In vivo optical imaging using fluorescently labeled self-quenched monoclonal antibodies, activated through binding and internalization within target cells, results in excellent target-to-background ratios. We hypothesized that these molecular probes could be utilized to accurately report on cellular internalization with fluorescence lifetime imaging (FLI). Two imaging probes were synthesized, consisting of the antibody trastuzumab (targeting HER2/neu) conjugated to Alexa Fluor750 in ratios of either 1:8 or 1:1. Fluorescence intensity and lifetime of each conjugate were initially determined at endosomal pHs. Since the 1:8 conjugate is self-quenched, the fluorescence lifetime of each probe was also determined after exposure to the known dequencher SDS. In vitro imaging experiments were performed using 3T3/HER2(+) and BALB/3T3 (HER2(-)) cell lines. Changes in fluorescence lifetime correlated with temperature- and time-dependent cellular internalization. In vivo imaging studies in mice with dual flank tumors [3T3/HER2(+) and BALB/3T3 (HER2(-))] detected a minimal difference in FLI. In conclusion, fluorescence lifetime imaging monitors the internalization of target-specific activatable antibody-fluorophore conjugates in vitro. Challenges remain in adapting this methodology to in vivo imaging.

中文翻译：

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可激活目标特异性分子探针的荧光寿命成像。


使用荧光标记的自猝灭单克隆抗体进行体内光学成像，通过靶细胞内的结合和内化而激活，可产生出色的靶标与背景比。我们假设这些分子探针可用于通过荧光寿命成像（FLI）准确报告细胞内化。合成了两种成像探针，由抗体曲妥珠单抗（靶向 HER2/neu）以 1:8 或 1:1 的比例与 Alexa Fluor750 缀合组成。每种缀合物的荧光强度和寿命最初在内体 pH 值下测定。由于 1:8 缀合物是自猝灭的，因此每个探针的荧光寿命也在暴露于已知的去猝灭剂 SDS 后测定。使用3T3/HER2(+)和BALB/3T3(HER2(-))细胞系进行体外成像实验。荧光寿命的变化与温度和时间依赖性细胞内化相关。对双侧肿瘤小鼠 [3T3/HER2(+) 和 BALB/3T3 (HER2(-))] 进行的体内成像研究发现 FLI 存在微小差异。总之，荧光寿命成像可在体外监测目标特异性可激活抗体-荧光团缀合物的内化。将这种方法应用于体内成像仍然存在挑战。