Three-dimensional, tomographic super-resolution fluorescence imaging of serially sectioned thick samples.

Three-dimensional, tomographic super-resolution fluorescence imaging of serially sectioned thick samples.
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DOI:
10.1371/journal.pone.0038098
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发表时间:
2012
期刊:
影响因子:
3.7
通讯作者:
Kuner T
Kuner T
中科院分区:
综合性期刊3区
文献类型:
--
作者:
Nanguneri S;Flottmann B;Horstmann H;Heilemann M;Kuner T

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具有近分子分辨率的厚组织样品的三维荧光成像仍然是生命科学中的基本挑战。为了解决这个问题,我们开发了tomoSTORM,这是一种将基于单分子定位的超分辨率显微镜与结构完整脑组织的阵列断层扫描相结合的方法。组织成带状的连续切片连续成像,横向分辨率为28 nm,轴向分辨率为40 nm,组织体积高达50 µm×50 µm×2.5 µm。使用有针对性的表达膜结合(m)GFP和免疫组化在萼的举行,模型突触中枢神经传递,我们划定了膜和线粒体的精细结构的过程。该方法允许在大组织体积中进行多路复用超分辨率成像,其分辨率比共聚焦显微镜好三个数量级。
Three-dimensional fluorescence imaging of thick tissue samples with near-molecular resolution remains a fundamental challenge in the life sciences. To tackle this, we developed tomoSTORM, an approach combining single-molecule localization-based super-resolution microscopy with array tomography of structurally intact brain tissue. Consecutive sections organized in a ribbon were serially imaged with a lateral resolution of 28 nm and an axial resolution of 40 nm in tissue volumes of up to 50 µm×50 µm×2.5 µm. Using targeted expression of membrane bound (m)GFP and immunohistochemistry at the calyx of Held, a model synapse for central glutamatergic neurotransmission, we delineated the course of the membrane and fine-structure of mitochondria. This method allows multiplexed super-resolution imaging in large tissue volumes with a resolution three orders of magnitude better than confocal microscopy.
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