Fusion 200

In a typical experiment, we used the below protocol to generate bisulfite sequencing libraries on ~2000 single cells. Three 0.5 m-long PFA tubing (IDEX Health & Science, 1622L) connected to three syringes were loaded with 100 μl of the nuclei suspension, 160 μl of 2x lysis buffer (2% Triton x-100, 100 mM Tris-HCl pH 8.0, 300 mM NaCl, 0.2% sodium deoxycholate (Sigma-Aldrich, 302-95-4), 10 mM sodium butyrate (Sigma-Aldrich, B5887-1G), 0.375 mM CaCl₂, 0.0375 U/μl MNase (Thermo Fisher Scientific, 88216), 0.2% Sarkosyl (Sigma-Aldrich, L7414), with freshly added 1.6 μl of PIC and 1.6 μl of 100 mM PMSF), and 1 ml oil (Bio-Rad, 1864006), respectively. The three syringes were mounted on three separate syringe pumps (Chemyx, FUSION 200) and the reagents were flowed simultaneously into the three inlets of the droplet generation device (Fig. 2a) at the flow rates of 2 μl/min for the nuclei suspension, 2 μl/min for the lysis buffer, and 14 μl/min for the oil to generate droplets with a size of ~35 μm in diameter. We continuously collected the generated droplets into a 0.2 ml tube that was placed on ice for 10 min to gather about 1.78 million droplets. We incubated the droplets on ice for 10 min, then at room temperature for 15 min, and finally at 65 °C for 30 min. After incubation, excessive oil at the bottom of the tube was removed and the tube was put on ice until use.

Evaporative loss of water immersion fluid at 37°C was countered by microfluidics. Briefly, a Chemyx Fusion 200 syringe pump was used to perfuse water at a rate of 10 μl min⁻¹ through Tygon microtube with a 0.01″ internal diameter. Water formed a droplet at the end of the tubing, and was pulled into the interface between the glass coverslip and the objective by capillary forces. This design permits water immersion microscopy for over 10 h at 37°C.

To test the flow-imaging ability of the developed device, we prepared a scattering flow phantom. To simulate an optically turbid medium, such as a tissue background, the phantom was made of polydimethylsiloxane mixed with 0.15% (15 g/100 mL) TiO₂. A 2.5% microparticle solution was then pumped into a light-transparent 1 mm diameter silicon tube immersed in the phantom at constant flow rates ranging from 0.023 mL/min (0.5 mm/s flow speed) to 0.094 mL/min (2.0 mm/s flow speed) using a high-precision infusion syringe pump (Fusion 200, Chemyx Inc., Stafford, TX, USA). This effectively simulated the blood flow of a superficial vessel within stationary tissue. The device probe was positioned linearly 5 mm in front of the surface of the perfused phantom. The light from the probe tip illuminated the phantom for the LSCI measurement.

Polyacrylamide gels with a stiffness gradient were generated as described [29 (link)] with mild modifications. 65 μL acrylamide mix (19 μL 40% acrylamide, 19 μL 2% bis-acrylamide, 27 μL 10 mM HEPES with 2 mg/mL Irgacure2959, Sigma-Aldrich, 410896, Saint Louis, MO, USA) was applied to glutaraldehyde-modified 24 mm glass coverslip, covered with a glass coverslip made hydrophobic by treatment with Repel-Silane. Gradients were generated by initially covering the acrylamide mix solution with an opaque mask and then slowly sliding it at a controlled speed while irradiating with a UV bench lamp. The mask was slid with the help of an automatic syringe pump (Chemyx Fusion 200). To ensure complete polymerization, the whole acrylamide mix solution was first exposed to UV light for 12 min without covering, and then mask was slid at 40 μm/s for 10 min to produce the steep stiffness gradient gels. After gel photo-polymerization, the hydrophobic glass coverslip was removed and the gel was washed with PBS thoroughly to remove unreacted reagents. The stiffness was measured with AFM. To promote cell adhesion, fibronectin was covalently linked to the gels as described below. Uniform gels were made from 40% acrylamide and 2% bis-acrylamide mixed with 10% ammonium persulfate and 1% TEMED and received 20 min UV light explosion without any covering.

Figure 2c shows the schematic diagram of the EIS measurement setup. Before feeding a cell sample, the microchannel was coated with a solution of bovine serum albumin (HiMEDIA, Thane, India) in deionized water. The cell suspension was fed into the microfluidic device through inlet B, while the medium without cells was fed into inlet A. The outlet of the device was connected to a syringe pump (Fusion 200, Chemyx, Stafford, TX, USA) to draw the cell sample and the blank solution into the device at a flow rate of 0.003 µL/min. Cells were forced to flow through the measurement channel by the sheath flow. The impedance between the electrodes was measured by an impedance analyzer (E4990A, Keysight, Santa Rosa, CA, USA). The impedance analyzer was controlled by an in-house MATLAB program, and the measured data were sent to a computer via a universal serial bus (USB) connection for subsequent analysis.