Interdisciplinary Research Institute, Science and Technology University of Lille, USR 3078 CNRS, BCF, Villeneuve d'Ascq, France.
Cytometry A. 2009 Dec;75(12):1007-14. doi: 10.1002/cyto.a.20802.
Multispectral fluorescence lifetime imaging microscopy is a promising and powerful technique for discriminating multiply labeled samples and for detecting molecular interactions inside thick, heterogeneous, and light-scattering milieu such as tissue. The fast and correct analysis of the spectral and lifetime images constitutes a major challenge, which requires a high level of expertise. We present here a new approach that considerably simplifies this analysis avoiding complex fitting algorithm strategies and permitting a fast and visual graphical representation of the fluorescence lifetimes. By transforming the experimental data from time domain to frequency domain for each spectral channel, we calculate the multispectral polar representation and demonstrate its interest on multiply fluorescent labeled sample. We further apply it on Förster resonance energy transfer (FRET) experiments and demonstrate that FRET measurements with a high level of precision can be performed. With addition of emission wavelength as third dimension in the polar representation, autofluorescence emitted by the sample is thus clearly identified. Analysis artifacts induced by the sample or by fitting algorithm choice become then totally inexistent.
多光谱荧光寿命成像显微镜是一种很有前途和强大的技术，可用于区分多重标记的样品，并用于检测组织等厚、异质和光散射环境中的分子相互作用。快速正确地分析光谱和寿命图像是一个主要挑战，这需要高水平的专业知识。我们在这里提出了一种新方法，通过避免复杂的拟合算法策略，大大简化了这种分析，并允许快速直观地显示荧光寿命的图形表示。通过将每个光谱通道的实验数据从时域转换到频域，我们计算了多光谱极坐标表示，并证明了其在多重荧光标记样品上的应用。我们进一步将其应用于Förster 共振能量转移（FRET）实验，并证明可以进行高精度的 FRET 测量。在极坐标表示中添加发射波长作为第三维，因此可以清楚地识别样品发出的自发荧光。由样品或拟合算法选择引起的分析伪影变得完全不存在。
