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.
An optical two-beam trap composed from two counter propagating laser beams is an interesting setup due to the ability of the system to trap, hold, and stretch soft biological objects like vesicles or single cells. Because of this functionality, the system was also named the optical stretcher by Jochen Guck, Josep Kaas and co-workers almost 20 years ago. In a favorable setup, the two opposing laser beams meet with equal intensities in the middle of a fluidic channel in which cells may ow past, be trapped, stretched, and allowed to move on, giving the promise of a high throughput device. Yet, single beam optical traps, aka optical tweezers, by far outnumber the existing optical stretchers in research labs throughout the world. The ability to easily construct an optical stretcher setup in a low-cost material would possibly imply more frequent use of the optical stretching technique. Here, we discuss advantages and disadvantages of choice of material and methodology for chip assembly and chip production. For high throughput investigations of stretching deformation of single cells, optical stretching is, however, out-performed by hydrodynamic deformability assays. As we will discuss, injection molded polymer chips may with advantage be applied both for optical stretching and for hydrodynamic deformability experiments.
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