Congenital cardiovascular defects are the leading cause of birth defect related death. It has been hypothesized that fluid mechanical forces of embryonic blood flow affect cardiovascular development and play a role in congenital malformations. Studies in small animal embryos can improve our understanding of congenital malformations and can lead to better treatment. We have developed an ultrafast optical coherence tomography (OCT) technique for multidimensional imaging of the beating heart in a chicken embryo in vivo. Preliminary imaging experiments have been conduct to obtain high-resolution image sequences. Computational fluid dynamics (CFD) simulations were performed to provide quantitative analysis of the embryonic flow mechanics and the associated anatomy.

We proposed to use cellular spheroids of co-cultured lung cancer cells and fibroblasts as a platform to evaluate the efficacy of anti-cancer drug combinations. We labelled the cancer cells and fibroblast with dyes of different colors, and employed selective plane illumination microscopy (SPIM) [1, 2] to provide a three-dimensional (3D) perspective of relative positions and amounts of the co-cultured cells. Therefore we were able to evaluate the drug effects on individual types of cells in the 3D co-culture environment.

The size of the spheroids strongly influences the evaluation of the drug effects. In order to unify the spheroid size, we used a microfluidic culture device that contained cubic chambers for confining the cellular spheroids [3]. Figure 1 shows the scheme and photo of the device used in the present work. The side wall of these culture chambers was flat such that the illuminating light sheet could propagate through without distortion. In the present work, the spheroids were kept in cubic chambers with a side length of 250 µm.

We found that the co-culture of CL1-0 lung cancer cells and MRC-5 fibroblasts could form a spheroid (diameter ~ 200 µm) much easier than the cancer cell alone. The fibroblasts were enclosed by the cancer cells in a spheroid, regardless of the seeding sequences. In contrast, while the cancer cells were co-cultured with bronchial epithelial cells BEAS-2B, the latter did not invade into the cancer cell aggregation (Fig. 2). This result implied that fibroblasts could play an essential role in the early stage of tumor formation.

Next, we used the co-culture spheroids to test the efficacy of a common anti-cancer drug cisplatin (CDDP) in combination with chloroquine (CQ), an inhibitor of cellular autophagy. We first used the total intensity of the fluorescent dye-labelled cells as a parameter to judge the drug efficacy. The addition of CQ enhanced the CDDP efficacy at 0.2 and 2.0 µM. With the SPIM images, we further realized that the survival rate of cancer cells in the co-culture spheroids was reduced by the addition of CQ, in comparison with the cases with CDDP only. In other words, CQ might selectively enhance the injury to the cancer cells in the co-culture spheroids. This result could not be revealed with the conventional cell viability test on the whole spheroid. We will report the results of other drug combinations in the conference.

The co-culture spheroids consist of cancer cells and stromal cells combined with 3D SPIM imaging could serve as a useful platform to investigate the tumor formation process and to test the drug combination efficacy. For the evaluation of therapeutic strategies, the results from experiments in 3D microenvironments could be more relevant than those from 2D experiments.

REFERENCES:

1. Huisken et al., "Optical sectioning deep inside live embryos by selective plane illumination microscopy," Science 2004, 305, 1007-1009.
2. J. Verveer et al., "High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy," Nat. Methods 2007, 4, 311-313.
3. Patra et al., "Migration and vascular lumen formation of endothelial cells in cancer cell spheroids of various sizes," Biomicrofluidics 2014, 8, 052109.

Super-resolution focused light spots are always expected in scanning optical microscopy. There are bright focused light spots and dark focused spots in stimulated emission depletion (STED) microscopy. The latter dark spot is normally generated by focusing a linearly polarized beam with spiral phase. In this work, we generated a super-resolution optical tube by tightly focusing a binary phase modulated azimuthally polarized laser beam. The binary phase modulation is achieved by a glass substrate with multi-belt concentric ring grooves. We also characterized the 3D beam profile by using a cross-shaped knife-edge fabricated on a silicon photo-detector. The size of the super-resolution dark spot in the tube is 0.32λ, which remains unchanged for ~4λ within the tube. This optical tube may find applications in super-resolution microscopy, optical trapping and particle acceleration.

The three-dimensional (3D) structure of chromatin is important for gene regulation and transcription. It is very challenging to visualize different genomic loci in 3D space at large scale. Traditional Fluorescence in situ hybridization (FISH) can only label and visualize several (with the help of multi-color FISH) or very limited genomic loci or chromosomes (with the help of combination of different colors), because of the overlapping emission fluorescence signals. However, upconversion nanocrystals (UCNPs) convert infrared radiation to visible luminescence, thus are promising for bringing another dimension to visualize genomic loci in three-dimensional space. With the help of low-power super-resolution stimulated emission depletion (STED) microscopy, one can obtain sub-30nm resolution in images of the highly-doped UCNPs. Here we at first try to develop a novel method to label and visualize different genomic loci with UCNPs in fixed cell nuclei, then combine this method with traditional FISH, to visualize multiple genomic loci in genome. This strategy offers the new possibility to image 3D genome with high-throughput imaging.

In Lead Zirconium Titanate Pb[Zr_1-xTi_x]O₃ based piezoelectric transducers, silicon film has been used as a passive structural layer to enhance the mechanical stability and reliability of the system due to its excellent mechanical properties. Previously reported piezoelectric unimorph transducers consisting of silicon (single crystalline or polycrystalline) structures are formed with SOI wafers or deposited silicon film. To date, PZT thin film bimorph or multimorph configurations consists of PZT/ZnO/PZT [1] or PZT/PZT stacks with Pt as the elastic shim [2]. Brittleness of the PZT thin film poses limitations in its use as bimorph micro-actuators. Bimorph actuators with PZT thin films alone are insufficient to provide reasonable structural strength required to move loads such as micro-lens and micro-mirrors. With thicker PZT films to form a bimorph actuator, it is more susceptible to form large cracks and pores which poses reliability issue. Bimorph actuators consisting of just two active layers (PZT/PZT) without a passive structure are not practical for micro-lens or micro-mirror actuation. This kind of configuration are usually seen in energy harvesting devices instead. Formation of bimorph or multimorph arrangements with low stress silicon as the passive structural layer(s) in-between the piezoelectric active layers could potentially enhance the mechanical performance and reliability of such transducers. However, typical LPCVD and Epi-poly depositions require processing temperatures that are not suitable for PZT films to sustain its quality. Therefore, bimorph arrangements consisting of PZT/Poly-Si/PZT layers have never been reported before. For the first time, a bimorph thin film PZT micro-cantilever actuator with low thermal budget Ultra-High Vacuum E-beam Evaporated Polysilicon (UHVEEPoly) as a passive structural layer is fabricated.

The micro-actuator is 1000 µm long and 250 µm wide. Fig. 1 shows a simplified fabrication process flow for the bimorph actuator. The actuator consists of Ti(15nm)/Pt(100nm)/PZT(1.2µm)/Ti(15nm)/Pt(100nm)/Ti(15nm)/SiO₂(0.75µm)/Poly-Si(4µm)/SiO₂(0.75µm)/Ti(15nm)/Pt(100nm)/Ti(15nm)/PZT(1.2µm)/Ti(15nm)/Pt(100nm) layers to form approximately 8.3μm thick structure. The released bimorph structure is shown in Fig. 2a. It can be seen that the residual stress is well balanced on the top and bottom actuator to achieve a near flat curvature as oppose to an unimorph structure reported from our previous work [3]. From a cross-section view of a focused ion beam (FIB) milling in Fig. 2b, it can also be verified that the top and bottom active layers have equivalent thickness which result in the small initial deflection. The bimorph actuator has a measured resonance frequency of 6.3 kHz by exciting the top or the bottom actuator with a chrip signal (Fig. 4a and b). The bi-directional movement of the actuator can be confirmed by 180º phase difference between the top actuation and bottom actuation phase response plots (Fig. 4c and d), demonstrating an unique suitability for micro-actuation mechanisms or micro energy harvesters where a passive structural layer is required to enhance the mechanical performance and reliability of the system.

Progress Claims

This article presents a novel method to bond PLA, a 3D-printing material, and PMMA, a popular substrate material for microfluidic applications. With this technique, the tubing connectors can be fabricated by 3D printing and make this PLA/PMMA hybrid microfluidic chip extremely easy to use for experiments. The major challenge of bonding this hybrid chip is the high level surface roughness of PLA substrates with its significant influences on bonding strength [1]. After Ethanol treatment and UV irradiation of this hybrid chip, a post-annealing step was realized to facilitate the bonding. To further analyze the bonding quality leakage test, cross-sectional image by microscope, and pressure bursting test were conducted. The experiment results clearly showed that this method could successfully and rapidly form a strong bond (＞13 bars) between PLA and PMMA substrates.

Background

Nowadays, thermoplastics are used commonly in microfluidic applications. The bonding of PMMA/PLA can offer significant benefits taken advantages from 3D-printing process such as allowing producing incredibly complex products in a short time, while minimizing material waste.

Description of Bonding Procedure

Ethanol solution was distributed uniformly sandwiched between two substrates by spin-coating process (190 rpm, 10 sec). Following the UV irradiation (56 sec), an instantaneous and permanent bonding can be formed between PMMA and PLA. However, the bonding strength is significantly affected by the high level of surface roughness of PLA substrates (range from 4.5 to 6.5μm). It can cause the failure of bonding. To solve this critical issue, we employed a post-annealing strategy (55℃, 30 min) straightforwardly after UV exposure step. Its purpose is help create more contacting points between two bonded substrates because of surface degradation caused by coarsening phenomena [3]. Besides post annealing can help relieve stress and therefore improve the bonding strength. The temperature executed for post-annealing is below the glass transition temperature of PLA (Tg of PLA = 60~65℃), hence there has no significant channel deformation observed. The overall bonding procedure is described in Fig. 1.

After bonding, several experiments were conducted to characterize the bonding quality such as leakage tests, cross-sectional images by microscope, and burst tests.

Experimental Results

1. Comprehensive examination

Following the completion of the bonding experiments, leakage tests and the cross sectional investigation using microscope of bonded chip were conducted, the results of which are presented in Fig. 2. The figure clearly demonstrates the effectiveness of the proposed bonding method for heterogeneous substrates between PMMA/PLA. Figure 2(a) shows the bonded chip, Figure 2(b) shows the enlarged figure of the microchannel, both figures clearly showed that no leakage was observed. Figure 2(c) shows the cross-sectional image of bonded chip.

1. The influence of post-annealing conditions on bonding strength.

Fig.3 illustrates the set up for busting test and Table 1 lists the experiment results of bonding strength. In all of the experiments, the microchannels were fabricated on the PMMA substrates and the cover substrates were PLA. Table 1 clearly shows that the microfluidic chips have sufficient bonding strength above 10 bars.

Advances in the microfabrication of microfluidic and biochip devices have made polydimethylsiloxane (PDMS) the material of choice for biomedical, analytical and biotechnological applications.^1-3 And a variety of bonding technologies have been developed to seal the micro-channels on PDMS. But, most technologies aim for small and single chip functions. It is still a challenge to investigate new technologies for large-size and high-throughput PDMS microfluidic chip fabrication.

This paper reports a new bonding method for large size PDMS-based microfluidic chip fabrication, which is based on display technology. The fabrication process combines a process of first polaroid bonding commonly used in liquid crystal display (LCD) manufacturing production and then thermal bonding. Polaroid bonding provides specific advantages to the large size microchip including good alignment and bubble free. Thermal bonding is applied to improve the bonding strength between PDMS and glass.

 The microchannel design of the PDMS microfluidic chip is shown in Fig 1a. And Fig.1b is the photo of a large sized PDMS chip obtained by the new bonding method. Fig. 2a and 2b respectively shows the polaroid bonding machine used in this study and the schematic illustration of the machine’s working principle. The bonding strength of the PDMS chip obtained by different method is shown in Fig. 3. Leakage tests are another method to assess the adhesion strength of the large size PDMS-glass microchip. Fig. 4 exhibits the results of the leak tests.The maximum pressure between PDMS and the glass substrate is 739 Kpa, which is comparable to interference-assisted thermal bonding method.⁴ The new bonding method can also be used for 6×6 inch microchip bonding procedure. This provides an efficient method for manufacturing large size and high throughput PDMS microfluidic chip.

Paper-based microfluidics as a promising and powerful platform shows great potential in the past decades^1-4. As a crucial and essential function component to control fluid transport, the valve plays an important role in fluid manipulation, and enhancing the fluidic capability on μPADs. This paper describes a novel strategy to fabricate the movable valve on the paper-based microfluidic devices to manipulate capillary-driven fluids. The movable valve fabrication is firstly realized using hollow rivets as the holding center to control the paper channel in different layer movement that results in the channel's connection or disconnection. The relatively simple valve fabrication procedure is robust, versatile and compatible to the different levels of complexity μPADs. It is remarkable that the movable valve can be convenient, and free to control fluid without the timing setting that advantages make it user-friendly to the untrained users to carry out the complex multi-step operations. To verify the performance of the movable valve, several different designs of μPADs were tested and obtained with satisfactory results. In addition, in the proof-of-concept enzyme-linked immunosorbent assay (ELISA) experiments, we demonstrate the use of these valves in μPADs for the successful analysis of the samples of carcino-embryonic antigen (CEA) that showed good sensitivity and reproducibility. We hope this technique will open new avenues for the fabrication of paper-based valve in an easily adoptable and widely available way on μPADs and provide potential point-of-care applications in the future.

Superhydrophobic surface has been widely investigated by researchers in these years for its self-clean characteristics which can be used in various fields such as transport, medical treatment, energy and environment protection [1]. Although there are several methods to obtain the superhydrophobic surface, including etching, electrospinning, sol-gel method and template transfer etc, the fabrication process requires either expensive materials or equipment, complex manipulations or rigorous processing requirement [2][3].As a result, a large-area superhydrophobic surface could not be simply fabricated by these process. On the other hand, roll-to-roll ultraviolet nano-imprint (RtR UV NIL)  technology has been developed over the past years as a promising candidate for the large area fabrication, especially for the grating structures and solar cell films [4].In this paper, we proposed an equipment for fabrication of large area superhydrophobic materials based on RtR UV NIL and fast UV curing technology. Micro patterns could be transferred to a flexible substrate without complicated fabrication process in a clean room, and large area superhydrophobic film can be fabricated rapidly and efficiently.

The proposed RtR UV NIL equipment is composed of six parts: tension control part, film transmitting part, photoresist coating part, structure transferring part, curing part and surface treatment part. The RtR UV NIL equipment and its 3D structure modelare showed in Figure 1 and Figure 2, respectively. The whole fabrication process will be presented as following.

Firstly, the PDMS mold was obtained against the Si master fabricated via UV lithography (EVG 610, Austria) and deep reactive ion etching (DRIE) [5]. Then the UV resist (four types used are shown in Table 1) was coated on the polyethylene terephtalate (PET) substrate. After that, the microstructures on the PDMS mold were transferred to the UV resist using the RtR UV NIL equipment. After the UV exposure, the resist was cured and the film with the same micropillar pattern was obtained after demolding. At last, the fluoride treatment of the film was carried out  with fumigation of trimethylsilyl chloride, decreasing the surface energy. Also, it was found that it is possible to manufacture superhydrophobic microstructure arrays free of bubble defects using R2R UV imprinting technique through selecting processing parameters within the process window: the web speed between 0.5 and 0.7m/min, the pressure between 4 and 5kg/cm², the UV light lamp has a power of 7kW, its lamp watts is 260W/cm, and its wavelength range is 250–450nm, and the mold temperature between 57 and 65° C.

After process and recipe optimization, a typical micro structure array (20 μm-diameter, 40 μm-pitch,17 μm-height) was successfully transferred by using a customized UV resist (80% UA-232P, Shin-nakamura Chemical), as shown in Figure 3. The de-ionized water contact angle was measured up to 150° after fluorinated treatment as shown in Figure 4. For the future study, we will try more complicated T-type microstructure array using this equipment aiming at large-area superlyophobic film.

Microflow visualization (μ-FV) has been performed to study the silver nanoparticle droplet ejected from a drop-on-demand piezoelectric inkjet printhead and the equilibrium line characteristic of the nano-silver droplet deposition on PI substrates. The unipolar waveform with a frequency of 1000 Hz and an amplitude of 60 V has been adopted for ejecting the silver nanoparticle droplet with a solid content of 30%, surface tension of 30 mN/m and viscosity of 15 cps. The back pressure is modulated to prevent the formation of satellite droplets experimentally. The PI substrate was placed onto a computer-controlled three-axis moving stage capable of a movement accuracy of 30 μm. Therefore multiple prints of the nano-silver conductive lines have also been carried out based on the moving accuracy. The deposited silver nanoparticle conductive lines with the inter-dot spacings from 25 μm to 45 μm with single print to prints quadruple prints have also been investigated. Besides, the O₂ plasma treatment has been applied on the PI substrates with two durations. After the thermal treatment (sinter temperature of 200°C and sinter duration of 1 hour), the optical microscopic images of the deposited silver nanoparticle conductive lines before and after O₂ plasma treatment have been obtained. For the first time, the quadruple prints of the silver nanoparticle conductive lines on the PI films have been investigated to observe the line widths and electrical resistances. μ-FV has been successfully carried out to study the silver nanoparticle droplets ejected from a piezoelectric ink-jet printhead and the equilibrium film characteristic on PI substratse. The nano-silver conductive lines with quadruple prints on the PI substrate have a line width of 800 μm and a resistance of 1.4 Ω/cm which is also measured on the PI substrate with 2 min O₂ plasma treatment and single print (line width = 680 μm).