Droplet microfluidics for time-resolved serial crystallography

Microfluidic manipulation of droplet volume coupled with seeding can be used to precisely control crystal size. Droplet microfluidics also enables fast, millisecond-scale micromixing for advancing time-resolved serial crystallography.


Figure S2
Figure S2 Breaking the emulsion.Droplets are collected under a mineral oil overlay, with the buoyant nature causing an emulsion to form above the fluoro-oil carrier (A).The fluoro-oil is removed (B), and a ~10-fold volume of crystallisation buffer relative to emulsion volume is added to the emulsion (C).Next, a 1-fold volume of perfluoro-1-octanol (PFO) is added, and gently mixed to coalesce all droplets into a single aqueous volume (D).This volume and the crystals within it are retrieved for analysis (E).

Figure S3
Figure S3 Plots of figures of merit.(A) Mean signal-to-noise ratio 〈I/σ(I)〉, (B) Pearson's correlation coefficient CC1/2, (C) CC* and (D) Rsplit against resolution comparing lysozyme and Pdx1 crystals grown in batch with those grown in droplets.

Figure S4
Figure S4 Trypsin needles and confinement effects on lysozyme axial ratio.(A) Trypsin needles prepared in batch have various lengths with an axial ratio of ~10, (B) whereas crystallisation in droplets produces an axial ratio of ~5.(C) The axial ratio of lysozyme crystals grown in droplets tends to unity as droplets are miniaturised below 1 pL.

Figure S5
Figure S5 Viscosity effect on droplet generation throughput.Droplet microfluidics can manage high viscosity crystallisation buffers, although increased viscosity decreases the droplet generation frequency.

Figure S7
Figure S7Circulations within droplets provide convection to drive rapid micromixing.

Figure S8
Figure S8 Droplet synchronisation and 1:1 coupling followed by surfactant exchange using PFO to trigger droplet fusion and mixing (Video S2 and S3).The synchronisation channel is 75x40 m (w,h) with a droplet velocity of 120 mm/s.The droplet fuse and mix channel is 125x40 µm (w,h) with a droplet velocity of 85 mm/s.200 pL crystal droplets are fused with 225 pL dye droplets with mixing achieved in ~7 milliseconds.

Table S1
Droplet microfluidic conditions for preparing lysozyme crystals.

Table S2
Droplet microfluidic conditions for preparing Pdx1 crystals.

Table S3
Droplet microfluidic conditions for preparing trypsin type I needle crystals.