Dual-jet synchronization results in higher cooling rates for sample vitrification

Jet vitrification, a technique in use since the 1970s, has proven highly effective for cryo-fixation by delivering the rapid cooling rates needed to vitrify thicker samples. In the current study we designed a pre-conditioned jet system to address synchronization issues inherent in a dual jet setup. Using in silico simulations, we analyzed the morphology of the jet and observed how the pre-conditioning impacts its dynamics. By pre-filling the complete internal fluidic flow path, we minimized jetting inconsistencies and enhanced the accuracy and precision of the jet intersection point, thus providing more consistent cooling for cryo-EM samples during vitrification. High speed camera observations and temperature measurements confirmed the improvement of jet synchronization. An increased maximum cooling rate of 3.4 × 106 K/s represents a substantial improvement over a standard jetting setup. These findings underscore the critical importance of precise flow control for optimizing cooling efficiency and will further improve the vitrification quality of thicker samples.