Ix73 inverted fluorescence microscope

For both single-particle tracking and DDM experiments, we image the microspheres using an Olympus IX73 inverted fluorescence microscope with a 20× 0.4 NA objective and a Hamamatsu ORCA-Flash 2.8 CMOS camera (320 nm/pixel). For SPT, we collect 20 1920×1440 pixel videos of 2000 frames at 10 fps for each condition. For each of the videos, we observe >40 trackable particles, producing a total of >800 particles tracked. For DDM, we collect 12 512×512 pixel videos of 5000 frames at 10 fps across two samples. Videos are analyzed by examining regions of interest (ROI) of 256×256 pixels. For each sample condition, the experiment is carried out on two independently prepared samples. The sample-to-sample variation we observe and measure is indistinguishable from video-to-video variation within a single sample. Error bars presented throughout are determined by analyzing 3 random subsets of videos from each sample and calculating the standard error in values from all 6 subsets (3 from each sample). We see no statistically significant differences in the magnitudes or distributions of these subset values when comparing the two different samples.

Two syringe pumps (Fusion 200, Chemyx Inc., Stafford, TX) were used to infuse fluorescein and distilled H₂O through one inlet of the chip, respectively. The glass capillary was vibrated by the piezoelectric transducer. To determine the optimal frequency for mixing, we swept the excitation frequency from 10 kHz to 100 kHz in 100 Hz increment, and the fluorescent signals in the channel were monitored using an Olympus IX-73 inverted fluorescence microscope with a Hamamatsu sCMOS camera. To investigate factors that influence the mixing performance, different flow rates, driving voltages and channel dimension were applied to this system. Fluorescence signals were monitored and analyzed using Image J (v1.51s). Mixing performance was then characterized by analyzing the mixing index, mixing time and mixing length.
To test the generation of digital chemical waveforms, we applied a pulsing square waveform to the transducer, with periods of 1 s, 2 s, 5 s and 10 s. The flow rate was set as 30 μL/min and the input voltage is 4.0 V_pp. Detection window was set at the downstream of the glass capillary, and the fluorescent signal was monitored over time.