A simple tissue clearing method for increasing the depth penetration of STED microscopy of fixed brain slices
A simple tissue clearing method for increasing the depth penetration of STED microscopy of fixed brain slices
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DOI：
10.1088/1361-6463/ab6f1b
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发表时间：
2020-04-29
影响因子：
3.4
通讯作者：
Nagerl, U. Valentin
中科院分区：
文献类型：
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作者：
Angibaud, Julie;Mascalchi, Patrice;Nagerl, U. Valentin
STED显微镜已被世界各地的许多研究实验室和成像核心设施采用。它原则上为生物学家提供了比传统光学显微镜更高的空间分辨率，以揭示衍射屏障之外细胞的形态和分子细节。然而，在存在光学像差的情况下，STED用户通常很难获得这一优势，这会严重降低显微镜的性能。一个典型的罪魁祸首是STED显微镜的光学器件和生物样本之间的折射率不匹配，这通常会引起球面像差。不可避免地，它们涂抹出激发和STED激光器的焦点强度分布，并且这个问题随着成像深度而急剧增加。这意味着STED显微镜通常被限制在距盖玻片10 μ m以下的距离。然而，对于许多神经生物学问题，它是必不可少的图像更深入到组织，以避免边缘伪影，并提高数据throughput.Seeking，以减少这种球面像差和提高STED显微镜的深度穿透，我们检查了包埋介质CFM 3，它具有相同的折射率油，并被称为有一个快速的生物组织的清除效果。我们评估了CFM 3的STED成像超出直接表面的生物样品，并将其与常见的包埋介质Mowlets进行比较，使用荧光珠和树突棘在固定的脑切片作为测试samples.We表明，CFM 3使得有可能正确地解决树突棘在深度约40 μ m与标准的商业STED显微镜，不像Mowlets，其中的空间分辨率严重下降。由于CFM 3提供了一种经济而简单的方法来减少折射率失配以及光学透明脑组织而不扭曲组织的微结构，我们建议将其用作脑切片深处超分辨率成像的包埋介质。
STED microscopy has been adopted by many research laboratories and imaging core facilities around the world. It offers biologists in principle a much higher spatial resolution than conventional light microscopy to reveal morphological and molecular details of cells beyond the diffraction barrier. However, this advantage is oftentimes hard for STED users to attain in the presence of optical aberrations, which can severely degrade microscope performance.A classic culprit is the mismatch in refractive index between the optics of the STED microscope and the biological specimen, which normally induces spherical aberrations. Inevitably, they smear out the focal intensity distributions of the excitation and STED lasers, and this problem increases dramatically with imaging depth. This means that STED microscopy has typically been restricted to distances of under 10 mu m from the cover slip. However, for many neurobiological questions it is essential to image deeper into the tissue to avoid edge artifacts and to increase data throughput.Seeking to reduce such spherical aberrations and to improve the depth penetration of STED microscopy, we examined the embedding medium CFM3, which has the same refractive index as oil and is known to have a rapid clearing effect on biological tissue. We assessed CFM3 for STED imaging beyond the immediate surface of the biological sample and compared it with the common embedding medium Mowiol, using fluorescent beads and dendritic spines in fixed brain slices as test samples.We show that CFM3 makes it possible to properly resolve dendritic spines at a depth of around 40 mu m with a standard commercial STED microscope, unlike Mowiol, where the spatial resolution is severely degraded. As CFM3 provides an economical and easy way to reduce the refractive index mismatch as well as to optically clear brain tissue without distorting the micro-architecture of the tissue, we recommend its use as embedding medium for super-resolution imaging deep inside brain slices.