PMC

Preparation steps for micropipette based droplet generation. (a) The chip is first filled with continuous phase (HFE-7500 oil enriched with surfactant). Surface modification with a perfluoro-silane avoids to trap bubbles. The pipette tip containing oil is left connected to the chip. (b) A pipette tip filled with the phase to be dispersed is then connected to the chip. (c) An empty pipette tip, which is attached to the pipette with its plunger pressed at maximum, is finally connected to the chip. (d) Releasing the pipette plunger immediately starts droplet generation as the corresponding negative pressure induces the flow of both liquids through a flow-focusing device. (e) The emulsion droplets are collected into the pipette tip attached to the pipette that can be disconnected from the chip at any moment.

Step-by-step workflow. a) Set up the system. Pull plunger to 7.0 mL tick. b) Load aqueous and oil solutions to the reservoirs. c) Pull the plunger to 9.0 mL tick quickly and rest the syringe on the station. d) Droplets are formed in the chip and flow to collection vial.

(A). Photograph of the DEP separation chip. The chip size is 30 mm × 20 mm. (Inset) Red section shows a close-up of the chip, showing the electrodes along the inside of the wells. The section of the chip in the image is ∼5 mm square. (B) Schematic of the chip, showing the three modes of DEP behavior. Cells flow from top to bottom through the wells; in the left well, cells can be seen experiencing positive DEP, are attracted to the electrodes, and held; in the center well, cells experience negative DEP, are repelled into the center of the well, and pass through. In the third well, cells experience no DEP force. In reality, cells of the same type will experience the same mode of behavior in all wells on the chip, but two different cell populations can exhibit behaviors different from each other. If one subpopulation experiences positive DEP and the others exhibit negative or neutral behavior, they can be separated. (C) The chip is loaded into a fluidic cartridge comprising two parts; an upper part B contains both housing and plunger, whereas a lower part A collects the cell solution. The chip fits between the two, sealed on both sides by O rings, and is clamped together by three Allen bolts.

(a) A photograph of the mixing-spraying, TR cryo-EM apparatus, labeled to show all major components. The mixing-spraying microfluidic chip is mounted inside an environmentally controlled chamber. A syringe pump, which is controlled by a laboratory-written, Visual Basic and C++ software program called Howard5e, is used to inject the reactants from inlets 1 and 2 into the microfluidic chip. Once in the microfluidic chip, the reactants are mixed and allowed to react for the reaction time specific to the microfluidic chip being used. The EM grid is held at the end of the plunger by a pair of tweezers. The Howard5e software controls and synchronizes the syringe pump as well as the plunger that holds the tweezer-mounted EM grid. Thus, as the sprayers discharge the reaction from the microfluidic chip onto the EM grid, the plunger is activated to plunge the EM grid into cryogen. (b-f) Images of cryo-EM grids prepared by the mixing-spraying, TR cryo-EM apparatus, going from low to high magnification. (b) Grid-view depicting droplets of different sizes deposited on the grid. (c) Square-view depicting droplet distribution over the holes. (d) Hole-view depicting ice distribution over holes. For image acquisition, thin ice regions were selected. (e) A representative micrograph showing good particle density. (f) Power spectrum of the acquisition image of panel e.