Calibration of a wide-field frequency-domain fluorescence lifetime microscopy system using light emitting diodes as light sources
Calibration of a wide-field frequency-domain fluorescence lifetime microscopy system using light emitting diodes as light sources
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DOI：
10.1111/j.1365-2818.2006.01689.x
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发表时间：
2006-11-01
影响因子：
2
通讯作者：
Kaminski, C. F.
中科院分区：
文献类型：
--
作者：
Elder, A. D.;Frank, J. H.;Kaminski, C. F.
高亮度发光二极管是宽视场频域荧光寿命成像显微镜的一种廉价而通用的光源。本文首次对基于LED的荧光寿命成像显微系统进行了全面的标定。采用射频发生器对增强型电荷耦合器件（CCD）相机的发光二极管（LED）强度和增益进行同步调制。采用零差检测方案来测量所发射的荧光的解调和相移，由此在每个图像像素处确定相位和调制寿命。该系统的特点是它的灵敏度，以测量短寿命（500 ps至4 ns），其能力，区分图像特征与小的寿命差异。校准测量在淬灭的溶液中进行，含有罗丹明6G染料和几个独立的测量结果进行比较与其他测量方法，包括时间相关的单光子计数，时间门控检测，和声光调制器（AOM）的激发源的调制。结果从测量和模拟。详细讨论了有限信噪比、基线漂移和定标误差的影响。有限的调制带宽的高亮度，大面积的LED器件（类似于40 MHz的设备在这里使用）的影响。结果表明，相位寿命的测量是强大的子ns的水平，而调制寿命是容易出现错误，即使在大的信噪比。优化测量保真度的策略进行了讨论。荧光寿命成像显微镜系统的应用举例说明从微流控装置中的分子混合和靶向药物输送研究的研究。
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 (similar to 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.