Squire A, Bastiaens P I
Cell Biophysics Laboratory, Imperial Cancer Research Fund, London, U.K.
J Microsc. 1999 Jan;193(Pt 1):36-49. doi: 10.1046/j.1365-2818.1999.00427.x.
A microscope set-up and numerical methods are described which enable the measurement and reconstruction of three-dimensional nanosecond fluorescence lifetime images in every voxel. The frequency domain fluorescence lifetime imaging microscope (FLIM) utilizes phase detection of high-frequency modulated light by homodyne mixing on a microchannel plate image intensifier. The output signal at the image intensifier's phosphor screen is integrated on a charge coupled device camera. A scanning stage is employed to obtain a series of phase-dependent intensity images at equally separated depths in a specimen. The Fourier transform of phase-dependent data gives three-dimensional (3D) images of the Fourier coefficients. These images are deblurred using an Iterative Constrained Tikhonov-Miller (ICTM) algorithm in conjunction with a measured point spread function. The 3D reconstruction of fluorescence lifetimes are calculated from the deblurred images of the Fourier coefficients. An improved spatial and temporal resolution of fluorescence lifetimes was obtained using this approach to the reconstruction of simulated 3D FLIM data. The technique was applied to restore 3D FLIM data of a live cell specimen expressing two green fluorescent protein fusion constructs having distinct fluorescence lifetimes which localized to separate cellular compartments.
本文描述了一种显微镜设置和数值方法，可实现对每个体素的三维纳秒荧光寿命图像进行测量和重建。频域荧光寿命成像显微镜（FLIM）利用微通道板图像增强器上的零差混频对高频调制光进行相位检测。图像增强器荧光屏上的输出信号在电荷耦合器件相机上进行积分。采用扫描台在样品中以等间隔深度获取一系列与相位相关的强度图像。与相位相关数据的傅里叶变换给出了傅里叶系数的三维（3D）图像。这些图像使用迭代约束蒂霍诺夫 - 米勒（ICTM）算法结合测量的点扩散函数进行去模糊处理。荧光寿命的三维重建由去模糊后的傅里叶系数图像计算得出。使用这种方法对模拟的三维FLIM数据进行重建，获得了荧光寿命在空间和时间上的更高分辨率。该技术被应用于恢复表达两种具有不同荧光寿命且定位于不同细胞区室的绿色荧光蛋白融合构建体的活细胞样品的三维FLIM数据。
