Imaging of fast-moving single cells with adaptive single pixel detection

开发能够在单细胞水平上快速检测细胞群的非靶向技术是下一代技术的热门，但面临着各种挑战。因此，我们的愿景是开发一种有助于非标记的多组学分析的质谱系统，其设计标准如下：1.与低通量方法的能力相匹配的生化覆盖率（~500种蛋白质、200种代谢物和300种脂质物质）2.高达10，000个哺乳动物细胞（或其它单细胞生物体）/秒的分析因此，我们提出了一种新的设计，通过使用由流动聚焦机构产生的含细胞液滴来解决这些挑战，并且液滴内化学消化之后是皮秒共振红外激光蒸发，然后对所产生的气态有机离子进行质谱分析。待解决的一个主要研究问题是进入的细胞/生物体的准确蒸发，这些细胞/生物体预计以至少1 m/s的速度行进，并且需要验证液滴的内容物以避免“假阳性”，因此需要实时的空间信息。一种能够以至少10 μ m量级的速率捕获运动细胞的空间信息的超快成像装置，因此，将构建每秒4帧，以便于随后的质谱分析，并在单细胞水平上提供额外的生物物理信息。单像素检测（SPD）取代了通常可用性有限且在应用范围内性能特性不同的昂贵阵列检测器，为当前问题提供了一种稳健的替代解决方案。

Development of an untargeted technique capable of rapidly detecting cell populations at a single cell level is a sought after next-generation technology that faces various challenges.It is thus our vision that a mass spectrometric system that facilitates unlabelled, multi-omic analysis can be developed with the following design criteria: 1. the biochemical coverage to match the power of low throughput methods (~500 proteins, 200 metabolites and 300 lipid species)2. analysis of up to 10,000 mammalian cells (or other single-cell organisms)/second We have thus proposed a new design addressing these challenges by using cell-containing liquid droplets produced by a flow focusing mechanism, and the in-droplet chemical digestion is followed by picosecond resonant infrared laser evaporation prior to mass spectrometric analysis of the produced gaseous organic ions. A major research problem to be solved is the accurate evaporation of the incoming cells/organisms which is expected to travel at a speed of at least 1m/s and the content of the droplets needs to be verified to avoid 'false positives', hence spatial information in real-time is needed.An ultrafast imaging set-up capable of capturing the spatial information of the moving cells at a rate that is at least on the order of 10^4 frames per second will thus be constructed to facilitate the succeeding mass spectrometric analysis and to provide additional biophysical information at a single cell level. Instead of costly array detectors that generally have limited availability and differing performance characteristics across the application spectrum, single pixel detection (SPD) offers a robust alternative solution for the problem at hand.