Long-lived metal complexes open up microsecond lifetime imaging microscopy under multiphoton excitation: from FLIM to PLIM and beyond
Long-lived metal complexes open up microsecond lifetime imaging microscopy under multiphoton excitation: from FLIM to PLIM and beyond
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DOI：
10.1039/c3sc51875b
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发表时间：
2014-03-01
期刊：
影响因子：
8.4
通讯作者：
Weinstein, Julia A.
中科院分区：
文献类型：
--
作者：
Baggaley, Elizabeth;Botchway, Stanley W.;Weinstein, Julia A.
具有亚微米分辨率的寿命成像显微镜通过允许可视化它们的结构以及使用外部探针在体内感测与生物相关的Anatyte来提供对生命系统的基本了解。化学对于下一代生物工具的发展至关重要，在这些工具中，对比度、敏感性和分子特异性有助于观察生命的基本过程。目前的一个基本限制是传统荧光探针的纳秒寿命，这通常限制了对亚纳秒变化的敏感性，而纳秒背景自体荧光则影响了对比度。将时间分辨发射成像显微镜(TREM)与寿命为微秒(或更长)的探头结合使用，可以实现高分辨率可视化，完全排除背景，并同时绘制包括氧在内的生物相关分析物的地图-灵敏度比目前可以达到的高一个数量级。然而，到目前为止，微秒时间尺度与现有的多光子激发/探测技术不兼容。在这里，我们首次实现了多光子激发的微秒成像，同时保持了基本的亚微米空间分辨率。这种新方法是无背景的，并将可用的成像和传感时间扩大了1000倍。利用基于铂的微秒级显著发射探测器家族中的第一个工程水溶性成员，我们展示了(I)活细胞和组织组织的无背景多光子激发微秒深度成像的第一个实例，(Ii)体内探测器寿命随局部微环境的数量级变化。从有机分子的超快荧光到过渡金属络合物或镧系元素/吖系元素及其组合的较慢发射，双光子TREM的概念可以被视为“薄膜+PLIM”，因为它可以用于任何时间尺度。它将过渡金属络合物作为多功能发射探针与新的多光子激发/微秒检测方法结合在一起，为生物、医学和材料科学中的多光子成像和传感创建了一个变革性的框架。
Lifetime imaging microscopy with sub-micron resolution provides essential understanding of living systems by allowing both the visualisation of their structure, and the sensing of bio-relevant anatytes in vivo using external probes. Chemistry is pivotal for the development of the next generation of bio-tools, where contrast, sensitivity, and molecular specificity facilitate observation of processes fundamental to life. A fundamental limitation at present is the nanosecond lifetime of conventional fluorescent probes which typically confines the sensitivity to sub-nanosecond changes, whilst nanosecond background autofluorescence compromises the contrast. High-resolution visualization with complete background rejection and simultaneous mapping of bio-relevant analytes including oxygen - with sensitivity orders of magnitude higher than that currently attainable - can be achieved using time-resolved emission imaging microscopy (TREM) in conjunction with probes with microsecond (or longer) lifetimes. Yet the microsecond timescale has so far been incompatible with available multiphoton excitation/detection technologies. Here we realize for the first time microsecond-imaging with multiphoton excitation whilst maintaining the essential sub-micron spatial resolution. The new method is background-free and expands available imaging and sensing timescates 1000-fold. Exploiting the first engineered water-soluble member of a family of remarkably emissive platinum-based, microsecond-lived probes amongst others, we demonstrate (i) the first instance of background-free multiphoton-excited microsecond depth imaging of live cells and histological tissues, (ii) over an order-of-magnitude variation in the probe lifetime in vivo in response to the local microenvironment. The concept of two-photon TREM can be seen as "FLIM + PLIM" as it can be used on any timescale, from ultrafast fluorescence of organic molecules to slower emission of transition metal complexes or lanthanides/actinides, and combinations thereof. It brings together transition metal complexes as versatile emissive probes with the new multiphoton-excitation/microsecond-detection approach to create a transformative framework for multiphoton imaging and sensing across biological, medicinal and material sciences.