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TCSPC based approaches for multiparameter detection in living cells

机译：基于TCSPC的活细胞多参数检测方法

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In living cells a manifold of processes take place simultaneously. This implies a precise regulation of intracellular ion homeostasis. In order to understand their spatio-temporal pattern comprehensively, the development of multiplexing concepts is essential. Due to the multidimensional characteristics of fluorescence dyes (absorption and emission spectra, decay time, anisotropy), the highly sensitive and non-invasive fluorescence microscopy is a versatile tool for realising multiplexing concepts. A prerequisite are analyte-specific fluorescence dyes with low cross-sensitivity to other dyes and analytes, respectively. Here, two approaches for multiparameter detection in living cells are presented. Insect salivary glands are well characterised secretory active tissues which were used as model systems to evaluate multiplexing concepts. Salivary glands secrete a KCl-rich or NaCl-rich fluid upon stimulation which is mainly regulated by intracellular Ca~(2+) as second messenger. Thus, pairwise detection of intracellular Na~+, Cl~- and Ca~(2+) with the fluorescent dyes ANG2, MQAE and ACR were tested. Therefore, the dyes were excited simultaneously (2-photon excitation) and their corresponding fluorescence decay times were recorded within two spectral ranges using time-correlated single-photon counting (TCSPC). A second approach presented here is based on a new TCSPC-platform covering decay time detection from picoseconds to milliseconds. Thereby, nanosecond decaying cellular fluorescence and microsecond decaying phosphorescence of Ruthenium-complexes, which is quenched by oxygen, were recorded simultaneously. In both cases changes in luminescence decay times can be linked to changes in analyte concentrations. In consequence of simultaneous excitation as well as detection, it is possible to get a deeper insight into spatio-temporal pattern in living tissues.
机译：在活细胞中，多个过程同时发生。这意味着细胞内离子稳态的精确调节。为了全面了解其时空模式，多路复用概念的发展至关重要。由于荧光染料的多维特性（吸收和发射光谱，衰减时间，各向异性），因此高度灵敏且无创的荧光显微镜是实现多路复用概念的通用工具。先决条件是特定于分析物的荧光染料，分别对其他染料和分析物具有较低的交叉敏感性。在此，提出了两种在活细胞中进行多参数检测的方法。唾液腺是特征明确的分泌活性组织，被用作模型系统以评估多路复用概念。唾液腺在刺激时分泌富含KCl或NaCl的液体，其主要受细胞内Ca〜（2+）作为第二信使的调节。因此，测试了用荧光染料ANG2，MQAE和ACR对细胞内Na〜+，Cl〜-和Ca〜（2+）的成对检测。因此，染料被同时激发（2-光子激发），并使用时间相关的单光子计数（TCSPC）在两个光谱范围内记录了它们相应的荧光衰减时间。这里介绍的第二种方法基于一种新的TCSPC平台，该平台涵盖从皮秒到毫秒的衰减时间检测。因此，同时记录了被氧淬灭的钌配合物的纳秒衰减细胞荧光和微秒衰减磷光。在这两种情况下，发光衰减时间的变化都可以与分析物浓度的变化联系在一起。由于同时激发和检测，有可能更深入地了解活组织中的时空模式。

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