Serafino Michael J, Jo Javier A
Department of Electrical and Computer Engineering, University of Oklahoma, Stephenson Research and Technology Center, Suite 1108, 101 David L Boren Blvd, Norman, OK 73072, USA.
Biomed Opt Express. 2023 Mar 23;14(4):1608-1625. doi: 10.1364/BOE.480287. eCollection 2023 Apr 1.
Due to the complexity, limited practicality, and cost of conventional fluorescence lifetime imaging/microscopy (FLIM) instrumentation, FLIM adoption has been mostly limited to academic settings. We present a novel point scanning frequency-domain (FD) FLIM instrumentation design capable of simultaneous multi-wavelength excitation, simultaneous multispectral detection, and sub-nanosecond to nanosecond fluorescence lifetime estimation. Fluorescence excitation is implemented using intensity-modulated CW diode lasers that are available in a selection of wavelengths spanning the UV-VI-NIR range (375-1064 nm). Digital laser intensity modulation was adopted to enable simultaneous frequency interrogation at the fundamental frequency and corresponding harmonics. Time-resolved fluorescence detection is implemented using low-cost, fixed-gain, narrow bandwidth (100 MHz) avalanche photodiodes, thus, enabling cost-effective fluorescence lifetime measurements at multiple emission spectral bands simultaneously. Synchronized laser modulation and fluorescence signal digitization (250 MHz) is implemented using a common field-programmable gate array (FPGA). This synchronization reduces temporal jitter, which simplifies instrumentation, system calibration, and data processing. The FPGA also allows for the implementation of the real-time processing of the fluorescence emission phase and modulation at up to 13 modulation frequencies (processing rate matching the sampling rate of 250 MHz). Rigorous validation experiments have demonstrated the capabilities of this novel FD-FLIM implementation to accurately measure fluorescence lifetimes in the range of 0.5-12 ns. endogenous, dual-excitation (375nm/445nm), multispectral (four bands) FD-FLIM imaging of human skin and oral mucosa at 12.5 kHz pixel rate and room-light conditions was also successfully demonstrated. This versatile, simple, compact, and cost-effective FD-FLIM implementation will facilitate the clinical translation of FLIM imaging and microscopy.
由于传统荧光寿命成像/显微镜（FLIM）仪器的复杂性、实用性有限以及成本较高，FLIM的应用大多局限于学术环境。我们提出了一种新型的点扫描频域（FD）FLIM仪器设计，它能够进行多波长同步激发、多光谱同步检测以及亚纳秒至纳秒级的荧光寿命估计。荧光激发通过强度调制连续波二极管激光器实现，这些激光器有多种波长可供选择，涵盖紫外 - 可见 - 近红外范围（375 - 1064 nm）。采用数字激光强度调制，能够在基频和相应谐波处进行同步频率询问。时间分辨荧光检测使用低成本、固定增益、窄带宽（100 MHz）的雪崩光电二极管实现，从而能够同时在多个发射光谱带进行经济高效的荧光寿命测量。同步激光调制和荧光信号数字化（250 MHz）通过通用现场可编程门阵列（FPGA）实现。这种同步减少了时间抖动，简化了仪器、系统校准和数据处理。FPGA还允许在高达13个调制频率下对荧光发射相位和调制进行实时处理（处理速率与250 MHz的采样速率匹配）。严格的验证实验证明了这种新型FD - FLIM实现能够准确测量0.5 - 12 ns范围内的荧光寿命。还成功展示了在12.5 kHz像素速率和室内光条件下对人体皮肤和口腔黏膜进行内源性、双激发（375nm/445nm）、多光谱（四个波段）的FD - FLIM成像。这种通用、简单、紧凑且经济高效的FD - FLIM实现将促进FLIM成像和显微镜技术的临床转化。
