All-optical histology using ultrashort laser pulses
All-optical histology using ultrashort laser pulses
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DOI：
10.1016/s0896-6273(03)00370-2
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发表时间：
2003-07-03
期刊：
影响因子：
16.2
通讯作者：
Kleinfeld, D
中科院分区：
文献类型：
--
作者：
Tsai, PS;Friedman, B;Kleinfeld, D
作为一种手段，以自动化的脑组织的三维组织学分析，我们演示了使用飞秒激光脉冲迭代切割和图像固定以及新鲜组织。用1至10 μ J脉冲完成切割，以微米精度消融组织。我们发现，脉冲激光切割后保留了组织的渗透性，免疫反应性和光学清晰度。此外，来自表达荧光蛋白的转基因小鼠的样品将其荧光保留在切割表面的微米内。外源性或内源性荧光标记的成像下至100 μ m或更多的切割表面下完成0.1至1 nJ的脉冲和常规的双光子激光扫描显微镜。在一个实例中，以微米分辨率可视化新皮质柱的整个范围内的标记投射神经元。在第二个例子中，测量新皮质块内的微血管并以微米分辨率重建。
As a means to automate the three-dimensional histological analysis of brain tissue, we demonstrate the use of femtosecond laser pulses to iteratively cut and image fixed as well as fresh tissue. Cuts are accomplished with 1 to 10 muJ pulses to ablate tissue with micron precision. We show that the permeability, immunoreactivity, and optical clarity of the tissue is retained after pulsed laser cutting. Further, samples from transgenic mice that express fluorescent proteins retained their fluorescence to within microns of the cut surface. Imaging of exogenous or endogenous fluorescent labels down to 100 mum or more below the cut surface is accomplished with 0.1 to 1 nJ pulses and conventional two-photon laser scanning microscopy. In one example, labeled projection neurons within the full extent of a neocortical column were visualized with micron resolution. In a second example, the microvasculature within a block of neocortex was measured and reconstructed with micron resolution.