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

光声成像（OA）或光声成像是一种新兴的成像方式，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三维体内成像深度远远超出了光学方法。然而，由于OA在生命科学中的应用相对较新，标签和分子工具的范围有限。到目前为止，只有少数专用标签被提出，而基因编码的数量更少——这是体内靶向成像的先决条件。从分子的角度来看，荧光成像中已知的各种标记，特别是绿色荧光蛋白（GFP）之类的标记，都可以用于OA。然而，虽然荧光成像的目标是高分子亮度（即量子产率），但OA的概念主要需要一种无辐射的去激发途径。这一根本性的差异使得丰富多样的荧光成像工具在OA中的直接应用存在问题。此外，需要近红外标签来充分利用OA成像固有的更高穿透深度。因此，必须为OA设计一系列新的分子标记，以充分增强这种新的成像模式。除了设计原则之外，基于蛋白质的标签工程的一个基本先决条件是筛选标签所需属性的能力。在设计基因编码荧光标记的情况下，筛选可以依靠荧光信号的检测。然而，新的筛选策略必须应用于oa标签工程。本提案中概述的研究包括基于微流体的高通量OA细胞仪的设计，以及OA优化标签的概念验证定向进化和筛选。在初步的工作中，我们已经可以证明在微流体配置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，我们测试了OA中的几种红色gfp型色蛋白，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.