Uplanfln

Fluorinated oil HFE-7500 (3M) with Picosurf-1 (PS1, 2.5%, Sphere Fluidics) was used as the dispersed phase, while commercial RF was diluted to various concentrations (0–180 µM) to be the aqueous phase. Liquid flow in all the chips was driven with Harvard Apparatus 2000 syringe infusion pumps. Plastic syringes of 1 and 5 ml were used to load the ethanol solutions and the surfactant-enriched fluorous carrier, respectively. The syringes were connected to the microchips via fine bore polyethylene tubing (ID = 0.38 mm, OD = 1.09 mm, Smiths Medical International Ltd). When flow rates of 2000 µl h⁻¹ (continuous phase, fluorinated oil and surfactant) and 250 µl h⁻¹ (dispersed phase, being the RF solutions) were used within a flow-focusing nozzle of 80 × 75 (width × height) droplets of ≈100 µm were generated. Droplet formation was monitored through a 10× microscope objective (UPlanFLN, Olympus) and a Phantom V72 fast camera mounted on the microscope (IX71, Olympus). Fluorescence of each droplet was detected as they passed along a laser beam focused on the outlet channel after the droplet generation. A home-made LabVIEW script was used for the quantification of the fluorescence to be adjusted to a calibration curve (electronic supplementary material, figure S2).

Fresh or incubated human blood obtained from 30 healthy volunteers was deposited on a glass slide and observed without fixation or staining using a BX-51 optical microscope (Olympus, Tokyo, Japan) equipped with a 100× oil immersion objective with iris (UPlanFLN, Olympus) and a dark-field condenser (Cerbe Distribution, Sherbrooke, Canada). Each specimen was observed individually at a magnification of either 1,000 or 2,000× and images were acquired with a Spot Flex color camera (Diagnostic Instruments, Sterling Heights, MI). Two aliquots were observed from each specimen or treatment (incubation time).

The optical measurement of the sample was executed on an inverted microscope (Olympus IX81). Emission from the LED with metasurface-integrated electrode was collected by a 100 × microscope objective (Olympus UPlanFL N, NA = 0.95) and optical images were obtained by an SCMOS image sensor (INFINITY 3, Numenera Inc.). The collected emission was directed to a spectrometer with 350 μm entrance slit width (Spectro Pro 500i, Action Research Inc.) and detected by an electron-multiplying charge-coupled device (EMCCD) (Newton^EM, ANDOR).
The Fourier-space images^{36 ,37 (link)} were measured using the setup shown in Fig. 3a. To obtain the Fourier-space image, a convex lens was placed at the image plane of the microscope, and then an EMCCD detector was positioned at the focal plane of the lens (f). The image taken from the EMCCD is a Fourier-space image. By switching the mirror positioned in the light path, we could easily change the light path from the microscope. To convert the Fourier-space image into an optical image, we positioned another lens with the same f at the distance of 2 f from the first lens. The transmitted light was focused into another CCD device by the second lens, and the optical image was obtained in this CCD.

Femtosecond laser pulses (80 MHz repetition rate, 100 fs, up to 25 nJ per pulse) from a Ti:Sapphire oscillator (Tsunami, Spectra-Physics, Andover, MA, USA) were applied at an 8 MHz repetition rate using an acousto-optic modulator (Pulse select, APE, Berlin, Germany) and a frequency doubled in the BBO SHG crystal. Then, femtosecond pulses with a central wavelength of 490 nm were coupled with an inverted optical microscope (IX71, Olympus, Tokyo, Japan) using a dichroic mirror (DMLP505, Thorlabs, Newton, MA, USA) and successfully focused on a sample using an objective lens (40 × 0.75NA UPlanFLN, Olympus, Tokyo, Japan). The samples were prepared as droplets of the purified fluorescent proteins dissolved in buffered solution (pH 5.5–10.0), applied to a standard 24 × 24 mm cover glass (Heinz Herenz, Hamburg, Germany). The average laser power was tuned with a polarizing attenuator in the range of 0.1–10 µW. The fluorescence (>505 nm) passed back through the objective lens and laser coupling dichroic mirror was directed towards the input of an Acton SP300i monochromator with a hybrid single-photon detector on its output (HPM-100-6, Becker&Hickl, Berlin, Germany), registering the fluorescence decay kinetics in the 510–520 nm band. The fluorescence decay data were analyzed using SPCImage software (Becker&Hickl, Berlin, Germany) with the measured instrument response function (IRF).