National Institute of Optics National Research Council (INO-CNR), Largo Enrico Fermi 6, 50125, Florence, Italy.
European Laboratory for Non-linear Spectroscopy (LENS), Via Nello Carrara 1, 50019, Sesto Fiorentino, Italy.
Sci Rep. 2020 May 15;10(1):8116. doi: 10.1038/s41598-020-65218-3.
Autofluorescence spectroscopy has emerged in recent years as a powerful tool to report label-free contrast between normal and diseased tissues, both in vivo and ex vivo. We report the development of an instrument employing Single Photon Avalanche Diode (SPAD) arrays to realize real-time multispectral autofluorescence lifetime imaging at a macroscopic scale using handheld single-point fibre optic probes, under bright background conditions. At the detection end, the fluorescence signal is passed through a transmission grating and both spectral and temporal information are encoded in the SPAD array. This configuration allows interrogation in the spectral range of interest in real time. Spatial information is provided by an external camera together with a guiding beam that provides a visual reference that is tracked in real-time. Through fast image processing and data analysis, fluorescence lifetime maps are augmented on white light images to provide feedback of the measurements in real-time. We validate and demonstrate the practicality of this technique in the reference fluorophores and in articular cartilage samples mimicking the degradation that occurs in osteoarthritis. Our results demonstrate that SPADs together with fibre probes can offer means to report autofluorescence spectral and lifetime contrast in real-time and thus are suitable candidates for in situ tissue diagnostics.
近年来，自动荧光光谱技术作为一种强大的工具，已经出现，可以在体内和体外报告正常组织和病变组织之间无标记的对比。我们报告了一种仪器的开发，该仪器采用单光子雪崩二极管 (SPAD) 阵列，在手持单点光纤探头下，在明亮的背景条件下，实现宏观尺度的实时多光谱自动荧光寿命成像。在检测端，荧光信号通过传输光栅，光谱和时间信息都被编码在 SPAD 阵列中。这种配置允许在实时范围内对感兴趣的光谱范围进行询问。外部相机和引导光束提供空间信息，引导光束提供实时跟踪的视觉参考。通过快速图像处理和数据分析，将荧光寿命图叠加在白光图像上，实时提供测量反馈。我们验证并演示了这种技术在参考荧光团和模拟骨关节炎中发生的降解的关节软骨样本中的实用性。我们的结果表明，SPAD 与光纤探头相结合，可以实时报告自动荧光光谱和寿命对比，因此是原位组织诊断的合适候选者。
