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建立了一台基于新研制的高重复频率皮秒扫描相机的双光子激发荧光寿命显微成像系统,重点介绍所研制的高重复频率皮秒扫描相机。为了在高时间分辨力的同时扩大时间测量范围,实现大面积两维空间高时间分辨取样测量,从而提高采样速率和更有效地发挥扫描相机的作用,设计和研制了一种大面积、高时间分辨力扫描变像管和一种重复频率高达1MHz的斜坡电压扫描电路。基于上述关键部件所研制的扫描相机具有重复频率高、扫描速度可调、时间分辨力高、工作面积大、非线性低、触发晃动小等优点。用钛宝石飞秒激光器作为激光脉冲源,通过脉冲提取器将76MHz的高重复频率降低为1MHz,采用可调延时器和标准具对扫描相机的时间分辨力、扫描速度和非线性进行标定。该系统的时间分辨力达到6.5ps,非线性为2.60%,可测量的时间范围从十几皮秒到几十纳秒。测量了若丹明6G和香豆素314两种标准荧光染料的荧光寿命,取得了与参考文献一致的实验结果。
A two-photon excitation fluorescence lifetime microscopy system based on the newly developed picosecond high-repetition frequency picosecond camera was established, focusing on the picosecond high-repetition frequency scanning camera developed. In order to expand the measurement range of time while resolving with high time resolution and realize the time-resolved sampling measurement of large-area two-dimensional space, so as to improve the sampling rate and make more effective use of the scanning camera, a large-area and high-time Resolution scan tube and a variable frequency of up to 1MHz ramp voltage scanning circuit. The scanning camera developed based on the above key components has the advantages of high repetition frequency, adjustable scanning speed, high time resolution, large working area, low nonlinearity and small shaking. Using a titanium-germanium femtosecond laser as a laser pulse source, the high repetition rate of 76 MHz is reduced to 1 MHz by a pulse extractor, and the time resolution, scanning speed and nonlinearity of the scanning camera are calibrated with an adjustable delay device and an etalon. The system achieves a time resolution of 6.5 ps and a non-linearity of 2.60%. The measurable time range is from a dozen picoseconds to tens of nanoseconds. Fluorescence lifetime of two standard fluorescent dyes Rhodamine 6G and Coumarin 314 was measured and the experimental results were consistent with the references.