Fruhwirth Gilbert O, Ameer-Beg Simon, Cook Richard, Watson Timothy, Ng Tony, Festy Frederic
King's College London, The Richard Dimbleby Department of Cancer Studies, Division of Cancer, Guy's Medical School Campus, SE1 1UL, London, United Kingdom.
Opt Express. 2010 May 24;18(11):11148-58. doi: 10.1364/OE.18.011148.
Development of remote imaging for diagnostic purposes has progressed dramatically since endoscopy began in the 1960's. The recent advent of a clinically licensed intensity-based fluorescence micro-endoscopic instrument has offered the prospect of real-time cellular resolution imaging. However, interrogating protein-protein interactions deep inside living tissue requires precise fluorescence lifetime measurements to derive the Förster resonance energy transfer between two tagged fluorescent markers. We developed a new instrument combining remote fiber endoscopic cellular-resolution imaging with TCSPC-FLIM technology to interrogate and discriminate mixed fluorochrome labeled beads and expressible GFP/TagRFP tags within live cells. Endoscopic-FLIM (e-FLIM) data was validated by comparison with data acquired via conventional FLIM and e-FLIM was found to be accurate for both bright bead and dim live cell samples. The fiber based micro-endoscope allowed remote imaging of 4 microm and 10 microm beads within a thick Matrigel matrix with confident fluorophore discrimination using lifetime information. More importantly, this new technique enabled us to reliably measure protein-protein interactions in live cells embedded in a 3D matrix, as demonstrated by the dimerization of the fluorescent protein-tagged membrane receptor CXCR4. This cell-based application successfully demonstrated the suitability and great potential of this new technique for in vivo pre-clinical biomedical and possibly human clinical applications.
自20世纪60年代内窥镜检查开始以来，用于诊断目的的远程成像技术取得了巨大进展。最近，一种获得临床许可的基于强度的荧光显微内窥镜仪器的出现，为实时细胞分辨率成像带来了希望。然而，要深入研究活组织内部的蛋白质-蛋白质相互作用，需要精确测量荧光寿命，以确定两个标记荧光标记之间的Förster共振能量转移。我们开发了一种新仪器，将远程纤维内窥镜细胞分辨率成像与TCSPC-FLIM技术相结合，用于检测和区分活细胞内混合荧光染料标记的珠子以及可表达的GFP/TagRFP标签。通过与传统FLIM获取的数据进行比较，验证了内窥镜-FLIM（e-FLIM）数据，发现e-FLIM对于明亮珠子和暗淡活细胞样本均准确。基于纤维的显微内窥镜能够在厚基质胶基质中对4微米和10微米的珠子进行远程成像，并利用寿命信息可靠地区分荧光团。更重要的是，这项新技术使我们能够可靠地测量嵌入三维基质中的活细胞中的蛋白质-蛋白质相互作用，荧光蛋白标记的膜受体CXCR4的二聚化证明了这一点。这种基于细胞的应用成功地证明了这项新技术在体内临床前生物医学以及可能的人类临床应用中的适用性和巨大潜力。
