Prism Design for Spectral Flow Cytometry.

Flow cytometers are instruments used for the rapid quantitative analysis of cell suspension. Traditional flow cytometry uses multi-channel filters to detect fluorescence, whereas full-spectrum fluorescence based on dispersion detection is a more effective and accurate method. The application of various dispersion schemes in flow cytometry spectroscopy has been studied. From the perspective of modern detectors and demand for the miniaturization of flow cytometry, prism dispersion exhibits higher and more uniform light energy utilization, meaning that it is a more suitable dispersion method for small flow cytometers, such as microfluidic flow cytometers. Prism dispersion designs include the size, number, and placement of prisms. By deducing the formula of the final position of light passing through the prism and combining it with the formula of transmittance, the design criteria of the top angle and the incident angle of the prism in pursuit of the optimum transmittance and dispersion index can be obtained. Considering the case of multiple prisms, under the premise of pursuing a smaller size, the optimal design criteria for dispersion system composed of multiple prisms can be obtained. The design of prism dispersion fluorescence detection was demonstrated with a microfluidic flow cytometer, and the effectiveness of the design results was verified by microsphere experiments and practical biological experiments. This design criterion developed in this study is generally applicable to spectral flow cytometers.

Rapid switching and durable on-chip spark-cavitation-bubble cell sorter.

Precise and high-speed sorting of individual target cells from heterogeneous populations plays an imperative role in cell research. Although the conventional fluorescence-activated cell sorter (FACS) is capable of rapid and accurate cell sorting, it occupies a large volume of the instrument and inherently brings in aerosol generation as well as cross-contamination among samples. The sorting completed in a fully enclosed and disposable microfluidic chip has the potential to eliminate the above concerns. However, current microfluidic cell sorters are hindered by the high complexities of the fabrication procedure and the off-chip setup. In this paper, a spark-cavitation-bubble-based fluorescence-activated cell sorter is developed to perform fast and accurate sorting in a microfluidic chip. It features a simple structure and an easy operation. This microfluidic sorter comprises a positive electrode of platinum and a negative electrode of tungsten, which are placed on the side of the main channel. By applying a high-voltage discharge on the pair of electrodes, a single spark cavitation bubble is created to deflect the target particle into the downstream collection channel. The sorter has a short switching time of 150 µs and a long lifespan of more than 100 million workable actions. In addition, a novel control strategy is proposed to dynamically adjust the discharge time to stabilize the size of the cavitation bubble for continuous sorting. The dynamic control of continuously triggering the sorter, the optimal delay time between fluorescence detection and cell sorting, and a theoretical model to predict the ideal sorting recovery and purity are studied to improve and evaluate the sorter performance. The experiments demonstrate that the sorting rate of target particles achieves 1200 eps, the total analysis throughput is up to 10,000 eps, the particles sorted at 4000 eps exhibit a purity greater than 80% and a recovery rate greater than 90%, and the sorting effect on the viability of HeLa cells is negligible.

Diffractive Beam Shaper for Multiwavelength Lasers for Flow Cytometry.

Illumination spot in a flow cytometer is a crucial factor determining the measurement accuracy and stability. The traditional mechanism is to precisely calibrate multiple optical components to convert circular Gaussian beams into elliptical Gaussian beams, making it difficult to shape multiwavelength lasers simultaneously. A diffractive beam shaper for multicolor lasers with high simplicity, only containing one diffractive optical element and one focusing lens is created in this work. It can produce rectangular spots, of which the number, the sizes, and the positions are accurately determined by the incident wavelengths. Demonstrated in the customized microflow cytometer, the coefficient of variations (CV) of the optical signals by the beam shaper are 3.6-6.5%, comparable to those derived from the commercial instrument with 3.3-6.3% CVs. Benefiting from the narrow rectangular spots and the flexibility of diffractively shaped lasers, the measurement of bead sizes with 4-15 µm diameters and the real-time detection of flow velocity from 0.79 to 9.50 m/s with the CV of <5% are achieved. © 2020 International Society for Advancement of Cytometry.