Application of multiline two-photon microscopy to functional in vivo imaging
Application of multiline two-photon microscopy to functional in vivo imaging
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DOI：
10.1016/j.jneumeth.2005.12.003
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发表时间：
2006-03-15
影响因子：
3
通讯作者：
Sauer, M
中科院分区：
文献类型：
--
作者：
Kurtz, R;Fricke, M;Sauer, M
高空间分辨率和低光损伤风险使双光子激光扫描显微镜 (TPLSM) 成为生物成像的首选方法。然而，神经元钙调节等功能动力学的研究通常也需要高时间分辨率。迄今为止，采集速度通常通过线扫描来提高，这限制了沿单轴结构的空间分辨率。为了克服高空间分辨率和高时间分辨率之间的差距，我们使用光束复用器执行 TPLSM，以在样品内生成多个激光焦点。通过用电子倍增相机检测这些激光焦点发出的荧光，可以同时执行多个线扫描。除了多线扫描之外，最多 64 束激光束的阵列还可用于 x-y 扫描模式，以高帧速率收集整个图像。为了评估多线 TPLSM 在功能性体内成像中的适用性，对果蝇大脑中的视觉运动敏感神经元中的钙信号进行了监测。我们的方法同时采集不同细胞位置信号的能力通过对分支神经突和“脊柱”样结构的测量得到了例证。钙动态取决于分支大小，但“刺”与其“母神经突”并没有系统性的差异。通过与共焦显微镜进行比较，对我们的装置的空间分辨率进行了严格的评估，并且通过在成像和点扫描模式下运行装置来直接评估图像检测期间发射光散射的负面影响。 (c) 2005 Elsevier B.V. 保留所有权利。
High spatial resolution and low risks of photodamage make two-photon laser-scanning microscopy (TPLSM) the method of choice for biological imaging. However, the study of functional dynamics such as neuronal calcium regulation often also requires a high temporal resolution. Hitherto, acquisition speed is usually increased by line scanning, which restricts spatial resolution to structures along a single axis. To overcome this gap between high spatial and high temporal resolution we performed TPLSM with a beam multiplexer to generate multiple laser foci inside the sample. By detecting the fluorescence emitted from these laser foci with an electron-multiplying camera, it was possible to perform multiple simultaneous linescans. In addition to multiline scanning, the array of up to 64 laser beams could also be used in x-y scan mode to collect entire images at high frame rates. To evaluate the applicability of multiline TPLSM to functional in vivo imaging, calcium signals were monitored in visual motionsensitive neurons in the brain of flies. The capacity of our method to simultaneously acquire signals at different cellular locations is exemplified by measurements at branched neurites and 'spine'-like structures. Calcium dynamics depended on branch size, but 'spines' did not systematically differ from their 'parent neurites'. The spatial resolution of our setup was critically evaluated by comparing it to confocal microscopy and the negative effect of scattering of emission light during image detection was assessed directly by running the setup in both imaging and point-scanning mode. (c) 2005 Elsevier B.V. All rights reserved.