Microsecond cell triple-sorting enabled by multiple pulse irradiation of femtosecond laser.

Femtosecond-laser-assisted cell manipulation, as one of the high throughput cell sorting techniques, is tailored for single-step multiple sorting based on controllable impulsive force. In this paper, femtosecond laser pulses are focused within a pocket structure and they induce an impulse force acting on the flowing objects. The impulsive force is shown to be controllable by a new method to adjust the femtosecond pulse properties. This allows precise streamline manipulation of objects having various physical qualities (e.g., weight and volume). The pulse energy, pulse number, and pulse interval of the femtosecond laser are altered to determine the impulsive force strength. The method is validated in single cell or bead triple-sorting experiments and its capability to perform streamline manipulation in as little as 10 µs is shown. The shift profiles of the beads acting under the impulsive force are studied in order to better understand the sorting mechanism. Additionally, beads and cells with different fluorescence intensities are successfully detected and directed into different microchannels, with maximum success rates of 90% and 64.5%, respectively. To sum up, all results suggest that this method has the potential to sort arbitrary subpopulations by altering the number of femtosecond pulses and that it takes the first step toward developing a single-step multi-selective system.

Assessment of the electrical penetration of cell membranes using four-frequency impedance cytometry.

The electrical penetration of the cell membrane is vital for determining the cell interior via impedance cytometry. Herein, we propose a method for determining the conductivity of the cell membrane through the tilting levels of impedance pulses. When electrical penetration occurs, a high-frequency current freely passes through the cell membrane; thus, the intracellular distribution can directly act on the high-frequency impedance pulses. Numerical simulation shows that an uneven intracellular component distribution can affect the tilting levels of impedance pulses, and the tilting levels start increasing when the cell membrane is electrically penetrated. Experimental evidence shows that higher detection frequencies (>7 MHz) lead to a wider distribution of the tilting levels of impedance pulses when measuring cell populations with four-frequency impedance cytometry. This finding allows us to determine that a detection frequency of 7 MHz is able to pass through the membrane of Euglena gracilis (E. gracilis) cells. Additionally, we provide a possible application of four-frequency impedance cytometry in the biomass monitoring of single E. gracilis cells. High-frequency impedance (≥7 MHz) can be applied to monitor these biomass changes, and low-frequency impedance (<7 MHz) can be applied to track the corresponding biovolume changes. Overall, this work demonstrates an easy determination method for the electrical penetration of the cell membrane, and the proposed platform is applicable for the multiparameter assessment of the cell state during cultivation.

Dual-frequency impedance assays for intracellular components in microalgal cells.

Intracellular components (including organelles and biomolecules) at the submicron level are typically analyzed in situ by special preparation or expensive setups. Here, a label-free and cost-effective approach of screening microalgal single-cells at a subcellular resolution is available based on impedance cytometry. To the best of our knowledge, it is the first time that the relationships between impedance signals and submicron intracellular organelles and biomolecules are shown. Experiments were performed on Euglena gracilis (E. gracilis) cells incubated under different incubation conditions (i.e., aerobic and anaerobic) and 15 µm polystyrene beads (reference) at two distinct stimulation frequencies (i.e., 500 kHz and 6 MHz). Based on the impedance detection of tens of thousands of samples at a throughput of about 900 cells per second, three metrics were used to track the changes in biophysical properties of samples. As a result, the electrical diameters of cells showed a clear shrinkage in cell volume and intracellular components, as observed under a microscope. The morphology metric of impedance pulses (i.e., tilt index) successfully characterized the changes in cell shape and intracellular composition distribution. Besides, the electrical opacity showed a stable ratio of the intracellular components to cell volume under the cellular self-regulation. Additionally, simulations were used to support these findings and to elucidate how submicron intracellular components and cell morphology affect impedance signals, providing a basis for future improvements. This work opens up a label-free and high-throughput way to analyze single-cell intracellular components by impedance cytometry.

Parallel Impedance Cytometry for Real-Time Screening of Bacterial Single Cells from Nano- to Microscale.

The benefits of impedance cytometry include high-throughput and label-free detection, while long-term calibration is required to remove the effects of the detection circuits. This study presents a novel impedance cytometry system, called parallel impedance cytometry, to simplify the calibration and analysis of the impedance signals. Furthermore, target objects can be detected even when benchmarked against similar objects. Parallel dual microchannels allow the simultaneous detection of reference and target particles in two separate microchannels, without the premixing of reference and target suspensions. The impedance pulses of both can appear separately on the opposite sides of the same time series, which have been verified via simulation and experimental results. Raw impedance signals can easily distinguish target particles from reference ones. Polystyrene beads with different sizes ranging from nano- to microscale (e.g., 500, 750 nm, 1, 2, 3, and 4.5 µm) confirm the nanosensitivity of the system. In addition, the detection of antibiotic-treated Escherichia coli cells demonstrates that our system can be used for the quantitative assessment of the dielectric properties of individual cells, as well as for the proportion of susceptible cells. Through benchmarking against untreated E. coli cells in the other channel, our method enables the discrimination of susceptible cells from others and the comparison of susceptible and insusceptible cells in the target suspension. Those findings indicate that the parallel impedance cytometry can greatly facilitate the measurement and calibration of the impedances of various particles or cells and provide a means to compare their dielectric properties.

Focusing of Particles in a Microchannel with Laser Engraved Groove Arrays.

Continuous microfluidic focusing of particles, both synthetic and biological, is significant for a wide range of applications in industry, biology and biomedicine. In this study, we demonstrate the focusing of particles in a microchannel embedded with glass grooves engraved by femtosecond pulse (fs) laser. Results showed that the laser-engraved microstructures were capable of directing polystyrene particles and mouse myoblast cells (C2C12) towards the center of the microchannel at low Reynolds numbers (Re < 1). Numerical simulation revealed that localized side-to-center secondary flows induced by grooves at the channel bottom play an essential role in particle lateral displacement. Additionally, the focusing performance proved to be dependent on the angle of grooves and the middle open space between the grooves based on both experiments and simulation. Particle sedimentation rate was found to critically influence the focusing of particles of different sizes. Taking advantage of the size-dependent particle lateral displacement, selective focusing of micrometer particles was demonstrated. This study systematically investigated continuous particle focusing in a groove-embedded microchannel. We expect that this device will be used for further applications, such as cell sensing and nanoparticle separation in biological and biomedical areas.

Microscopic impedance cytometry for quantifying single cell shape.

In this work, we investigated the ability of impedance flow cytometry to measure the shape of single cells/particles. We found that the impedance pulses triggered by micro-objects that are asymmetric in morphology show a tilting trend, and there is no such a tilting trend for symmetric ones. Therefore, we proposed a new metric, tilt index, to quantify the tilt level of the impedance pulses. Through simulation, we found that the value of tilt index tends to be zero for perfectly symmetrical objects, while the value is greater than zero for asymmetrical ones. Also, this metric was found to be independent on the trajectories (i.e., lateral, and z-direction shift) of the target micro-object. In experiments, we adopted a home-made lock-in amplifier and performed experiments on 10 µm polystyrene beads and Euglena gracilis (E. gracilis) cells with varying shapes. The experimental results coincided with the simulation results and demonstrated that the new metric (tilt index) enables the impedance cytometry to characterize the shape single cells/particles without microscopy or other optical setups.