Laboratory of Biophysics of Synaptic Processes, Kazan Institute of Biochemistry and Biophysics, FRC Kazan Scientific Center of RAS, 420111Kazan, Russia.
Department of Radiophotonics and Microwave Technologies, Kazan National Research Technical University named after A.N. Tupolev, 420111Kazan, Russia.
Microsc Microanal. 2020 Apr;26(2):204-210. doi: 10.1017/S1431927620000148.
Here, we describe a method of acquisition of fast fluorescent signals with the help of the laser scanning confocal microscope (LSCM). Our method permits an increase in the temporal resolution of acquired signals. The method is based on LSCM recordings of fast fluorescent signals with the shortest achievable time sweep, which are performed with the help of a proprietary algorithm. A series of recordings is made in multiple steps; at each step, the fluorescent signal is incremented by a time interval smaller than the time sweep of the frame of LSCM. The size of the increment determines the achievable time resolution. The convolution of the recorded images results in a signal with the temporal resolution determined by the chosen time increment. This method was applied to register the change in fluorescence (calcium transient) of calcium dye preloaded into peripheral nerve endings by electrical stimulation of the motor nerve. Calculated parameters of the calcium transient were identical to the parameters obtained earlier with the help of a high-speed camera and photodiode. We conclude that the method described here can be applied for the registration of fast fluorescent signals by LSCM with a high spatial and temporal resolution.
在这里，我们描述了一种借助激光扫描共聚焦显微镜（LSCM）获取快速荧光信号的方法。我们的方法可以提高获取信号的时间分辨率。该方法基于 LSCM 以最短可实现的时间扫描记录快速荧光信号，这是通过专有算法完成的。一系列记录分多个步骤进行；在每个步骤中，荧光信号增加一个小于 LSCM 帧时间扫描的时间间隔。增量的大小决定了可实现的时间分辨率。记录的图像的卷积导致具有由所选时间增量确定的时间分辨率的信号。该方法应用于通过刺激运动神经来记录预先加载到周围神经末梢的钙染料的荧光（钙瞬变）的变化。钙瞬变的计算参数与使用高速相机和光电二极管获得的参数相同。我们得出的结论是，这里描述的方法可用于通过 LSCM 以高空间和时间分辨率记录快速荧光信号。
