Flow cytometry (FC) is a pivotal tool for studying the physical and chemical properties of particles. State-of-the-art FC systems are highly advanced, yet they are expensive, bulky, and require high sample volume, qualified operators, and periodic maintenance. The manipulation of particles suspended in viscoelastic fluids has received increasing attention, especially for miniaturized flow cytometry technologies. This study presents a miniaturized optical capillary FC device using the viscoelastic focusing technique. A straight, one inlet/outlet microcapillary device is precisely aligned to a fiber-coupled laser source and detectors. Forward scattered, side scattered, and fluorescently emitted light signals are collected and analyzed in a real-time environment. The developed platform fits onto an inverted microscope stage enabling real-time microscopy imaging of the particles of interest together with the flow cytometry analysis. We achieved stable viscoelastic focusing and performed FC measurements for rigid polystyrene beads (diameters: 2 – 15 μm), non-spherical human erythrocytes, and canonical shape metaphase human chromosomes. We performed cytometry measurements with a throughput of 100 events/s yielding a coefficient of variation of 2%. This newly developed FC device is a versatile tool and can be operated with any inverted microscope to get the mutual benefits of optical and imaging FC measurements. Furthermore, it is possible to extend these benefits by adding more back-end tools, such as optical trapping and Raman spectroscopy.
ABO blood typing is the determination of four different blood groups: type A, B, AB, or O. Clinically approved ABO blood typing methods are suffering from expensive reagents and multiple time-consuming cross-referencing steps, creating the need for fast, sustainable, sensitive, and label-free technologies. Raman spectroscopy techniques have shown potential to distinguish biomolecules and blood components such as purified serum proteins, albumin, and globulin. In combination with machine learning tools, the accuracy and specificity of Raman spectroscopic measurements can be improved and adapted to clinical applications. This study presents a multivariate analysis of human-blood samples for ABO blood typing using Raman spectroscopy and support vector machine (SVM) classification. A custom-built NIR Raman spectroscopy setup with a 785 nm wavelength laser is coupled into an inverted microscope to collect Raman spectra from each blood sample. Donor samples are drawn from EDTA tubes into a fused silica microcapillary without dilution and sample preparation steps. Raman measurements from more than 270 donor samples are analyzed to get accurate blood typing predictions. The blood types are distinguished pairwise by an average AUC score of 0.94, showing great potential of the developed system for future blood typing applications.
Miniaturized optofluidic platforms play an important role in bio-analysis, detection and diagnostic applications. The advantages of such miniaturized devices are extremely low sample requirement, low cost development and rapid analysis capabilities. Fused silica is advantageous for optofluidic systems due to properties such as being chemically inert, mechanically stable, and optically transparent to a wide spectrum of light. As a three dimensional manufacturing method, femtosecond laser scanning followed by chemical etching shows great potential to fabricate glass based optofluidic chips. In this study, we demonstrate fabrication of all-fiber based, optofluidic flow cytometer in fused silica glass by femtosecond laser machining. 3D particle focusing was achieved through a straightforward planar chip design with two separately fabricated fused silica glass slides thermally bonded together. Bioparticles in a fluid stream encounter with optical interrogation region specifically designed to allocate 405nm single mode fiber laser source and two multi-mode collection fibers for forward scattering (FSC) and side scattering (SSC) signals detection. Detected signal data collected with oscilloscope and post processed with MATLAB script file. We were able to count number of events over 4000events/sec, and achieve size distribution for 5.95μm monodisperse polystyrene beads using FSC and SSC signals. Our platform shows promise for optical and fluidic miniaturization of flow cytometry systems.
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