after reading this specification. Therefore, it should be understood that the present invention as disclosed herein is intended to cover such modifications that fall within the scope of the appended claims.

Claims

1. An ultra-high throughput scanning field-of-view imaging system, comprising: a platform for cell detection that rotates in real time, the platform having at least one circle of cell chambers, each chamber having a detection area, an inlet and outlet channel with an opening between a pair of ends, the inlet and outlet channel for delivering a fluid medium of a cell sample introduced into the opening to the detection area, the opening facing an inner radial position of the rotating platform compared to the detection area so as to prevent liquid in the detection area from being extruded by centrifugal force when the platform rotates, while remaining in communication with the external environment before, during and after rotation; and an imaging system for passing light through the cell chamber and receiving an image of the cell sample.

2. The ultra-high throughput scanning field-of-view system according to claim 1, wherein the chambers are arranged in a plurality of concentric rings, and relative movement is possible between the imaging system and the platform so as to move the imaging area of the system from one ring to another.

3. The ultra-high throughput scanning field-of-view system according to claim 1, wherein the platform has at least two layers, each layer having chambers, the chambers of one layer being laterally offset from the chambers of the other layer so as to closely arrange the detection areas of the chambers without overlap, only non-detection areas (such as inlet and outlet channels) overlapping.

4. The ultra-high throughput scanning field-of-view system according to claim 1, wherein the cell chambers are made of a plurality of stacked, ultraviolet-cured adhesive-bonded and laser-patterned fused silica plates, each chamber having an upper plate with two holes, an intermediate plate with a detection area and an inlet and outlet channel with an opening laser-machined between a pair of ends, and a flat bottom plate without any cutouts, the channel leading from the opening at the inner radius of the platform to the detection area at the outer radius of the platform; and the silica plates are bonded together by ultraviolet-cured adhesive.

5. The ultra-high throughput scanning field-of-view system according to claim 1, wherein the detection sample can be a two-dimensional monolayer adherent cell, a three-dimensional cell culture or a three-dimensional tissue / organoid.

6. The ultra-high throughput scanning field-of-view system according to claim 1, wherein the chambers are designed to allow direct contact with the external environment, which is beneficial for the growth of cells at any time to ensure that the sample cells remain healthy in the long term, can continuously contact the culture medium and sample cells in the chamber, facilitate the addition or removal of substances, and recover the sample cells after detection, while maintaining no leakage during high-speed rotational movement.

7. The ultra-high throughput scanning field-of-view system according to claim 1, for a platform with a diameter of 120 mm, the rotational speed range is from 100 revolutions per minute to 6000 revolutions per minute.

8. The ultra-high throughput scanning field-of-view system according to claim 1, the thickness variation of the 120 mm diameter platform is less than 100 μm.

9. The ultra-high throughput scanning field-of-view system according to claim 5, wherein the opening diameter is 0.5 - 1.5 mm, the top layer thickness is 0.5 mm, the middle layer thickness is 1 mm, the bottom layer thickness is 0.5 mm, and the total chamber thickness is 1 mm.

10. The ultra-high throughput scanning field-of-view system according to claim 2, wherein the platform has four (4) concentric rings, with a total of 96 chambers evenly distributed.

11. The ultra-high throughput scanning field-of-view system according to claim 4, wherein the platform has twelve (12) concentric rings with a total of 384 chambers evenly distributed.

12. A method for manufacturing a chamber for an ultra-high throughput scanning field-of-view system, comprising the following steps: drawing a design pattern in CAD software; cutting the pattern on a fused silica wafer using a laser; bonding a bottom wafer and a middle wafer; repeating bonding step 602 to bond a top wafer to the middle-bottom assembly; wiping the top-middle-bottom assembly with a Kimwipe soaked in acetone to remove excess glue; irradiating the assembly with a light source to fully cure the adhesive; and storing the assembly to age the adhesive, thereby enhancing the bonding strength and chemical resistance.

13. The method according to claim 13, wherein the bonding step comprises the following steps: cleaning the wafers successively with mild detergent, acetone, and isopropyl alcohol; assembling the wafers; thoroughly drying the wafers and then gently wiping them with a Kimwipe; applying an ultraviolet curable adhesive to the bonding surface of one wafer; aligning and pasting one wafer with another wafer; clamping the two wafers between two glass plates and applying uniform compressive stress to fix the wafers together; and irradiating the wafers with a 365-nanometer light source to cure the adhesive.

Images