Becker Wolfgang, Bergmann Axel, Biskup Christoph
Becker & Hickl, Nahmitzer Damm 30, 12277 Berlin, Germany.
Microsc Res Tech. 2007 May;70(5):403-9. doi: 10.1002/jemt.20432.
We present a fluorescence lifetime imaging technique with simultaneous spectral and temporal resolution. The technique is fully compatible with the commonly used multiphoton microscopes and nondescanned (direct) detection. An image of the back-aperture of the microscope lens is projected on the input of a fiber bundle. The input of the fiber bundle is circular, and the output is flattened to match the input slit of a spectrograph. The spectrum at the output of the spectrograph is projected on a 16-anode PMT module. For each detected photon, the encoding logics of the PMT module deliver a timing pulse and the number of the PMT channel in which the photon was detected. The photons are accumulated by a multidimensional time-correlated single photon counting (TCSPC) process. The recording process builds up a four-dimensional photon distribution over the times of the photons in the excitation pulse period, the wavelengths of the photons, and the coordinates of the scan area. The method delivers a near-ideal counting efficiency and is capable of resolving double-exponential decay functions. We demonstrate the performance of the technique for autofluorescence imaging of tissue.
我们提出了一种具有同时光谱和时间分辨率的荧光寿命成像技术。该技术与常用的多光子显微镜和非扫描（直接）检测完全兼容。显微镜镜头后孔径的图像投射到光纤束的输入端。光纤束的输入端是圆形的，其输出端被展平以匹配光谱仪的输入狭缝。光谱仪输出端的光谱投射到一个16阳极光电倍增管模块上。对于每个检测到的光子，光电倍增管模块的编码逻辑会提供一个定时脉冲以及检测到该光子的光电倍增管通道编号。光子通过多维时间相关单光子计数（TCSPC）过程进行累积。记录过程在激发脉冲周期内光子的时间、光子的波长以及扫描区域的坐标上建立起一个四维光子分布。该方法具有近乎理想的计数效率，并且能够分辨双指数衰减函数。我们展示了该技术在组织自发荧光成像方面的性能。
