Zhang Lixin, Zheng Xianlin, Deng Wei, Lu Yiqing, Lechevallier Severine, Ye Zhiqiang, Goldys Ewa M, Dawes Judith M, Piper James A, Yuan Jingli, Verelst Marc, Jin Dayong
Advanced Cytometry Labs, ARC Centre of Excellence for Nanoscale BioPhotonics (CNBP), Macquarie University, Sydney, NSW 2109, Australia.
Centre d'Élaboration de Matériaux et d'Etudes Structurales (CERMES - CNRS), Paul Sabatier University, France.
Sci Rep. 2014 Oct 13;4:6597. doi: 10.1038/srep06597.
Time-gated luminescence microscopy using long-lifetime molecular probes can effectively eliminate autofluorescence to enable high contrast imaging. Here we investigate a new strategy of time-gated imaging for simultaneous visualisation of multiple species of microorganisms stained with long-lived complexes under low-background conditions. This is realized by imaging two pathogenic organisms (Giardia lamblia stained with a red europium probe and Cryptosporidium parvum with a green terbium probe) at UV wavelengths (320-400 nm) through synchronization of a flash lamp with high repetition rate (1 kHz) to a robust time-gating detection unit. This approach provides four times enhancement in signal-to-background ratio over non-time-gated imaging, while the average signal intensity also increases six-fold compared with that under UV LED excitation. The high sensitivity is further confirmed by imaging the single europium-doped Y₂O₂S nanocrystals (150 nm). We report technical details regarding the time-gating detection unit and demonstrate its compatibility with commercial epi-fluorescence microscopes, providing a valuable and convenient addition to standard laboratory equipment.
使用长寿命分子探针的时间门控发光显微镜可以有效消除自发荧光，实现高对比度成像。在此，我们研究了一种新的时间门控成像策略，用于在低背景条件下同时可视化用长寿命配合物染色的多种微生物。这是通过将高重复率（1kHz）的闪光灯与强大的时间门控检测单元同步，在紫外波长（320 - 400nm）下对两种致病生物体（用红色铕探针染色的贾第鞭毛虫和用绿色铽探针染色的微小隐孢子虫）进行成像来实现的。与非时间门控成像相比，这种方法的信背比提高了四倍，而平均信号强度与紫外LED激发下相比也增加了六倍。通过对单个铕掺杂Y₂O₂S纳米晶体（150nm）进行成像，进一步证实了其高灵敏度。我们报告了关于时间门控检测单元的技术细节，并展示了它与商用落射荧光显微镜的兼容性，为标准实验室设备提供了一种有价值且方便的补充。
