Becker Wolfgang, Shcheslavskiy Vladislav, Rück Angelika
Becker & Hickl GmbH, Berlin, Germany.
Adv Exp Med Biol. 2017;1035:19-30. doi: 10.1007/978-3-319-67358-5_2.
TCSPC FLIM/PLIM is based on a multi-dimensional time-correlated single-photon counting process. The sample is scanned by a high-frequency-pulsed laser beam which is additionally modulated on/off synchronously with the pixels of the scan. FLIM is obtained by building up the distribution of the photons over the scanning coordinates and the times of the photons in the excitation pulse sequence, PLIM is obtained by building up the photon distribution over the scanning coordinates and the photon times in the modulation period. FLIM and PLIM data are thus obtained simultaneously within the same imaging process. Since the technique uses not only one but many excitation pulses for every phosphorescence signal period the sensitivity is much higher than for techniques that excite with a single pulse only. TCSPC FLIM/PLIM works both with one-photon and two-photon excitation, does not require a reduction of the laser pulse repetition rate by a pulse picker, and eliminates the need of high pulse energy for phosphorescence excitation.
时间相关单光子计数荧光寿命成像/磷光寿命成像（TCSPC FLIM/PLIM）基于多维时间相关单光子计数过程。样品由高频脉冲激光束扫描，该激光束与扫描像素同步进行额外的开/关调制。通过建立光子在扫描坐标上的分布以及光子在激发脉冲序列中的时间分布来获得荧光寿命成像（FLIM），通过建立光子在扫描坐标上的分布以及光子在调制周期内的时间分布来获得磷光寿命成像（PLIM）。因此，在同一成像过程中可同时获得FLIM和PLIM数据。由于该技术在每个磷光信号周期使用多个而非单个激发脉冲，其灵敏度远高于仅用单个脉冲激发的技术。TCSPC FLIM/PLIM可用于单光子和双光子激发，无需通过脉冲选择器降低激光脉冲重复率，且无需高脉冲能量进行磷光激发。
