Sorting Sounds - A high-throughput microfluidics screening platform for the development of genetically encoded labels for Optoacoustic imaging

光声(OA)或光声成像是一种新兴的成像方式，可以在高分辨率下提供无与伦比的穿透深度。这提供了更全面的时间分辨率3D活体成像，其深度远远超出了光学方法的范围。然而，由于OA在生命科学中的应用相对较新，标记和分子工具的范围有限。到目前为止，只有少量的专用标记被提出，还有更少的标记是基因编码的--这是进行体内靶向成像的先决条件。从分子的角度来看，从荧光成像中已知的广泛的标记--特别是类似绿色荧光蛋白(GFP)的标记--可以用于OA。然而，尽管荧光成像的目标是高分子亮度(即量子产额)，但OA的概念需要一种主要的无辐射去激发途径。这种根本的不同使得丰富多样的荧光成像工具在办公自动化中的立即应用成为问题。此外，需要近红外标签才能充分利用OA成像固有的更高穿透深度的优势。因此，必须为OA设计一系列新的分子标记，以充分支持这一新的成像模式。除了设计原则之外，基于蛋白质的标签工程的一个基本前提是能够筛选所需的标签属性。在设计基因编码的荧光标签的情况下，筛选可以依赖于对荧光信号的检测。然而，新的筛选策略必须应用于OA标签工程。该提案中概述的研究包括设计一种基于微流控技术的高通量OA细胞仪，以及概念验证定向进化和筛选OA优化的标记。在前期工作中，我们已经证明了在微流体结构中忠实地检测单个细胞中的OA信号的可行性，并概述了技术先决条件。此外，我们在OA中测试了几个红色GFP类型的染色质蛋白，以确定最适合作为文库生成起点的候选蛋白。

Optoacoustic (OA) or Photoacoustic imaging is an emerging imaging modality providing unsurpassed penetration depth at high resolutions. This provides for more comprehensive time resolved 3D in vivo imaging at depths far beyond the reach of optical methods. However, with the application of OA in life sciences being relatively new the range of labels and molecular tools is limited. Thus far only a small number of dedicated labels has been put forward with an even smaller number being genetically encoded - a prerequisite for targetable in vivo imaging. From the molecular perspective a wide range of labels known from fluorescence imaging - especially green fluorescent protein (GFP) like labels - could be utilized for OA. However, while fluorescence imaging aims for a high molecular brightness (i.e. quantum yield) the concept of OA requires a predominantly radiation-less deexcitation pathway. This fundamental difference makes an immediate application of the rich variety of fluorescence imaging tools in OA problematic. Furthermore, near-infrared labels are required to access the full advantage of the higher penetration depth intrinsic to OA imaging. Consequently, a new range of molecular labels has to be designed for OA to fully empower this new imaging modality. Next to the design principles a fundamental prerequisite to protein based label engineering is the ability to screen for the desired properties of the label. In the case of the design of genetically encoded fluorescence labels screening could rely on the detection of fluorescence signals. However, new screening strategies have to be applied for OA-label engineering. The research outlined in this proposal includes design of a microfluidics based high-throughput OA cytometry device as well as proof-of-concept directed-evolution and screening for OA-optimized labels. In preliminary work we could already demonstrate the feasibility of faithfully detecting OA signals in single cells in a microfluidics configuration and outlined the technical prerequisites. Furthermore, we tested several red GFP-type chromo-proteins in OA to identify candidates most suitable as starting points for library generation.