Development of the next generation of SWIR emItting probes for bioimaging

目前的挑战和目标，研究生理病理现象和疾病相关的过程中活的生物体与非侵入性光学生物成像是实现深组织穿透，同时保持高检测灵敏度和高的空间和时间分辨率。在小动物成像中用于高对比度、高灵敏度和高空间分辨率的有希望的光谱窗口是在~900-2500 nm之间的短波红外（SWIR），其中与可见光（400-700 nm）和NIR（~700-900 nm）相比，组织的散射、吸收和自发荧光强烈降低。充分利用SWIR光致发光（PL）成像（PLI）目前受到以下因素的阻碍：i.）缺乏可以在体内安全使用的具有高PL量子产率（PL QY）和高亮度的合适造影剂，以及ii.）许多短波红外发射器的光学特性缺乏定量和可靠的数据。这限制了可实现的对比度、空间和时间分辨率以及发射器性能的可比性，阻碍了新SWIR对比剂的合理设计。针对高空间和时间分辨率（即，为了实现动物PLI血管网络的快速图像采集（快速图像采集），德国和法国定量光谱学、生物物理学、探头设计和光学成像领域的专家联手开发了新型SWIR造影剂和先进的图像分析方法。因此，我们将利用原子级精确的金纳米团簇（AuNC）和具有改进光学性质的Ag 2S量子点等无重金属量子点（QD）合理设计明亮的SWIR报告分子，并在溶液、模拟组织的人工模型和小动物中对其进行光谱表征。提高检测灵敏度和分辨率的策略将得到解决。通过比较SWIR中的不同光谱窗口，从而考虑组织的波长相关吸收和散射特性，以及ii.）通过制备多聚体AuNC来调节PL和亮度。同时，将研究与这些发射器的短波红外光谱复用。将在小鼠中评价性能最佳和“生物安全”的造影剂，以确定其用于器官血管形成的SWIR PLI的潜力。将使用不同的图像分析方法对空间分辨率和信噪比进行量化，从而增强PL数据信息内容以解析血液和淋巴流。结果i.）将提供明亮且“安全”的SWIR发射器和先进的图像分析方法，使得能够对活体动物中的血管网络和3D流体流动绘图进行非侵入性真实的时间深度成像，以更好地理解正常和病理性血管生成的机制，ii.）产生关于生物相关环境中不同SWIR报告子的信号相关光谱关键特征的定量和可靠数据，iii.）允许将这些特征与SWIR PLI中的探头性能相关联，以及iv.）有助于标准化SWIR PL测量。

Current challenges and objectives for studying physio-pathological phenomena and disease-related processes in living organisms with non-invasive optical bioimaging are to realize a deep tissue penetration while maintaining a high detection sensitivity and a high spatial and temporal resolution. A promising spectral window for high contrast, high sensitivity, and high spatial resolution in small animal imaging is the shortwave infrared (SWIR) between ~900–2500 nm where scattering, absorption and autofluorescence of tissue are strongly reduced compared to the visible (400–700 nm) and NIR (~700–900 nm). Full exploitation of SWIR photoluminescence (PL) imaging (PLI) is currently hampered by i.) a lack of suitable contrast agents with a high PL quantum yield (PL QY) and a high brightness, that can be used safely in vivo and ii.) a lack of quantitative and reliable data on the optical properties of many SWIR emitters. This limits achievable contrast, spatial and temporal resolution and comparability of emitter performance, hampering the rational design of new SWIR contrast agents. Aiming at high spatial and temporal resolution (i.e., fast image acquisition) of the vascular network in PLI of animals, German and French experts in quantitative optical spectroscopy, photophysics, probe design, and optical imaging joined forces to develop new SWIR contrast agents and advanced image analysis methods. Therefore, we will rationally design bright SWIR reporters utilizing atomically precise gold nanoclusters (AuNCs) and heavy metal-free quantum dots (QDs) like Ag2S QDs with improved optical properties and spectroscopically characterize them in solution, artificial models mimicking tissues, and small animals. Strategies to improve detection sensitivity and resolution will be addressed i.) by comparing different spectral windows in the SWIR, thus considering wavelength-dependent absorption and scattering properties of tissue, and ii.) by preparing multimeric AuNCs to tune PL and brightness. In parallel, SWIR spectral multiplexing with these emitters will be studied. Best performing and “biologically safe” contrast agents will be evaluated in mice to determine their potential for SWIR PLI of organ vascularization. Spatial resolution and signal-to-noise ratios will be quantified, using different methods of image analysis, thereby enhancing PL data information content to resolve blood and lymphatic flows. The results i.) will provide bright and “safe” SWIR emitters and advanced methods of image analysis, enabling non-invasive real time deep imaging of vascular networks and 3D fluid flow mapping in live animals to better understand mechanisms of normal and pathological angiogenesis, ii.) yield quantitative and reliable data on the signal-relevant spectroscopic key features of different SWIR reporters in biologically relevant environments, iii.) allow to correlate these features and probe performance in SWIR PLI, and iv.) help to standardize SWIR PL measurements.