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Abstract
Single-cell-scale selective manipulation and targeted capture play a vital role in cell behavior analysis. However, selective microcapture has primarily been performed in specific circumstances to maintain the trapping state, making the subsequent in situ characterization and analysis of specific particles or cells difficult and imprecise. Herein, we propose a novel method that combines femtosecond laser two-photon polymerization (TPP) micromachining technology with the operation of optical tweezers (OTs) to achieve selective and targeted capture of single particles and cells. Diverse ordered microcages with different shapes and dimensions were self-assembled by micropillars fabricated via TPP. The micropillars with high aspect ratios were processed by single exposure, and the parameters of the micropillar arrays were investigated to optimize the capillary-force-driven self-assembly process of the anisotropic microcages. Finally, single microparticles and cells were selectively transported to the desired microcages by manipulating the flexibly of the OTs in a few minutes. The captured microparticles and cells were kept trapped without additional forces.
© 2021 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
1. Introduction
Owing to the development of biomedicine and cellular behavior, cell manipulation and capture have attracted extensive research attention [1–3]. Because of the heterogeneity of cells, it is imperative to achieve selective manipulation and targeted capture of single cells to identify the individual functional characteristics of specific cells. For example, cancer is one of the major diseases affecting human development [4]. The effective analysis of carcinogens is important in the early diagnosis and prevention of cancer [5–8].
Benefiting from the advantages of high throughput, high portability, and miniaturization, microfluidic-technology-based hydrodynamic capture has been the most frequently used approach for particle and cell capture [9–14]. However, because the micro-trap requires constant pressure to keep the particles or cells captured, even a slight disturbance affects the capture efficiency. Although various optimization schemes have been proposed to improve the capture efficiency [15–17], most of these designs still require constant pressure. When the pressure decreases or vanishes, the particles or cells escape easily. In addition, because the trapping process cannot be artificially interfered with, the particles or cells are captured randomly, which makes it difficult to achieve targeted capture of specific particles or cells and hinders flexibility.
Magnetic-driven [18,19], dielectrophoresis [20,21], and optically induced dielectrophoresis [22,23] techniques have the advantages of easy control, flexible operation, and high flux, and are widely used in particle trapping. Experiments have proven that these technologies can be used in selective manipulation and to capture single particles or cells. However, they are highly dependent on a specific operating environment; therefore, in the absence of this operating environment, the particles return to a disordered state, complicating the subsequent characterization and analysis. Therefore, an approach that can achieve selective manipulation of individual particles and cells while realizing stable capture without continuous additional force is expected to open a new route to relevant single cell research.
Inspired by natural phenomena, researchers have attempted to capture particles reliably by introducing new physical mechanisms. For example, owing to the presence of capillary forces, most filaments will adhere to each other when they are pulled out of a liquid. Accordingly, many researchers have introduced the capillary-force-driven self-assembly to create functional microstructures [24]. Previous studies have been conducted on the use of self-assembly to form plasma nanogap arrays for surface enhancement of Raman scattering [25], fabrication of 2D “grippers” self-assembled into a 3D fixture through residual stress to capture red blood cells [26], and the use of capillary force to make bionic microchannels [27]. In this study, we aimed to capturing particles and cells reliably via the self-assembly of a microcage structure without additional force.
As a high-precision 3D micromachining method, femtosecond laser two-photon polymerization (TPP) with good controllability and flexibility can be applied to the rapid machining of microstructures [28–32]. Owing to the suppression of the heat-affected zone and unique nonlinear electron absorption, TPP can enable flexible fabrication of microstructures in various transparent materials with ultra-high resolution and complex 3D structures [33,34]. Optical tweezers (OTs), a special means of microdisplacement control, are widely used in single-scale particle and cell manipulation with outstanding characteristics such as no mechanical contact, no mechanical damage, no interference with the surrounding environment, and flexible and efficient operation [35–39]. In addition, OTs do not require additional functional materials, avoiding the destruction of samples, which is crucial in the field of biomedicine.
This report demonstrates a novel method for selective manipulation and targeted capture of individual particles and cells using self-assembly microcages with the assistance of OTs. As the basic unit of the microcages, micropillars with different heights and diameters were completed by femtosecond laser TPP from bottom to top. By systematically analyzing the effects of the capillary force on the self-assembled structure, various ordered anisotropic microcages composed of different numbers of micropillars were fabricated to achieve the effective fixation of the target object. To further study the behavior of single particles or cells, OTs were used to focus a shaped laser beam onto the particle surface, enabling the object to move rapidly and in a directed manner. Then, we assembled the target into the structure using OTs to achieve the selective capture of microparticles and NIH-3T3 cells on a single-scale. We expect that this method could be used in extensive applications, including single-cell analysis, drug screening, and targeted therapy.
2. Results and discussion
2.1 System combining femtosecond laser TPP with OTs
The investigated system combining selective manipulation and targeted capture is composed of two parts: a femtosecond laser TPP system for the processing of self-assembly microcage structures and an OT-based selective manipulation system [Fig. 1(a)]. Briefly, microstructure arrays were fabricated using the TPP system, and microcage arrays were generated by capillary force during the photoresist development. A femtosecond laser with a wavelength of 1030 nm was focused into the photoresist through a homemade inverted micromachining system. Micropillar arrays of different heights, diameters, and gaps were directly written with the help of a 3D motion platform, as shown in Fig. 1(b). After the micropillar arrays were developed and the sample was exposed to air, the capillary force generated by the evaporation of the residual developer on the surface resulted in the bending of the micropillars. Then, self-assembly microcages were formed between neighboring micropillars, as shown in Fig. 1(c). The rate of self-assembly can be effectively improved by increasing the micropillar height and shortening the gap between micropillars. Compared with other micromachining technologies with physical masks, the parameters of the microcage arrays can be dynamically modulated in real time by controlling the femtosecond laser energy and the movement of the 3D platform.
Fig. 1. Two-photon polymerization (TPP) processing with optical tweezer (OT) operating system. (a) Schematic of microstructure fabrication and microsphere capture via TPP and OTs. (b) Micropillar arrays fabricated via femtosecond laser TPP. (c) Self-assembly of microcage arrays driven by capillary forces occurs between adjacent micropillars. (d) Process of trapping polystyrene (PS) microspheres using OTs. Scale bars: 15 μm.
The OT system consisted of a continuous laser with a wavelength of 532 nm, a polarized beam splitter to tune the laser energy, a spatial light filter to modulate the shape of the incident light beam, and an objective lens to focus the laser on the manipulated samples. Owing to the 3D gradient force light trap generated by the tightly focused laser around the focus point, particles and cells were trapped near the focus point, as shown in Fig. 1(d). The position of the trapped single particle (covered by the green laser) will change with the movement of the laser focus to achieve directional movement, while other particles will remain in place. The 3D gradient force light trap could be tuned continuously and quickly by adjusting the laser power. Consequently, by manipulating the OTs, specific particles and cells were transported into the microcages and captured by them. Furthermore, the manipulation had little impact on the surrounding environment. By focusing a beam of suitable energy on the particle, no mechanical damage to the particle was caused; therefore, the manipulation was harmless. This trait is very valuable in biological fields such as cell manipulation. The dynamic and selective manipulation and targeted capture of single cells were demonstrated through the combination of TPP fabrication and OT-based manipulation.
2.2 Fabrication and characterization of micropillar arrays
Each Micropillar was fabricated by a single-pulse femtosecond laser TPP. The height and diameter of each micropillar were controlled by adjusting the laser power and the distance between the sample and objective lens. Figure 2 shows the variation tendencies of key experimental micropillar parameters during processing. During TPP processing, the glass-photoresist interface was regarded as the zero-height position of the micropillar. The theoretical micropillar height (Hth) is defined as the distance between the platform and the zero-height position along the Z-axis direction. Theoretically, when the exposure power increases, the area where the processing energy exceeds the polymerization threshold becomes larger, which results in the generation of larger micropillars (either in diameter or height). To verify the effect of exposure power on micropillar parameters, we processed micropillars with heights ranging from 5 to 40 μm at 5-μm intervals by moving the 3D piezoelectric platform a corresponding distance and characterized the actual micropillar heights (Hac) via scanning electron microscopy (SEM) [Figs. 2(a) and 2(b)]. Figure 2(c) depicts the relationship between the Hth and Hac processed with different laser power levels. The diagram clearly shows that the theoretical values are basically consistent with the actual values. These results show that, at the same theoretical height, the polymerization area change caused by the variation in exposure power is small (less than 1 μm). Compared with Hth (5–40 μm), different laser power levels have a minor effect on Hac. The relationship between Hth and the actual micropillar diameters (Dac) under different laser power levels was also analyzed, as shown in Fig. 2(d). Evidently, the diameter increased with increasing micropillar height and laser exposure power. The former phenomenon results from the larger laser spot projecting onto the glass-photoresist interface when the micropillar height increases. The latter can be explained by the fact that, when the exposure power increases, the area where the processing energy exceeds the energy threshold becomes larger, leading to a larger TPP area. Simultaneously, owing to bottom-up processing, the area near the substrate is more likely to initiate polymerization and gradually decrease with increasing depth. Therefore, the micropillar morphology is similar to that of a “tower,” with a larger diameter at the bottom and slender top, which is especially obvious for higher micropillars.
Fig. 2. Characterization and analysis of micropillars with different heights and diameters. (a) and (b) Micropillar arrays processed at 0.4 W power with heights of (a) 10 μm and (b) 30 μm. Scale bars: 50 μm. The insets show SEM images of a single micropillar. Scale bars: 5 μm. (c) and (d) Dependence of the actual micropillar processing height and diameter on the theoretical setting height at power levels of 0.4, 0.6, and 0.8 W.
2.3 Microparticle manipulation using OT
The OT system was exploited for the micromanipulation of free-floating polystyrene (PS) microspheres in deionized water. Owing to the pressure of the optical gradient light trap, transparent particles were captured instantaneously when the continuous laser was focused on the upper surface of the particles. When the particles are much larger than the wavelength of the incident beam, the beam force on the particle can be explained using a geometrical optical model. As shown in Fig. 3(a), the incident laser beam is decomposed into countless small rays. Here, we consider rays 1 and 2 as examples to focus on the upper surface of the microsphere. When the laser acts on the surface of the particle, reflection (scattering force), refraction (gradient force), and other phenomena occur following accordance with Snell’s law. Furthermore, because photons carry momentum, when rays 1 and 2 pass through a particle, a downward momentum is produced, which is expressed as the force of the laser acting on the particle. According to the conservation of momentum, the microsphere will generate upward momentum, corresponding to opposing forces F1 and F2. The net opposing force (Fnet) will pull the microsphere into the focus of the laser, and when the scattering and gradient forces reach a balance, stable microsphere capture is achieved [40].
Fig. 3. Principle analysis and microsphere operation of optical tweezers. (a) Qualitative view of microsphere trapping by the optical tweezer. (b)–(d) Trajectory of particles operated via optical tweezers. The motion states are initial position (b), moving path (c), and target position (d). Scale bars: 10 μm.
Figures 3(b)–3(d) show the trapping results of a 10-μm PS microsphere achieved using the OT system. With marks 1 and 2 as reference, the focused beam was applied to the surface of the microsphere to complete its trapping. Meanwhile, the moving path of the microsphere could be completed by moving the motion platform. By properly controlling the laser energy and focus position, the entire motion process was completed within seconds. To ensure that the particles were stably captured and moved to the specified position quickly, we chose a larger laser power to increase the restraint of the optical trap to the microspheres. Moreover, if the laser power continuously increases, the attraction of the light trap to the surrounding microspheres will also be enhanced, enabling multiple microspheres to be trapped and transported simultaneously, which in turn significantly improves the directional transport efficiency of the microspheres.
2.4 Parameter investigations of capillary self-assembly
The capillary force usually acts at fluid–air–solid interfaces to minimize the surface energy. When the distance between two adjacent micropillars is sufficiently short, with the evaporation of the solution, capillary force interaction occurs between the micropillars, and the force is negatively related to the spacing between the micropillars. The smaller the micropillar spacing, the greater the capillary force generated by adjacent micropillars. When the capillary force is greater than the straight force that maintains the micropillar stiffness, the micropillars converge with the guidance of the capillary force. This process, as depicted in Fig. 4(a), is called the capillary-force-driven self-assembly of micropillars. After self-assembly, the tips of the micropillars come into contact with each other and generate van der Waals forces so that the self-assembled micropillars maintain contact with each other. The capillary-force-driven self-assembly phenomenon is especially obvious when the microstructure has a high aspect ratio. It should be noted that the micropillar tip becomes slender with increasing height, which results from the ellipsoidal distribution of the femtosecond laser energy at the focus, making the laser energy decrease and the polymerization area becomes smaller when it is far from the focal plane. Interestingly, we found that the slender micropillar tips make the self-assembly of microcages much easier.
Fig. 4. Diverse ordered self-assembled microstructure fabrication and analysis. (a) Schematic of the capillary-force-driven self-assembly process of micropillars. (b)–(d) Self-assembly microstructures composed of multiple micropillars. The numbers of micropillars are (b) three, (c) four, and (d) five. Scale bars: 20 μm. (e) “SIA” logo patterning through self-assembly microstructures composed of four micropillars. Scale bars: 50 μm.
Based on the above analysis, the optimizing parameters for capillary-driven self-assembly were investigated by controlling the height and spacing between adjacent micropillars. As a result, we achieved various directionally ordered self-assembly microstructures composed of different numbers of micropillars flexibly. We designed an isosceles triangular self-assembled structure array [Fig. 4(b)] with a side length of 10 μm and a height of 5 μm. Similarly, we also designed a square self-assembly structure array [Fig. 4(c)] with a side length of 10 μm, and a pentagonal array [Fig. 4(d)] with a side length of 10 μm and a height of 10 μm. Furthermore, the patterning of the logo “SIA” was realized by the fabrication of square self-assembly microstructure units with a side length of 10 μm, as shown in Fig. 4(e). The experimental results show a 100% probability of the controllable self-assembly effect when the parameters of the micropillar arrays are optimized, demonstrating the feasibility and stability of the method in microcage fabrication.
2.5 Use of self-assembly structures to capture particles with OTs
After demonstrating the flexibility of OT manipulation as well as the controllability and stability of the self-assembled microstructures, we examined the selective capture of individual microparticles in self-assembly structures under OT manipulation. Figure 5(a) shows the schematic of a microparticle captured by a “butterfly” microstructure consisting of four micropillars. Initially, a relatively simple triangular microcage structure was utilized for microparticle capture [See Visualization 1]. The distribution of the three micropillars is depicted in Fig. 5(b), as well as the capture result imaged by SEM. The isosceles triangular structure ensures that the particle can only be captured when it enters from the long side of the triangular structure. Thus, it is impossible for the particle to escape from the microcage from the two short sides of the structure. However, the small 3D space among the three micropillars enables the particle to escape easily. To obtain a better capture effect, an anisotropic “butterfly” structure was created, as shown in Fig. 5(c). Here, 5-μm-diameter SiO2 microspheres were manipulated by the OT system to capture them in the anisotropic structure. According to the size of the anisotropic structure, the microspheres can only enter on one side of the structure but not on the others, showing good anisotropy of the self-assembled structure. By combining the high efficiency and flexibility of OT manipulation, the target particle can be captured in the specified microcage within seconds. The proposed approach was demonstrated to be capable of selective manipulation with high efficiency as well as targeted capture, which is difficult to achieve using other methods.
Fig. 5. OTs manipulate microspheres for microcage capture. (a) 3D schematic of microcage capturing microspheres. Anisotropic capture of a single microsphere by microcages with (b) three and (c) four pillars. (d) A single microcage captures both microspheres simultaneously. Scale bars: 5 μm. Anisotropy of a single microsphere captured by a microcage array with (e) three and (f) four pillars. Scale bars: 20 μm.
In addition, multiple particles can be inserted into the same microcage, as shown in Fig. 5(d), which is important for studying the interactions between particles. We also performed particle capture using different microcage arrays, and the results are shown in Figs. 5(e) and 5(f). The capture efficiency can be defined by the formula:
where E is the capture efficiency, N is the number of microcages in the microcage array, and N’ is the number of microspheres retained in the microcage array after liquid evaporation.Interestingly, compared with the isosceles triangular microcage, the butterfly structure shows higher capture efficiency (∼70%) because of its more enclosed space. Moreover, we believe that the capture efficiency can be further improved by optimizing the structural parameters. This method provides a new route for in situ single-cell-scale observation.
2.6 Self-assembled structure based single-cell capture and observation
The high efficiency capture capability for microparticles was demonstrated using the anisotropic microcage structure. Moving forward, the selective manipulation and targeted capture of single cells were revealed accordingly [See Visualization 2]. As an important cell material, mouse fibroblasts (NIH-3T3) have been widely used in the study of in vitro carcinogenesis caused by tumor viruses and carcinogens because of their obvious contact inhibition [41,42]. Here, the microcage structure was designed according to the size of NIH-3T3 cells (about 15 μm), as shown in Fig. 6(a). We dropped the digested cells onto the microcage structure designed in advance. To better observe the relative distributions of cells and microcages before OT manipulation, we labeled the cells and microcages via fluorescent staining in vivo, as shown in Figs. 6(b) and 6(c). The microcage structures and NIH-3T3 cells are depicted in green and red, respectively. As can be seen, the cells distribute randomly around the structure. Next, we transported the specified NIH-3T3 cells to the butterfly structure using OTs [See Visualization 3]. The entire process is shown in Figs. 6(d)–6(f). The cells were pretreated to prevent them from sticking to the substrate and affecting the efficiency of OT manipulations. The results show that the method achieves directional selective capture of single cells. Furthermore, the subsequent in situ analysis of a single cell captured using this platform can effectively prevent interference from circumstances such as adding nutrients or changing the culture medium, and no continuous external force is required to maintain the captured state. On this basis, it would be interesting to analyze cell behavior in the restricted area. For example, in vivo fluorescent staining of captured cells, since the designed microcage do not completely cover the cell, the growth and apoptosis at a single-cell scale can be observed continuously, which is useful in targeted therapy. Furthermore, the microcage structure can be regarded as an independent biological scaffold, and the migration and adhesion of captured cells can be analyzed by cytoskeleton staining as well. Moreover, trapping other types of cells or immune substances surrounding the microcage that captures the single cell could be useful for the real-time in situ observation of the interactions between different types of cells. Because the peripheral cells do not have a shielding problem, most cell analysis methods are suitable for such applications.
Fig. 6. Single-cell manipulation and capture. (a) Optical image of the four-pillar microcage array. Fluorescence images of NIH-3T3 cell distribution in microcage arrays with (b) four and (c) three pillars. Scale bars: 50 μm. Relative distribution of a single NIH-3T3 cell and microcage (d) before and (e) after capture. (f) SEM image of a single NIH-3T3 cell in the microcage after capture. Scale bars: 10 μm.
3. Experiments
3.1 Experimental system
The TPP processing system was based on a femtosecond pulsed laser (central wavelength: 1030 nm, pulse duration: 240 fs, repetition frequency: 200 kHz), equipped with a 50× objective lens (NA = 0.65, Nikon, Japan). A 3D piezoelectric motion platform (nPBio300, nPoint, USA) and inverted laser lithography were selected to write microscale characteristic structures. The OT manipulating system was based on a continuous laser (central wavelength: 532 nm, average power range: 0–300 mW), equipped with a 40× objective lens (NA = 1.0, Nikon, Japan). The laser energy was modulated by a polarizing beam splitter, and a spatial beam filter was adopted to obtain a high-quality Gaussian beam. This beam was then focused into the liquid environment using the objective lens for fast directional particle manipulation. A dichromatic mirror (DMLP638, Thorlabs, USA) was adopted to reflect the green laser and transmit the illumination and imaging light. A charge-coupled device (Point Gray, Canada) was used to realize real-time observation of the TPP processing and OT manipulation.
3.2 Micropillar fabrication
The photoresist (SZ2080, FORTH, Greece) was drop-casted onto the cover glass and baked for 1 h before micropillar fabrication. The micropillar arrays (2×200) with a height of 5–40 μm (Δ = 5 μm) were prepared by fixing the spacing between the micropillars at 30 μm. Next, the above structures were processed by setting the laser output power to 0.4, 0.6, and 0.8 W. Each micropillar was fabricated via single-pulse with a pulse duration of 240 fs. The number and frequency of laser pulses can be preset by a customized program. The micropillar arrays were fabricated by synchronizing the laser pulses and the piezoelectric platform. Finally, they were developed for 6 min with n-propanol:isopropanol = 1:1, soaked in isopropanol for 20 min, and dried.
3.3 OT manipulating particles
PS microspheres with a diameter of 10 μm were selected as the operation objects. A 10-μL commercial PS pellet suspension was diluted with deionized water until the particles could be completely dispersed in the solution. After sitting for 2 min, some of the microspheres settled at the bottom of the solution. The continuous laser with a power of 200 mW at 532 nm wavelength was focused on the surface of the particles, and the particles fell into the optical trap. Two target points were selected as the reference frame, and the rapid directional movement of the captured microspheres was achieved by adjusting the moving platform. By adjusting the laser energy and the position of the focus, the speed of the moving microspheres can reach tens of microns per second.
3.4 Fabrication of self-assembled structures
Micropillars with a height of 30 μm were selected as the basic unit of the self-assembled process because of their good self-assembly performance. An isosceles triangle pattern with a side length of 10 μm and a height of 5 μm, a square with a side length of 10 μm, and a pentagonal pattern with a base of 10 μm and a height of 10 μm were printed. The horizontal and vertical pitch of each pattern were 20 and 40 μm, respectively. After development, as the solution evaporates, directional self-assembly occurs between the closely spaced micropillars owing to the capillary driving force.
3.5 OT manipulation for microparticle capture
SiO2 microspheres with a diameter of 5 μm were selected as the operating objects. Compared with the 5-μm-diameter PS microspheres, the 5-μm-diameter SiO2 microspheres settled at the bottom of the solution faster because of their larger mass, which was beneficial for the capture by the microcage. Through systematic analysis, the anisotropic triangular and butterfly microcage structures were designed in advance to fit the particle size. The bottom size of the triangular microcage was 8.5 μm, and both sides measured 4.5 μm, whereas the butterfly microcage had a relatively larger opening of 8.5 μm and smaller opening of 3 μm and both sides measured 3 μm. A continuous laser with a wavelength of 532 nm and a power of 200 mW was focused on the target particle surface, and one or more microspheres were transported to the specified microcage through the movement of the platform. The entire process was conducted in a water environment, and selective capture of the microsphere array could be completed within a few minutes.
3.6 Cell pretreatment
The NIH-3T3 was cultured in high glucose Dulbecco’s Modified Eagle’s Medium (DMEM) (HyClone, USA) supplemented with 1% penicillin/streptomycin (HyClone, USA) and 10% fetal bovine serum (HyClone, USA). The cells were incubated at 37 °C in a humidified atmosphere with 5% CO2. After treating the cells with trypsin (Invitrogen, USA) for 3 min, the excess solution was removed with a pipette. Then, the DMEM was removed with a pipette, and the cells were blown onto the substrate to obtain a cell suspension. Before the experiment, 2 mL of NIH-3T3 cell suspension was taken directly out of the culturing flask, and centrifuged (SC-3610, Anhui USTC Zonkia Scientific Instruments Co. Ltd., China) at 800 rpm for 5 min, and the supernatant was discarded. Then, a pipette was used to remove the residual culture medium. The resulting NIH-3T3 cells were resuspended in 1 mL of phosphate-buffered saline (PBS) (Invitrogen, USA) isotonic solution for further experiments.
3.7 Cell fluorescence staining
To further observe the effect of microcage structures on cells, 30 µL of propidium iodide and 20 µL of Calcein-AM were mixed in 12 mL of PBS. The cells on the microstructures were covered with dye solution and incubated for 20 min. The dyeing process was performed at room temperature (20–25 °C) in the dark. After washing the chip with PBS three times, the distribution of cells was visualized with an inverted fluorescence microscope.
3.8 OTs to manipulate cells
The remaining NIH-3T3 cells were washed twice with an isosmotic solution consisting of 50 mL of PBS and 0.5 g of bovine serum albumin (Sigma-Aldrich, USA) to reduce the affinity force between the cells and substrate, enhancing the operational performance of the laser-trapping cells. A continuous laser with a wavelength of 532 nm and an output power of 270 mW was focused on the cell surface for capture and operation, and the entire process was conducted in PBS.
3.9 Characterization and analysis
After depositing approximately 20 nm of Au on the chip, SEM images (ZEISS, EVO MA10, Germany) were obtained using a secondary scanning electron microscope at an accelerating voltage of 20 keV. The structure and size of the micropillar and the results before and after capture by the microcage were characterized. An eclipse Ti microscope (TIE, Nikon, Tokyo, Japan) was used to record the bright-field and fluorescence images of the microcage structure to obtain the comparison results before and after growing cells. The processing height and diameter of the micropillar were analyzed using ImageJ (image analysis software), and the data were plotted using Origin (data processing software).
4. Conclusion
In summary, a novel method is proposed to manipulate and capture single particles and cells selectively through the combination of capillarity-driven self-assembled microcages and OTs. Diverse ordered micropillar arrays with different parameters were designed and fabricated based on the femtosecond laser TPP with high flexibility, and stable and controllable capillary-force-driven self-assembly of microcages was achieved by optimizing the height and spacing between adjacent micropillars. Benefiting from optimal parameters, the triangular and butterfly microcages achieved the anisotropic capture of single microparticles with high capture efficiency. The number of captured particles can also be flexibly adjusted. The butterfly microcage was adopted to achieve the OT-assisted selective capture of a single cell, demonstrating the capability of this platform for the selective manipulation and targeted capture of single cells. The entire process can be completed within a few minutes, saving time. Because a single cell does not require continuous external force to remain captured by this platform and can effectively avoid the influence of external disturbances on the cell, subsequent in situ analysis could be conveniently performed, which shows extensive prospects in applications such as microfluidics, cell behavior analysis, and biomedical analysis.
Funding
National Natural Science Foundation of China (61727811, 61803366, 61821005, 61925307, 61973298); External Cooperation Program of the Chinese Academy of Sciences (173321KYSB20170015); CAS Interdisciplinary Innovation Team (JCTD-2019-09); Key Research Program of Frontier Science (QYZDB-SSW-JSC008); Liaoning Revitalization Talents Program (XLYC1807006); Youth Innovation Promotion Association of the Chinese Academy of Sciences (Y201943).
Disclosures
The authors declare no conflict of interest.
Data availability
No data were generated or analyzed in the presented research.
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