Chair of Biological Imaging (CBI) and Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technical University of Munich, Munich, Germany.
Institute of Biological and Medical Imaging (IBMI), Helmholtz Zentrum München, Neuherberg, Germany.
Sci Rep. 2021 Jan 26;11(1):2181. doi: 10.1038/s41598-021-81584-y.
Morphological and functional optoacoustic imaging is enhanced by dedicated transgene reporters, in analogy to fluorescence methods. The development of optoacoustic reporters using protein engineering and directed evolution would be accelerated by high-throughput in-flow screening for intracellular, genetically encoded, optoacoustic contrast. However, accurate characterization of such contrast is impeded because the optoacoustic signals depend on the cell's size and position in the flow chamber. We report herein an optoacoustic flow cytometer (OA-FCM) capable of precise measurement of intracellular optoacoustic signals of genetically-encoded chromoproteins in flow. The novel system records light-scattering as a reference for the detected optoacoustic signals in order to account for cell size and position, as well as excitation light flux in the focal volume, which we use to reference the detected optoacoustic signals to enhance the system's precision. The OA-FCM was calibrated using micrometer-sized particles to showcase the ability to assess in-flow objects in the size range of single-cells. We demonstrate the capabilities of our OA-FCM to identify sub-populations in a mixture of two E. coli stocks expressing different reporter-proteins with a precision of over 90%. High-throughput screening of optoacoustic labels could pave the way for identifying genetically encoded optoacoustic reporters by transferring working concepts of the fluorescence field such as directed evolution and activated cell sorting.
形态和功能光声成像是通过专用的转基因报告器增强的，类似于荧光方法。通过高通量的内流筛选，用于细胞内、基因编码的光声对比，可以加速光声报告器的发展。然而，由于光声信号取决于细胞在流动室中的大小和位置，因此对这种对比的准确描述受到阻碍。我们在此报告了一种能够精确测量流动中基因编码色素蛋白的细胞内光声信号的光声流动细胞仪（OA-FCM）。该新型系统记录光散射作为检测到的光声信号的参考，以说明细胞的大小和位置，以及焦体积中的激发光通量，我们使用该参考来检测光声信号，以提高系统的精度。使用微米大小的颗粒对 OA-FCM 进行了校准，以展示评估在单个细胞大小范围内的流动物体的能力。我们证明了我们的 OA-FCM 能够以超过 90%的精度识别两种表达不同报告蛋白的大肠杆菌混合物中的亚群。光声标签的高通量筛选可以为通过转移荧光领域的工作概念，如定向进化和激活细胞分选，来识别基因编码的光声报告器铺平道路。
