Az plan fluor

We performed all DNA imaging with an upright microscope (AZ100, Nikon, Tokyo, Japan) equipped with a 5× objective (NA 0.5, AZ Plan Fluor, Nikon, Tokyo, Japan) set to 3× optical zoom, an LED light source (Sola, Lumencor, Beaverton, OR) and a CMOS camera (Zyla, Andor, Belfast, UK). We used a Cy3 filter (TRITC-B-NTE, Semrock, Rochester, NY) for observing labelled DNA (focusing experiments), a Cy5 filter (49006, Chroma, Bellows Falls, VT) for detecting molecular beacons (hybridization experiments).
In the LVF chips, we chose a detection point 1.4 cm downstream from the end of the converging region. For the DNA focusing experiments, we calculated the final (focused) concentration from a calibration curve constructed by filling the channel with known concentrations of DNA solution. The total amount of focused sample could then be found by spatially integrating over the resulting concentration map and multiplying by the depth of the channel. In the integration, we used a threshold of 10% of the peak value. We confirmed that no cross-contamination occurred between runs by comparing the fluorescence signature after ITP focusing in a new device in the absence of DNA to that in a device previously used to focus a high concentration of DNA. For the hybridization experiments we calculated the signal by integrating over a fixed 100 × 200 μm² region in the channel.

We used 1-μm-diameter pink carboxyl polystyrene particles (Spherotech Inc., USA) mixed in a buffer composed of 10 mM acetic acid and 1 mM NaOH (Sigma-Aldrich, Switzerland) as tracer particles to visualize the flow. For visualization of the particles, we used an upright fluorescence microscope (AZ100, Nikon, Germany) equipped with a solid-state light source (Mira, Lumencor, USA), a 5× objective (AZ-Plan Fluor, Nikon, Germany), and a mCherry filter cube (562/40 excitation, 641/75 nm emission, and 593 nm dichroic mirror, Nikon, Germany). We imaged using a CCD camera (Clara, Andor-Oxford Instrument, UK) with an exposure time of 100 ms. The gate electrodes and the driving electrode were actuated with two power supplies (Keithley 2410, Tektronix, USA), producing square-wave signals (−200, 200 V) at a frequency of 25 Hz. We used an in-house MATLAB code (R2019b, Mathworks) that sets the alternating voltages of the power supplies (−200, 200 V) and a pulse generator (Stanford Research Systems, DG535) that sends a square-wave signal to the power supplies and triggers the switching of the alternating voltages. The photoconductive switches had a 100-µm gap and were illuminated with dedicated LEDs (RND 135–00129, RND Electronics, China) controlled by manual switches (RND 210-00189, RND Electronics, China).

Macroconfocal imaging was conducted on a Nikon AZ-100 stand equipped with a C2 scan head with the 2X objective (NA 0.2, WD 45, AZ-Plan Fluor, Nikon). Videomicroscopy (brightfield and epifluorescence) images were acquired on a Nikon Ti Eclipse stand, collected with an Orca R2 camera (Hamamatsu, Japan) through the following set of objectives: 4X (NA 0.13, WD 17.2, CFI Plan Fluor, Nikon), 10X (NA 0.3, WD 16, CFI Plan Fluor, Nikon) and a 20X (NA 0.45, ELWD 6.9–8.2, CFI S Plan Fluor, Nikon). Confocal images and movies were made with a Nikon Eclipse Ti equipped with a C2Si scan head and a 20X objective (NA 0.75, WD 1, PlanApo VC, Nikon). All of these scopes are equipped with computer controlled motorized stages (Nikon and Prior) that permit repetitive and reproducible scanning of all the wells within the chambers. All microscopes are equipped with the Nikon NIS Elements package software.