Elder A D, Frank J H, Swartling J, Dai X, Kaminski C F
Department of Chemical Engineering, University of Cambridge, Pembroke Street, Cambridge, UK.
J Microsc. 2006 Nov;224(Pt 2):166-80. doi: 10.1111/j.1365-2818.2006.01689.x.
High brightness light emitting diodes are an inexpensive and versatile light source for wide-field frequency-domain fluorescence lifetime imaging microscopy. In this paper a full calibration of an LED based fluorescence lifetime imaging microscopy system is presented for the first time. A radio-frequency generator was used for simultaneous modulation of light emitting diode (LED) intensity and the gain of an intensified charge coupled device (CCD) camera. A homodyne detection scheme was employed to measure the demodulation and phase shift of the emitted fluorescence, from which phase and modulation lifetimes were determined at each image pixel. The system was characterized both in terms of its sensitivity to measure short lifetimes (500 ps to 4 ns), and its capability to distinguish image features with small lifetime differences. Calibration measurements were performed in quenched solutions containing Rhodamine 6G dye and the results compared to several independent measurements performed with other measurement methodologies, including time correlated single photon counting, time gated detection, and acousto optical modulator (AOM) based modulation of excitation sources. Results are presented from measurements and simulations. The effects of limited signal-to-noise ratios, baseline drifts and calibration errors are discussed in detail. The implications of limited modulation bandwidth of high brightness, large area LED devices ( approximately 40 MHz for devices used here) are presented. The results show that phase lifetime measurements are robust down to sub ns levels, whereas modulation lifetimes are prone to errors even at large signal-to-noise ratios. Strategies for optimizing measurement fidelity are discussed. Application of the fluorescence lifetime imaging microscopy system is illustrated with examples from studies of molecular mixing in microfluidic devices and targeted drug delivery research.
高亮度发光二极管是用于宽场频域荧光寿命成像显微镜的一种廉价且通用的光源。本文首次介绍了基于发光二极管（LED）的荧光寿命成像显微镜系统的全面校准。使用射频发生器同时调制发光二极管（LED）强度和增强型电荷耦合器件（CCD）相机的增益。采用零差检测方案来测量发射荧光的解调与相移，由此在每个图像像素处确定相位寿命和调制寿命。该系统的特点在于其测量短寿命（500皮秒至4纳秒）的灵敏度，以及区分具有微小寿命差异的图像特征的能力。在含有罗丹明6G染料的猝灭溶液中进行了校准测量，并将结果与使用其他测量方法（包括时间相关单光子计数、时间选通检测以及基于声光调制器（AOM）的激发源调制）进行的几次独立测量结果进行了比较。给出了测量和模拟结果。详细讨论了有限信噪比、基线漂移和校准误差的影响。介绍了高亮度、大面积LED器件有限调制带宽（此处使用的器件约为40兆赫兹）的影响。结果表明，相位寿命测量在亚纳秒水平下依然稳健，而调制寿命即使在高信噪比情况下也容易出现误差。讨论了优化测量保真度的策略。通过微流控器件中分子混合研究和靶向药物递送研究的实例说明了荧光寿命成像显微镜系统的应用。
