High-speed, low-photodamage nonlinear imaging using passive pulse splitters
High-speed, low-photodamage nonlinear imaging using passive pulse splitters
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DOI：
10.1038/nmeth.1175
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发表时间：
2008-02-01
期刊：
影响因子：
48
通讯作者：
Betzig, Eric
中科院分区：
文献类型：
--
作者：
Ji, Na;Magee, Jeffrey C.;Betzig, Eric
脉冲激光器是非线性生物成像技术（如双光子荧光激发（TPE）显微镜）中的关键元件。然而，通常情况下，只有百分之一或更少的可用激光功率可以传递到样品之前，光致损伤变得过度。在这里，我们描述了一个被动的脉冲分裂器，每个激光脉冲转换成一个固定数量的子脉冲的相等的能量。我们将分裂器应用于标记有GFP的固定小鼠脑切片的TPE成像，并表明，在不同的功率制度中，分裂器可以用于增加信号速率超过100倍或减少光漂白速率超过四倍。在活体标本中，收益甚至更大：在秀丽隐杆线虫幼虫的体内成像过程中，光漂白减少了9倍，在大鼠海马脑切片的钙成像过程中，光损伤率减少了6至20倍。
Pulsed lasers are key elements in nonlinear bioimaging techniques such as two-photon fluorescence excitation (TPE) microscopy. Typically, however, only a percent or less of the laser power available can be delivered to the sample before photoinduced damage becomes excessive. Here we describe a passive pulse splitter that converts each laser pulse into a fixed number of sub-pulses of equal energy. We applied the splitter to TPE imaging of fixed mouse brain slices labeled with GFP and show that, in different power regimes, the splitter can be used either to increase the signal rate more than 100-fold or to reduce the rate of photobleaching by over fourfold. In living specimens, the gains were even greater: a ninefold reduction in photobleaching during in vivo imaging of Caenorhabditis elegans larvae, and a six- to 20-fold decrease in the rate of photodamage during calcium imaging of rat hippocampal brain slices.