通过时间选通改善脑组织中活神经元的双光子成像。
Improved two-photon imaging of living neurons in brain tissue through temporal gating.
作者信息
Gautam Vini, Drury Jack, Choy Julian M C, Stricker Christian, Bachor Hans-A, Daria Vincent R
机构信息
John Curtin School of Medical Research, The Australian National University, Canberra, ACT 2601, Australia.
John Curtin School of Medical Research, The Australian National University, Canberra, ACT 2601, Australia ; Medical School, The Australian National University, Canberra, ACT 2601, Australia.
出版信息
Biomed Opt Express. 2015 Sep 17;6(10):4027-36. doi: 10.1364/BOE.6.004027. eCollection 2015 Oct 1.
We optimize two-photon imaging of living neurons in brain tissue by temporally gating an incident laser to reduce the photon flux while optimizing the maximum fluorescence signal from the acquired images. Temporal gating produces a bunch of ~10 femtosecond pulses and the fluorescence signal is improved by increasing the bunch-pulse energy. Gating is achieved using an acousto-optic modulator with a variable gating frequency determined as integral multiples of the imaging sampling frequency. We hypothesize that reducing the photon flux minimizes the photo-damage to the cells. Our results, however, show that despite producing a high fluorescence signal, cell viability is compromised when the gating and sampling frequencies are equal (or effectively one bunch-pulse per pixel). We found an optimum gating frequency range that maintains the viability of the cells while preserving a pre-set fluorescence signal of the acquired two-photon images. The neurons are imaged while under whole-cell patch, and the cell viability is monitored as a change in the membrane's input resistance.
我们通过对入射激光进行时间选通来优化脑组织中活神经元的双光子成像，以减少光子通量，同时优化从采集图像中获得的最大荧光信号。时间选通产生一束约10飞秒的脉冲，并且通过增加束脉冲能量来改善荧光信号。使用声光调制器实现选通，其可变选通频率被确定为成像采样频率的整数倍。我们假设降低光子通量可使对细胞的光损伤最小化。然而，我们的结果表明，尽管产生了高荧光信号，但当选通频率和采样频率相等时（或实际上每个像素一个束脉冲），细胞活力会受到损害。我们发现了一个最佳选通频率范围，该范围在保持采集的双光子图像预设荧光信号的同时维持细胞的活力。在全细胞膜片钳状态下对神经元进行成像，并将细胞膜输入电阻的变化作为细胞活力的监测指标。
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