Electrospray sample injection for single-particle imaging with X-ray lasers

Abstract

The possibility of imaging single proteins constitutes an exciting challenge for X-ray lasers. Despite encouraging results on large particles, imaging small particles has proven to be difficult for two reasons: not quite high enough pulse intensity from currently available X-ray lasers and, as we demonstrate here, contamination of the aerosolised molecules by non-volatile contaminants in the solution. The amount of contamination on the sample depends on the initial droplet-size during aerosolisation. Here we show that with our electrospray injector we can decrease the size of aerosol droplets and demonstrate virtually contaminant-free sample delivery of organelles, small virions, and proteins. The results presented here, together with the increased performance of next generation X-ray lasers, constitute an important stepping stone towards the ultimate goal of protein structure determination from imaging at room temperature and high temporal resolution.

Ultracompact 3D microfluidics for time-resolved structural biology

“…Achieving jet diameters below one hundred nanometers for single-particle imaging should be possible by combining smaller gas orifices with features that stabilize the liquid meniscus, such as hypodermic needle-shaped liquid orifices 55 . Breaking the same volume flow into smaller droplets improves the density of the single-particle beam while also preventing clustering of multiple particles in a single droplet 20,51,52 . Low particle densities from aerosol injectors for single-particle imaging may be overcome through stacked jet arrays.…”

“…GDVNs achieve this by focusing the jet down to such diameters 9 . However, a further reduction to submicron jet diameters is desired to improve signal to noise ratios, in particular for weakly scattering samples, such as nanocrystals 18 , single fibers 19 or single, isolated particles 20 . New megahertz X-ray sources require jets that are fast enough to replenish undamaged sample in time for the next X-ray pulse 21 .…”

A statistical approach to detect protein complexes at X-ray free electron laser facilities

“…If we manage to improve sample injection and tune it so to increase the chance of having single-particle hits, we could have enough single RNA polymerase II hits to reconstruct its 3D structure at 5 Å resolution or better. The most critical improvement of injection would be to reduce the initial size of the aerosol droplets by using an electrospray injector 35 . At that point, the ongoing research efforts in extending existing 3D reconstruction methods to model the full conformational landscape of the sample becomes even more pressing, a concern that so far has remained largely theoretical in FXI applications.…”

A guide to sample delivery systems for serial crystallography

“…The aerosol injector is a device utilizing a nozzle to jet aerosol particles under high pressure at a high velocity, including electrically driven aerosol injectors and gas driven aerosol injectors . Atomized droplets containing sample generated by aerosol injectors pass through a series of separators, apertures, and graded pumps, eventually entering the vacuum environment to deliver the sample to the X‐ray beam.…”