Standing Surface Acoustic Wave (SSAW)‐Based Fluorescence‐Activated Cell Sorter

Microfluidic fluorescence‐activated cell sorters (μFACS) have attracted considerable interest because of their ability to identify and separate cells in inexpensive and biosafe ways. Here a high‐performance μFACS is presented by integrating a standing surface acoustic wave (SSAW)‐based, 3D cell‐focusing unit, an in‐plane fluorescent detection unit, and an SSAW‐based cell‐deflection unit on a single chip. Without using sheath flow or precise flow rate control, the SSAW‐based cell‐focusing technique can focus cells into a single file at a designated position. The tight focusing of cells enables an in‐plane‐integrated optical detection system to accurately distinguish individual cells of interest. In the acoustic‐based cell‐deflection unit, a focused interdigital transducer design is utilized to deflect cells from the focused stream within a minimized area, resulting in a high‐throughput sorting ability. Each unit is experimentally characterized, respectively, and the integrated SSAW‐based FACS is used to sort mammalian cells (HeLa) at different throughputs. A sorting purity of greater than 90% is achieved at a throughput of 2500 events s^?1. The SSAW‐based FACS is efficient, fast, biosafe, biocompatible and has a small footprint, making it a competitive alternative to more expensive, bulkier traditional FACS.     

CD34~+造血祖细胞及其亚群（CD34~+CD38~+和CD34~+CD38~-

分离纯化造血干祖细胞具有十分重要的理论和应用价值。本文利用吸附免疫微球的单克隆抗体分离系统，对不同来源造血组织的ＣＤ３４＋细胞进行纯化分离，经流式细胞仪检测，其纯度可达９５—９９％。在外源性生长因子的刺激下，ＣＤ３４＋细胞可形成大量各系造血集落，而ＣＤ３４－组分则几乎不含造血集落形成细胞。进一步的研究则是利用免疫荧光激活的流式细胞分选系统，将ＣＤ３４＋细胞群分为ＣＤ３４＋ＣＤ３８＋和ＣＤ３４＋ＣＤ３８－两个亚群，并比较正常人骨髓、脐带血、外周血来源的亚群细胞造血性能。结果表明，不同细胞亚群造血性能不均一，同一亚群不同来源的细胞也同样具有不均一性，从而为造血干细胞的基因治疗、建库、移植等提供了理论依据。     

Microfluidic sorting with a moving array of optical traps

Optical sorting was demonstrated by selective trapping of a set of microspheres (having specific size or composition) from a flowing mixture and guiding these in the desired direction by a moving array of optical traps. The approach exploits the fact that whereas the fluid drag force varies linearly with particle size, the optical gradient force has a more complex dependence on the particle size and also on its optical properties. Therefore, the ratio of these two forces is unique for different types of flowing particles. Selective trapping of a particular type of particles can thus be achieved by ensuring that the ratio between fluid drag and optical gradient force on these is below unity whereas for others it exceeds unity. Thereafter, the trapped particles can be sorted using a motion of the trapping sites towards the output. Because in this method the trapping force seen by the selected fraction of particles can be suitably higher than the fluid drag force, the particles can be captured and sorted from a fast fluid flow (about 150 μm/s). Therefore, even when using a dilute particle suspension, where the colloidal trafficking issues are naturally minimized, due to high flow rate a good throughput (about 30 particles/s) can be obtained. Experiments were performed to demonstrate sorting between silica spheres of different sizes (2, 3, and 5 μm) and between 3 μm size silica and polystyrene spheres.     

A Trachea‐Inspired Bifurcated Microfilter Capturing Viable Circulating Tumor Cells via Altered Biophysical Properties as Measured by Atomic Force Microscopy

Circulating tumor cells (CTCs), though exceedingly rare in the blood, are nonetheless becoming increasingly important in cancer diagnostics. Despite this keen interest and the growing number of potential clinical applications, there has been limited success in developing a CTC isolation platform that simultaneously optimizes recovery rates, purity, and cell compatibility. Herein, a novel tracheal carina‐inspired bifurcated (TRAB) microfilter system is reported, which uses an optimal filter gap size satisfying both 100% theoretical recovery rate and purity, as determined by biomechanical analysis and fluid–structure interaction (FSI) simulations. Biomechanical properties are also used to clearly discriminate between cancer cells and leukocytes, whereby cancer cells are selectively bound to melamine microbeads, which increase the size and stiffness of these cells. Nanoindentation experiments are conducted to measure the stiffness of leukocytes as compared to the microbead‐conjugated cancer cells, with these parameters then being used in FSI analyses to optimize the filter gap size. The simulation results show that given a flow rate of 100 μL min^?1, an 8 μm filter gap optimizes the recovery rate and purity. MCF‐7 breast cancer cells with solid microbeads are spiked into 3 mL of whole blood and, by using this flow rate along with the optimized microfilter dimensions, the cell mixture passes through the TRAB filter, which achieves a recovery rate of 93% and purity of 59%. Regarding cell compatibility, it is verified that the isolation procedure does not adversely affect cell viability, thus also confirming that the re‐collected cancer cells can be cultured for up to 8 days. This work demonstrates a CTC isolation technology platform that optimizes high recovery rates and cell purity while also providing a framework for functional cell studies, potentially enabling even more sensitive and specific cancer diagnostics.     

Multitarget dielectrophoresis activated cell sorter

The ability to rapidly and efficiently isolate specific viruses, bacteria, or mammalian cells from complex mixtures lies at the heart of biomedical applications ranging from in vitro diagnostics to cell transplantation therapies. Unfortunately, many current selection methods for cell separation, such as magnetic activated cell sorting (MACS), only allow the binary separation of target cells that have been labeled via a single parameter (e.g., magnetization). This limitation makes it challenging to simultaneously enrich multiple, distinct target cell types from a multicomponent sample. We describe here a novel approach to specifically label multiple cell types with unique synthetic dielectrophoretic tags that modulate the complex permittivities of the labeled cells, allowing them to be sorted with high purity using the multitarget dielectrophoresis activated cell sorter (MT-DACS) chip. Here we describe the underlying physics and design of the MT-DACS microfluidic device and demonstrate approximately 1000-fold enrichment of multiple bacterial target cell types in a single-pass separation.     

Intuitive, image-based cell sorting using optofluidic cell sorting

We present a microfluidic cell-sorting device which augments microscopy with the capability to perform facile image-based cell sorting. This combination enables intuitive, complex phenotype sorting based on spatio-temporal fluorescence or cell morphology. The microfluidic device contains a microwell array that can be passively loaded with mammalian cells via sedimentation and can be subsequently inspected with microscopy. After inspection, we use the scattering force from a focused infrared laser to levitate cells of interest from their wells into a flow field for collection. First, we demonstrate image-based sorting predicated on whole-cell fluorescence, which could enable sorting based on temporal whole-cell fluorescence behavior. Second, we demonstrate image-based sorting predicated on fluorescence localization (nuclear vs whole-cell fluorescence), highlighting the capability of our approach to sort based on imaged subcellular events, such as localized protein expression or translocation events. We achieve postsort purities up to 89% and up to 155-fold enrichment of target cells. Optical manipulation literature and a direct cell viability assay suggest that cells remain viable after using our technique. The architecture is highly scalable and supports over 10 000 individually addressable trap sites. Our approach enables sorting of significant populations based on subcellular spatio-temporal information, which is difficult or impossible with existing widespread sorting technologies.     

Continuous sorting of microparticles using dielectrophoresis

Sorting of particles such as cells is a critical process for many biomedical applications, and it is challenging to integrate it into an analytical microdevice. We report an effective and flexible dielectrophoresis (DEP)-based microfluidic device for continuous sorting of multiple particles in a microchannel. The particle sorter is composed of two components-a DEP focusing unit and a Movable DEP Trap (MDT). The trap is formed by an array of microelectrodes at the bottom of the channel and a transparent electrode plate placed at the top. The location of the trap is dependent on the configuration of voltages on the array and therefore is addressable. Flowing particles are first directed and focused into a single particle stream by the focusing unit. The streamed particles are then sorted into different fractions using the movable trap by rapidly switching the applied voltage. The performance of the sorter is demonstrated by successfully sorting microparticles in a continuous flow. The proposed DEP-based microfluidic sorter can be implemented in applications such as sample preparation and cell sorting for subsequent analytical processing, where sorting of particles is needed.     

Optical breakdown in fused silica and argon gas: application to Nd:YAG laser limiter

A gas cell filed with argon gas under pressure is placed in a tightly focused laser beam to provide a limiter for laser pulses above a certain peak power, corresponding to the optical breakdown threshold for the creation of a laser-induced plasma. Measurements of the threshold intensity as a function of argon gas pressure are given for a laser wavelength of 1.064 microm (Nd:YAG) and a pulse length of 6.4 ns. Threshold intensities for optical breakdown in fused silica were measured with the same optical system, enabling a relative comparison of breakdown thresholds, of interest for protecting fused-silica optical components in fiber-optic delivery systems for laser material processing applications. The threshold intensity was measured to 220 GW/cm2 in Ar at 1.0 x 10(5) N/m2 (1 atm), 80 GW/cm2 in Ar at 8.0 x 10(5) N/m2 (7.9 atm), and 55 GW/cm2 in fused silica. Even though the threshold in argon is higher than that in fused silica, the limiter will protect the optical components if the laser beam is focused to a tighter spot in the gas cell than at the input end of the fiber.     

Aptamer-enabled efficient isolation of cancer cells from whole blood using a microfluidic device

Circulating tumor cells (CTC) in the peripheral blood could provide important information for diagnosis of cancer metastasis and monitoring treatment progress. However, CTC are extremely rare in the bloodstream, making their detection and characterization technically challenging. We report here the development of an aptamer-mediated, micropillar-based microfluidic device that is able to efficiently isolate tumor cells from unprocessed whole blood. High-affinity aptamers were used as an alternative to antibodies for cancer cell isolation. The microscope-slide-sized device consists of >59,000 micropillars, which enhanced the probability of the interactions between aptamers and target cancer cells. The device geometry and the flow rate were investigated and optimized by studying their effects on the isolation of target leukemia cells from a cell mixture. The device yielded a capture efficiency of ~95% with purity of ~81% at the optimum flow rate of 600 nL/s. Further, we exploited the device for isolating colorectal tumor cells from unprocessed whole blood; as few as 10 tumor cells were captured from 1 mL of whole blood. We also addressed the question of low throughput of a typical microfluidic device by processing 1 mL of blood within 28 min. In addition, we found that ~93% of the captured cells were viable, making them suitable for subsequent molecular and cellular studies.     

Vibration Phase‐Based Ordering of Vibration Patterns Acquired With a Shearing Speckle Interferometer and Pulsed Illumination

Abstract: A method for both temporal and spatial characterisation of harmonic vibrations is presented. The method is based on simultaneous acquisition of phase‐stepped speckle interference patterns using a shearing speckle interferometer and the vibration phase for a series of vibration states within the vibration period. An unsynchronised free‐running pulse laser is used for illuminating a vibrating object yielding speckle interference patterns in random vibration phase order. Two π/2 phase‐stepped speckle interference patterns are acquired simultaneously for each recorded vibration state. The data set is sorted using vibration phase as the sorting key. The sorted speckle interference patterns are processed using a two‐bucket algorithm for the calculation of phase difference and by applying temporal phase unwrapping to finally obtain unwrapped phase distributions for any vibration state of the vibration cycle.     

Microfluidic, label-free enrichment of prostate cancer cells in blood based on acoustophoresis

Circulating tumor cells (CTC) are shed in peripheral blood at advanced metastatic stages of solid cancers. Surface-marker-based detection of CTC predicts recurrence and survival in colorectal, breast, and prostate cancer. However, scarcity and variation in size, morphology, expression profile, and antigen exposure impairs reliable detection and characterization of CTC. We have developed a noncontact, label-free microfluidic acoustophoresis method to separate prostate cancer cells from white blood cells (WBC) through forces generated by ultrasonic resonances in microfluidic channels. Implementation of cell prealignment in a temperature-stabilized (±0.5 °C) acoustophoresis microchannel dramatically enhanced the discriminatory capacity and enabled the separation of 5 μm microspheres from 7 μm microspheres with 99% purity. Next, we determined the feasibility of employing label-free microfluidic acoustophoresis to discriminate and divert tumor cells from WBCs using erythrocyte-lysed blood from healthy volunteers spiked with tumor cells from three prostate cancer cell-lines (DU145, PC3, LNCaP). For cells fixed with paraformaldehyde, cancer cell recovery ranged from 93.6% to 97.9% with purity ranging from 97.4% to 98.4%. There was no detectable loss of cell viability or cell proliferation subsequent to the exposure of viable tumor cells to acoustophoresis. For nonfixed, viable cells, tumor cell recovery ranged from 72.5% to 93.9% with purity ranging from 79.6% to 99.7%. These data contribute proof-in-principle that label-free microfluidic acoustophoresis can be used to enrich both viable and fixed cancer cells from WBCs with very high recovery and purity.     

Subwavelength focusing using a hyperbolic medium with a single slit

A hyperbolic dispersion medium with a planar surface that can be used for subwavelength focusing is proposed. By combining the hyperbolic medium in a single slit with diffraction limit width, a laser beam could be focused to a subwavelength spot in the near field. Compared to a conventional superlens, the subdiffraction focusing in this work has higher optical throughput. Using a planar hyperbolic medium, which is actually alternating silver/dielectric multilayers, we showed that the focusing resolution of the designed device is down to ~λ/5 using green light illumination (at a wavelength of 514.5 nm).     

Engineering Supramolecular Polymer Conformation for Efficient Carbon Nanotube Sorting

Supramolecular polymer sorting is a promising approach to separating single‐walled carbon nanotubes (CNTs) by electronic type. Unlike conjugated polymers, they can be easily removed from the CNTs after sorting by breaking the supramolecular bonds, allowing for isolation of electronically pristine CNTs as well as facile recycling of the sorting polymer. However, little is understood about how supramolecular polymer properties affect CNT sorting. Herein, chain stoppers are used to engineer the conformation of a supramolecular sorting polymer, thereby elucidating the relationship between sorting efficacy and polymer conformation. Through NMR and UV–vis spectroscopy, small‐angle X‐ray scattering (SAXS), and thermodynamic modeling, it is shown that this supramolecular polymer exhibits ring–chain equilibrium, and that this equilibrium can be skewed toward chains by the addition of chain stoppers. Furthermore, by controlling the stopper–monomer ratio, the sorting yield can be doubled from 7% to 14% without compromising the semiconducting purity (>99%) or properties of sorted CNTs.     

An integrated microfabricated cell sorter

We have developed an integrated microfabricated cell sorter using multilayer soft lithography. This integrated cell sorter is incorporated with various microfluidic functionalities, including peristaltic pumps, dampers, switch valves, and input and output wells, to perform cell sorting in a coordinated and automated fashion. The active volume of an actuated valve on this integrated cell sorter can be as small as 1 pL, and the volume of optical interrogation is approximately 100 fL. Different algorithms of cell manipulation, including cell trapping, were implemented in these devices. We have also demonstrated sorting and recovery of Escherichia coli cells on the chip.     

Optical forces for noninvasive cellular analysis

A novel, noninvasive measurement technique for quantitative cellular analysis is presented that utilizes the forces generated by an optical beam to evaluate the physical properties of live cells in suspension. In this analysis, a focused, near-infrared laser line with a high cross-sectional intensity gradient is rapidly scanned across a field of cells, and the interaction of those cells with the beam is monitored. The response of each cell to the laser depends on its size, structure, morphology, composition, and surface membrane properties; therefore, with this technique, cell populations of different type, treatment, or biological state can be compared. To demonstrate the utility of this cell analysis platform, we evaluated the early stages of apoptosis induced in the U937 cancer cell line by the drug camptothecin and compared the results with established reference assays. Measurements on our platform show detection of cellular changes earlier than either of the fluorescence-based Annexin V or caspase assays. Because no labeling or additional cell processing is required and because accurate assays can be performed with a small number of cells, this measurement technique may find suitable applications in cell research, medical diagnostics, and drug discovery.     

Optical and acoustic detection of laser-generated microbubbles in single cells

Acoustically monitored laser-induced optical breakdown (LIOB) has potential as an important tool to diagnose and treat living cells. Laser-induced intracellular microbubbles are readily detectable using high-frequency ultrasound, and LIOB can be controlled to operate within two distinct regimes. In the nondestructive regime, a single, short-lived bubble can be generated within a cell, without affecting its immediate viability. In the destructive regime, the induced photodisruption quickly can kill a targeted cell. To generate and monitor this range of bioeffects in real time, we have developed a system integrating an ultrafast laser source with optical and acoustic microscopy. Experiments were performed on monolayers of Chinese hamster ovary (CHO) cells. A 793 nm, 100 fs laser pulsed at 3.8 kHz was tightly focused within each cell to produce the photodisruption, and a 50 MHz ultrasonic transducer monitored the resultant bubble via continuous pulse-echo recordings. Photodisruption was also observed using bright field microscopy, and cell viability was assessed following laser exposure with a trypan blue assay. By controlling laser pulse fluence and exposure duration, either nondestructive or destructive LIOB could be produced. The intracellular position of the laser focus was also varied to demonstrate that cell viability was affected by the specific location of material breakdown.     

Fluorescent nanoswitch for monitoring specific pluripotency-related microRNAs of induced pluripotent stem cells: Development of polyethyleneimine-oligonucleotide hybridization probes

The isolation of high-grade (i.e.high-pluripotency) human induced pluripotent stem cells (hiPSCs) is a decisive factor for enhancing the purity of hiPSC populations or differentiation efficiency.A non-invasive imaging system that can monitor microRNA (miRNA) expression provides a useful tool to identify and analyze specific cell populations.However,previous studies on the monitoring/isolation of hiPSCs by miRNA expression have limited hiPSCs' differentiation system owing to long-term incubation with miRNA imaging probe-nanocarriers.Therefore,we focused on monitoring high-grade hiPSCs without influencing the pluripotency of hiPSCs.We reduced nanoparticle transfection time,because hiPSCs are prone to spontaneous differentiation under external factors during incubation.The fluorescent nanoswitch ("ON" with target miRNA),which can be applied for either imaging or sorting specific cells by fluorescence signals,contains an miRNA imaging probe (miP) and a PEI-PEG nanoparticle (miP-P).Consequently,this nanoswitch can sense various endogenous target miRNAs within 30 min in vitro,and demonstrates strong potential for not only imaging but also sorting pluripotent hiPSCs without affecting pluripotency.Moreover,miP-P-treated hiPSCs differentiate well into endothelial cells,indicating that miP-P does not alter the pluripotency of hiPSCs.We envisage that this miRNA imaging system could be valuable for identifying and sorting high-grade hiPSCs for improved practical applications.     

Negative enrichment of target cells by microfluidic affinity chromatography

A three-dimensional microfluidic channel was developed for high-purity cell separations. This system featured high capture affinity using multiple vertical inlets to an affinity surface. In cell separations, positive selection (capture of the target cell) is usually employed. Negative enrichment, the capture of nontarget cells and elution of target cells, has distinct advantages over positive selection. In negative enrichment, target cells are not labeled and are not subjected to strenuous elution conditions or dilution. As a result, negative enrichment systems are amenable to multistep processes in microfluidic systems. In previous work (Li, P.; Tian, Y.; Pappas, D. Anal. Chem.2011, 83, 774-781), we reported cell capture enhancement effects at vertical inlets to the affinity surface. In this study, we designed a chip that has multiple vertical and horizontal channels, forming a three-dimensional separation system. Enrichment of target cells showed separation purities of 92-96%, compared with straight-channel systems (77% purity). A parallelized chip was also developed for increased sample throughput. A two-channel system showed similar separation purity with twice the sample flow rate. This microfluidic system, featuring high separation purity and ease of fabrication and use is suitable for cell separations when subsequent analysis of target cells is required.     

Demonstration for an Optoelectronic Switching Network

Abstract

The concept for building a multistage switching network with optoelectronic components for several hundred parallel channels is investigated. The key components for an experiment are: laser arrays, microlens arrays, holographic optical permutation elements and a monolithic optoelectronic application specific integrated circuit with integrated photodetectors and electronic logic for switching. The demonstration presented in this contribution is a non-blocking and selfrouting shuffle exchange network implementing Batcher's sorting algorithm. It consists of three cascaded stages each having four parallel channels running at a clock rate of 1 MBit s^?1. Scaling towards larger number of channels and higher overall data throughput is considered.     

Intensity, polarization, and phase information in optical disk systems

Digital information in optical data storage systems can be encoded in the intensity, in the polarization state, or in the phase of a carrier laser beam. Intensity modulation is achieved at the surface of the storage medium either through destructive interference from surface-relief features (e.g., CD or DVD pits) or through reflectivity variations (e.g., alteration of optical constants of phase-change media). Magneto-optical materials make use of the polar magneto-optical Kerr effect to produce polarization modulations of the focused beam reflected from the storage medium. Both surface-relief structures and material-property variations can create, at the exit pupil of the objective lens of the optical pickup, a phase modulation (this, in addition to any intensity or polarization modulation or both). Current optical data storage systems do not make use of this phase information, whose recovery could potentially increase the strength of the readout signal. We show how all three mechanisms can be exploited in a scanning optical microscope to reconstruct the recorded (or embedded) data patterns on various types of optical disk.