Squire A, Verveer P J, Bastiaens P I
Cell Biophysics Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London, WC2A 3PX, U.K.
J Microsc. 2000 Feb;197(Pt 2):136-49. doi: 10.1046/j.1365-2818.2000.00651.x.
The experimental configuration and the computational algorithms for performing multiple frequency fluorescence lifetime imaging microscopy (mfFLIM) are described. The mfFLIM experimental set-up enables the simultaneous homodyne detection of fluorescence emission modulated at a set of harmonic frequencies. This was achieved in practice by using monochromatic laser light as an excitation source modulated at a harmonic set of frequencies. A minimum of four frequencies were obtained by the use of two standing wave acousto-optic modulators placed in series. Homodyne detection at each of these frequencies was performed simultaneously by mixing with matching harmonics present in the gain characteristics of a microchannel plate (MCP) image intensifier. These harmonics arise as a natural consequence of applying a high frequency sinusoidal voltage to the photocathode of the device, which switches the flow of photoelectrons 'on' and 'off' as the sinus voltage swings from negative to positive. By changing the bias of the sinus it was possible to control the duration of the 'on' state of the intensifier relative to its 'off' state, enabling the amplitude of the higher harmonic content in the gain to be controlled. Relative modulation depths of 400% are theoretically possible from this form of square-pulse modulation. A phase-dependent integrated image is formed by the sum of the mixed frequencies on the phosphor of the MCP. Sampling this signal over a full period of the fundamental harmonic enables each harmonic to be resolved, provided that the Nyquist sampling criterion is satisfied for the highest harmonic component in the signal. At each frequency both the phase and modulation parameters can be estimated from a Fourier analysis of the data. These parameters enable the fractional populations and fluorescence lifetimes of individual components of a complex fluorescence decay to be resolved on a pixel-by-pixel basis using a non-linear fit to the dispersion relationships. The fitting algorithms were tested on a simulated data set and were successful in disentangling two populations having 1 ns and 4 ns fluorescence lifetimes. Spatial invariance of the lifetimes was exploited to improve the accuracy significantly. Multiple frequency fluorescence lifetime imaging microscopy was then successfully applied to resolve the fluorescence lifetimes and fluorescence intensity contributions in a rhodamine dye mixture in solution, and green fluorescent protein variants co-expressed in live cells.
本文描述了用于执行多频荧光寿命成像显微镜（mfFLIM）的实验配置和计算算法。mfFLIM实验装置能够同时对在一组谐波频率下调制的荧光发射进行零差检测。在实际中，这是通过使用在一组谐波频率下调制的单色激光作为激发源来实现的。通过串联放置两个驻波声光调制器，至少可获得四个频率。通过与微通道板（MCP）图像增强器增益特性中存在的匹配谐波进行混频，同时对这些频率中的每一个进行零差检测。这些谐波是向该器件的光电阴极施加高频正弦电压的自然结果，随着正弦电压从负向正摆动，它会使光电子流“开启”和“关闭”。通过改变正弦波的偏置，可以控制增强器“开启”状态相对于其“关闭”状态的持续时间，从而控制增益中更高谐波含量的幅度。从这种方波调制形式理论上可以实现400%的相对调制深度。由MCP荧光屏上混合频率的总和形成一个与相位相关的积分图像。只要信号中最高谐波分量满足奈奎斯特采样准则，在基波的整个周期内对该信号进行采样就能分辨出每个谐波。在每个频率下，都可以通过对数据进行傅里叶分析来估计相位和调制参数。这些参数能够使用对色散关系的非线性拟合，逐像素地分辨复杂荧光衰减的各个成分的分数占比和荧光寿命。在一个模拟数据集上对拟合算法进行了测试，成功地分辨出了荧光寿命分别为1 ns和4 ns的两个群体。利用寿命的空间不变性显著提高了准确性。然后，多频荧光寿命成像显微镜成功应用于分辨溶液中罗丹明染料混合物以及活细胞中共表达的绿色荧光蛋白变体的荧光寿命和荧光强度贡献。
