Three-dimensional, tomographic super-resolution fluorescence imaging of serially sectioned thick samples.
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.
Institute of Anatomy and Cell Biology, Heidelberg University, Heidelberg, Germany.
Siddharth
S
Nanguneri
Benjamin
B
Flottmann
Heinz
H
Horstmann
Mike
M
Heilemann
Thomas
T
Kuner
D019412:Anatomy, Cross-Sectional / Q000379:methods*; D000818:Animals; D001921:Brain / Q000166:cytology / Q000648:ultrastructure; D021621:Imaging, Three-Dimensional / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D008928:Mitochondria / Q000648:ultrastructure; D051381:Rats; D017207:Rats, Sprague-Dawley; D013569:Synapses / Q000648:ultrastructure; D014054:Tomography / Q000379:methods*