A parallel microfluidic flow cytometer for high-content screening.
ABSTRACT: A parallel microfluidic cytometer (PMC) uses a high-speed scanning photomultiplier-based detector to combine low-pixel-count, one-dimensional imaging with flow cytometry. The 384 parallel flow channels of the PMC decouple count rate from signal-to-noise ratio. Using six-pixel one-dimensional images, we investigated protein localization in a yeast model for human protein misfolding diseases and demonstrated the feasibility of a nuclear-translocation assay in Chinese hamster ovary (CHO) cells expressing an NF?B-EGFP reporter.
Project description:The effects of global warming, pollution in river effluents, and changing ocean currents can be studied by characterizing variations in phytoplankton populations. We demonstrate the design and fabrication of a Microflow Cytometer for characterization of phytoplankton. Guided by chevron-shaped grooves on the top and bottom of a microfluidic channel, two symmetric sheath streams wrap around a central sample stream and hydrodynamically focus it in the center of the channel. The lasers are carefully chosen to provide excitation light close to the maximum absorbance wavelengths for the intrinsic fluorophores chlorophyll and phycoerythrin, and the excitation light is coupled to the flow cytometer through the use of an optical fiber. Fluorescence and light scatter are collected using two multimode optical fibers placed at 90-degree angles with respect to the excitation fiber. Light emerging from these collection fibers is directed through optical bandpass filters into photomultiplier tubes. The cytometer measured the optical and side scatter properties of Karenia b., Synechococcus sp., Pseudo-Nitzchia, and Alexandrium. The effect of the sheath-to-sample flow-rate ratio on the light scatter and fluorescence of these marine microorganisms was investigated. Reducing the sample flow rate from 200 μL/min to 10 μL/min produced a more tightly focused sample stream and less heterogeneous signals.
Project description:We describe a fluorescent detection technique for a lab-on-a-chip flow cytometer. Fluorescent emission is encoded into a time-dependent signal as a fluorescent cell or bead traverses a waveguide array with integrated spatial filters and color filters. Different from conventional colored filters with well-defined transmission spectral window, the integrated color filters are designed to have broad transmission characteristics, similar to the red-green-blue photoreceptors in the retina of human eye. This unique design allows us to detect multiple fluorescent colors with only three color filters based on the technique of color-space-time coding using only one single photomultiplier tube or avalanche photodetector.
Project description:This work develops a microflow cytometer, based on a microfluidic chip for three-dimensional (3D) hydrodynamic focusing and a binary optical element (BOE) for shaping and homogenizing a laser beam. The microfluidic chip utilizes sheath flows to confine the sample flow along the channel centerline with a narrow cross section. In addition to hydrodynamic focusing, secondary flows are generated to strengthen the focusing in the vertical direction. In experiments, the chip was able to focus the sample flow with cross sections of 15 ?m high and 8-30 ?m wide at 5 m/s, under the condition of the sample flow rates between 10 and 120 ?L/min. Instead of using the conventional elliptical Gaussian spot for optical detection, we used a specially designed BOE and obtained a 50 ?m × 10 ?m rectangular quasi-flat-top spot. The microflow cytometer combining the chip and the BOE was tested to count 3, 5, and 7 ?m fluorescence microbeads, and the experimental results were comparable to or better than those derived from two commercial instruments.
Project description:Three-dimensional, fluorescence imaging methods with ~1?MHz frame rates are needed for high-speed, blur-free flow cytometry and capturing volumetric neuronal activity. The frame rates of current imaging methods are limited to kHz by the photon budget, slow camera readout, and/or slow laser beam scanners. Here, we present line excitation array detection (LEAD) fluorescence microscopy, a high-speed imaging method capable of providing 0.8 million frames per second. The method performs 0.8?MHz line-scanning of an excitation laser beam using a chirped signal-driven longitudinal acousto-optic deflector to create a virtual light-sheet, and images the field-of-view with a linear photomultiplier tube array to generate a 66?×?14 pixel frame each scan cycle. We implement LEAD microscopy as a blur-free flow cytometer for Caenorhabditis elegans moving at 1?m?s-1 with 3.5-µm resolution and signal-to-background ratios >200. Signal-to-noise measurements indicate future LEAD fluorescence microscopes can reach higher resolutions and pixels per frame without compromising frame rates.
Project description:A microfabricated flow cytometer was used to demonstrate multiplexed detection of bacteria and toxins using fluorescent coded microspheres. Antibody-coated microspheres bound biothreat targets in a sandwich immunoassay format. The microfluidic cytometer focused the microspheres in three dimensions within the laser interrogation region using passive groove structures to surround the sample stream with sheath fluid. Optical analysis at four different wavelengths identified the coded microspheres and quantified target bound by the presence of phycoerythrin tracer. The multiplexed assays in the microflow cytometer had performance approaching that of a commercial benchtop flow cytometer. The respective limits of detection for bacteria (Escherichia coli, Listeria, and Salmonella) were found to be 10(3), 10(5), and 10(4) cfu/mL for the microflow cytometer and 10(3), 10(6), and 10(5) cfu/mL for the commercial system. Limits of detection for the toxins (cholera toxin, staphylococcal enterotoxin B, and ricin) were 1.6, 0.064, and 1.6 ng/mL for the microflow cytometer and 1.6, 0.064, and 8.0 ng/mL for the commercial system.
Project description:An accurate and affordable CD4+ T cells count is an essential tool in the fight against HIV/AIDS. Flow cytometry (FCM) is the "gold standard" for counting such cells, but this technique is expensive and requires sophisticated equipment, temperature-sensitive monoclonal antibodies (mAbs) and trained personnel. The lack of access to technical support and quality assurance programs thus limits the use of FCM in resource-constrained countries. We have tested the accuracy, the precision and the carry-over contamination of Partec CyFlow MiniPOC, a portable and economically affordable flow cytometer designed for CD4+ count and percentage, used along with the "CD4% Count Kit-Dry".Venous blood from 59 adult HIV+ patients (age: 25-58 years; 43 males and 16 females) was collected and stained with the "MiniPOC CD4% Count Kit-Dry". CD4+ count and percentage were then determined in triplicate by the CyFlow MiniPOC. In parallel, CD4 count was performed using mAbs and a CyFlow Counter, or by a dual platform system (from Beckman Coulter) based upon Cytomic FC500 ("Cytostat tetrachrome kit" for mAbs) and Coulter HmX Hematology Analyzer (for absolute cell count).The accuracy of CyFlow MiniPOC against Cytomic FC500 showed a correlation coefficient (CC) of 0.98 and 0.97 for CD4+ count and percentage, respectively. The accuracy of CyFlow MiniPOC against CyFlow Counter showed a CC of 0.99 and 0.99 for CD4 T cell count and percentage, respectively. CyFlow MiniPOC showed an excellent repeatability: CD4+ cell count and percentage were analyzed on two instruments, with an intra-assay precision below ± 5% deviation. Finally, there was no carry-over contamination for samples at all CD4 values, regardless of their position in the sequence of analysis.The cost-effective CyFlow MiniPOC produces rapid, reliable and accurate results that are fully comparable with those from highly expensive dual platform systems.
Project description:The development of microfluidic chip-based cytometers has become an important area due to their advantages of compact size and low cost. Herein, we demonstrate a sheathless microfluidic cytometer which integrates a standing surface acoustic wave (SSAW)-based microdevice capable of 3D particle/cell focusing with a laser-induced fluorescence (LIF) detection system. Using SSAW, our microfluidic cytometer was able to continuously focus microparticles/cells at the pressure node inside a microchannel. Flow cytometry was successfully demonstrated using this system with a coefficient of variation (CV) of less than 10% at a throughput of ~1000 events s(-1) when calibration beads were used. We also demonstrated that fluorescently labeled human promyelocytic leukemia cells (HL-60) could be effectively focused and detected with our SSAW-based system. This SSAW-based microfluidic cytometer did not require any sheath flows or complex structures, and it allowed for simple operation over a wide range of sample flow rates. Moreover, with the gentle, bio-compatible nature of low-power surface acoustic waves, this technique is expected to be able to preserve the integrity of cells and other bioparticles.
Project description:Flow cytometry is currently the gold standard for analysis of cells in the medical laboratory and biomedical research. Fuelled by the need of point-of-care diagnosis, a significant effort has been made to miniaturize and reduce cost of flow cytometers. However, despite recent advances, current microsystems remain less versatile and much slower than their large-scale counterparts. In this work, an all-silica fibre microflow cytometer is presented that measures fluorescence and scattering from particles and cells. It integrates cell transport in circular capillaries and light delivery by optical fibres. Single-stream cell focusing is performed by Elasto-inertial microfluidics to guarantee accurate and sensitive detection. The capability of this technique is extended to high flow rates (up to 800?µl/min), enabling a throughput of 2500 particles/s. The robust, portable and low-cost system described here could be the basis for a point-of-care flow cytometer with a performance comparable to commercial systems.
Project description:Increasing resistance by malaria parasites to currently used antimalarials across the developing world warrants timely detection and classification so that appropriate drug combinations can be administered before clinical complications arise. However, this is often challenged by low levels of infection (referred to as parasitemia) and presence of predominantly young parasitic forms in the patients' peripheral blood. Herein, we developed a simple, inexpensive and portable image-based cytometer that detects and numerically counts Plasmodium falciparum infected red blood cells (iRBCs) from Giemsa-stained smears derived from infected blood. Our cytometer is able to classify all parasitic subpopulations by quantifying the area occupied by the parasites within iRBCs, with high specificity, sensitivity and negligible false positives (~ 0.0025%). Moreover, we demonstrate the application of our image-based cytometer in testing anti-malarial efficacy against a commercial flow cytometer and demonstrate comparable results between the two methods. Collectively, these results highlight the possibility to use our image-based cytometer as a cheap, rapid and accurate alternative for antimalarial testing without compromising on efficiency and minimal processing time. With appropriate filters applied into the algorithm, to rule out leukocytes and reticulocytes, our cytometer may also be used for field diagnosis of malaria.
Project description:Microfluidic inertial focusing has been shown as a simple and effective method to localize cells and particles within a flow cell for interrogation by an external optical system. To enable portable point of care optical cytometry, however, requires a reduction in the complexity of the large optical systems that are used in standard flow cytometers. Here, we present a new design that incorporates optical waveguides and focusing elements with an inertial focusing flow cell to make a compact robust cytometer capable of enumerating and discriminating beads, cells, and platelets.