Hantke Max F, Bielecki Johan, Kulyk Olena, Westphal Daniel, Larsson Daniel S D, Svenda Martin, Reddy Hemanth K N, Kirian Richard A, Andreasson Jakob, Hajdu Janos, Maia Filipe R N C
Chemistry Research Laboratory, Department of Chemistry, Oxford University, 12 Mansfield Rd, Oxford OX1 3TA, UK.
Laboratory of Molecular Biophysics, Department of Cell and Molecular Biology, Uppsala University, Husargatan 3 (Box 596), Uppsala SE-75124, Sweden.
IUCrJ. 2018 Sep 11;5(Pt 6):673-680. doi: 10.1107/S2052252518010837. eCollection 2018 Nov 1.
Ultra-bright femtosecond X-ray pulses generated by X-ray free-electron lasers (XFELs) can be used to image high-resolution structures without the need for crystallization. For this approach, aerosol injection has been a successful method to deliver 70-2000 nm particles into the XFEL beam efficiently and at low noise. Improving the technique of aerosol sample delivery and extending it to single proteins necessitates quantitative aerosol diagnostics. Here a lab-based technique is introduced for Rayleigh-scattering microscopy allowing us to track and size aerosolized particles down to 40 nm in diameter as they exit the injector. This technique was used to characterize the 'Uppsala injector', which is a pioneering and frequently used aerosol sample injector for XFEL single-particle imaging. The particle-beam focus, particle velocities, particle density and injection yield were measured at different operating conditions. It is also shown how high particle densities and good injection yields can be reached for large particles (100-500 nm). It is found that with decreasing particle size, particle densities and injection yields deteriorate, indicating the need for different injection strategies to extend XFEL imaging to smaller targets, such as single proteins. This work demonstrates the power of Rayleigh-scattering microscopy for studying focused aerosol beams quantitatively. It lays the foundation for lab-based injector development and online injection diagnostics for XFEL research. In the future, the technique may also find application in other fields that employ focused aerosol beams, such as mass spectrometry, particle deposition, fuel injection and three-dimensional printing techniques.
由X射线自由电子激光（XFEL）产生的超亮飞秒X射线脉冲可用于对高分辨率结构进行成像，而无需结晶。对于这种方法，气溶胶注入一直是一种成功的方法，可将70 - 2000纳米的颗粒高效且低噪声地输送到XFEL光束中。改进气溶胶样品输送技术并将其扩展到单蛋白需要进行定量气溶胶诊断。本文介绍了一种基于实验室的瑞利散射显微镜技术，使我们能够在气溶胶颗粒离开注射器时追踪并测量直径低至40纳米的雾化颗粒大小。该技术用于表征“乌普萨拉注射器”，它是用于XFEL单颗粒成像的开创性且常用的气溶胶样品注射器。在不同操作条件下测量了颗粒束焦点、颗粒速度、颗粒密度和注入产率。结果还表明，对于大颗粒（100 - 500纳米），如何能够实现高颗粒密度和良好的注入产率。研究发现，随着颗粒尺寸减小，颗粒密度和注入产率会变差，这表明需要不同的注入策略才能将XFEL成像扩展到更小的目标，如单蛋白。这项工作展示了瑞利散射显微镜在定量研究聚焦气溶胶束方面的强大功能。它为基于实验室的注射器开发和XFEL研究的在线注入诊断奠定了基础。未来，该技术还可能在其他使用聚焦气溶胶束的领域找到应用，如质谱分析、颗粒沉积、燃料喷射和三维打印技术。
