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

• Published in: PloS one 2012-05, Vol.7 (5), p.e38098-e38098
• Main Authors: Nanguneri, Siddharth, Flottmann, Benjamin, Horstmann, Heinz, Heilemann, Mike, Kuner, Thomas
• Format: Article
• Language: English
• Subjects: Anatomy, Cross-Sectional - methods; Animals; Biology; Brain; Brain - cytology; Brain - ultrastructure; Chemistry; Confocal; Confocal microscopy; Fluorescence; Geometry; Glass substrates; Glutamatergic transmission; Image resolution; Imaging, Three-Dimensional - methods; Immunohistochemistry; Localization; Medical imaging; Medicine; Methods; Microscopy; Microscopy, Fluorescence - methods; Mitochondria; Mitochondria - ultrastructure; Multiplexing; Neuroimaging; Neurotransmission; Physics; Proteins; Rats; Rats, Sprague-Dawley; Resins; Synapses; Synapses - ultrastructure; Tomography - methods; Topography
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0038098

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.