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

PloS one (2012-06-05)
Siddharth Nanguneri, Benjamin Flottmann, Heinz Horstmann, Mike Heilemann, Thomas Kuner
ABSTRACT

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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Atto 520-Biotin, BioReagent, suitable for fluorescence, ≥95.0% (HPCE)
Supelco
Atto 520 NHS ester, BioReagent, suitable for fluorescence, ≥80.0% (coupling to amines)
Sigma-Aldrich
Atto 520 azide, suitable for fluorescence
Sigma-Aldrich
Anti-Cytochrome c antibody produced in sheep, ~0.5 mg/mL, affinity isolated antibody, buffered aqueous solution
Sigma-Aldrich
Atto 520 maleimide, for fluorescence, ≥90% (coupling rate)

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