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首页> 外文期刊>Biomedical Optics Express >Methanol immersion reduces spherical aberration of water dipping lenses at long wavelengths used in multi-photon laser scanning microscopy
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Methanol immersion reduces spherical aberration of water dipping lenses at long wavelengths used in multi-photon laser scanning microscopy

机译:甲醇浸没可降低多光子激光扫描显微镜中使用的长波长水浸透镜的球差

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摘要

Dipping objectives were tested for multi-photon laser scanning microscopy, since their large working distances are advantageous for thick specimens and the absence of a coverslip facilitates examination of living material. Images of fluorescent bead specimens, particularly at wavelengths greater than 850 nm showed defects consistent with spherical aberration. Substituting methanol for water as the immersion medium surrounding the beads corrected these defects and produced an increase in fluorescence signal intensity. The same immersion method was applied to two representative biological samples of fixed tissue: mouse brain labeled with FITC for tubulin and mouse gut in which the Peyer’s patches were labeled with Texas Red bilosomes. Tissue morphology was well preserved by methanol immersion of both tissues; the two-photon-excited fluorescence signal was six times higher than in water and the depth of penetration of useful imaging was doubled. No modification of the microscope was needed except the provision of a ring to retain a sufficient depth of methanol for imaging.
机译:浸渍物镜已通过多光子激光扫描显微镜进行了测试,因为其较大的工作距离对于厚样品是有利的,并且没有盖玻片有利于活体材料的检查。荧光珠标本的图像,特别是在大于850 nm的波长处,显示出与球差一致的缺陷。用甲醇代替水作为珠子周围的浸没介质可以纠正这些缺陷,并增加荧光信号强度。将相同的浸没方法应用于固定组织的两个代表性生物学样品:用FITC标记的微管蛋白小鼠脑和其中Peyer贴片标记有Texas Red Bilosomes的小鼠肠。甲醇浸入两个组织后,组织形态得到了很好的保留。双光子激发的荧光信号比水中高六倍,有用成像的穿透深度加倍。除了提供环以保留足够的甲醇深度以进行成像外,无需修改显微镜。

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