Fig. 1. Drop-on-drop experimental setup enables accelerated mixing in droplets.

a Overview of the setup. The protein crystals are ejected by an acoustic droplet ejector (ADE, white and dark gray column above tape and on the right-hand side) and synchronized to the XFEL master clock. The burst of substrate drops is ejected by a piezoelectric injector (PEI, blue box above tape, on the left-hand side) that can accommodate disposable cartridges with a range of orifices that dictate the size of the droplets. Reaction time is varied by the speed of the tape drive (10–600 mm s⁻¹) and depends upon the location of the PEI with respect to the ADE droplet intersection and the X-ray interaction points; in the case reported here, we selected time points between 0.1 and 6 s. b Relative fluorescence intensity (crosses) of the calcium-bound forms of Fura Red (salmon) and Fluo-5N (purple) dyes obtained from the scaling of the normalized spectral components and their errors of the fits (Supplementary Fig. 5) compared to reaction simulations of collision-driven hydrodynamic flow (inset) and equilibration by diffusion. Lines correspond to the fitted exponential functions of the experimental data and rise times are indicated. Fluorescence signal of Fura Red rises faster than Fluo-5N due to the lower dissociation constant.