Frequency domain (FD) fluorescence lifetime imaging (FLIM) involves the excitation of the sample of interest with a modulated light source and digitization of the fluorescence emission for further analysis. Traditional FD-FLIM systems use heterodyne or homodyne detection, where the excitation light source and detector are modulated at specific frequency(s). More recently, FD-FLIM systems that use reflection of the light source as a trigger or phase reference for lifetime calculations have been developed. These detection schemes, however, require extra components that increase the cost and complexity of the FD-FLIM system. Here, we report a novel FD-FLIM detection scheme whereby the light source modulation and emission digitization are implemented using Field Programmable Gate Arrays (FPGAs), and fixed gain avalanche photodiodes are used for fluorescence detection. The reported FD-FLIM system was designed for probing nanosecond lifetime fluorophores (2-10 ns) at three emission bands simultaneously. The system utilizes a 375 nm diode laser for excitation at multiple simultaneous modulation frequencies (between 1 MHz and 83 MHz, bandwidth limited intentionally by using a lowpass filter) and three fixed gain avalanche photodiodes for simultaneous detection of three emission bands: 405/20 nm, 440/40 nm, and 525/50 nm (center/FWHM). Real-time computation of the modulation and phase lifetimes is simply performed by direct application of the discrete Fourier transform (max. of 10 frequencies) to the digitized fluorescence emission signals. The accuracy and sensitivity of this novel FD-FLIM detection scheme was demonstrated by imaging standard fluorophores and ex vivo unfixed human coronary artery tissue samples.

中文翻译：

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使用现场可编程门阵列进行实时处理的直接频域荧光寿命成像

频域 (FD) 荧光寿命成像 (FLIM) 涉及用调制光源激发感兴趣的样品，并将荧光发射数字化以供进一步分析。传统的 FD-FLIM 系统使用外差或零差检测，其中激发光源和检测器以特定频率调制。最近，开发了使用光源反射作为触发器或相位参考来计算寿命的 FD-FLIM 系统。然而，这些检测方案需要额外的组件，增加了 FD-FLIM 系统的成本和复杂性。在这里，我们报告了一种新颖的 FD-FLIM 检测方案，其中使用现场可编程门阵列 (FPGA) 实现光源调制和发射数字化，固定增益雪崩光电二极管用于荧光检测。报道的 FD-FLIM 系统旨在同时探测三个发射波段的纳秒寿命荧光团 (2-10 ns)。该系统利用 375 nm 二极管激光器在多个同步调制频率（1 MHz 和 83 MHz 之间，通过使用低通滤波器有意限制带宽）和三个固定增益雪崩光电二极管同时检测三个发射波段：405/20 nm 、440/40 nm 和 525/50 nm（中心/FWHM）。通过将离散傅立叶变换（最多 10 个频率）直接应用于数字化荧光发射信号，可以简单地实时计算调制和相位寿命。