Tomographic STED microscopy to study bone resorption

Takahiro Deguchi; Sami V. Koho; Tuomas Näreoja; Juha Peltonen; Pekka Hänninen

doi:10.1117/12.2079157

9 March 2015 Tomographic STED microscopy to study bone resorption

Takahiro Deguchi, Sami V. Koho, Tuomas Näreoja, Juha Peltonen, Pekka Hänninen

Proceedings Volume 9330, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XXII; 93301M (2015) https://doi.org/10.1117/12.2079157
Event: SPIE BiOS, 2015, San Francisco, California, United States

Abstract

We present a tomographic Stimulated Emission Depletion (STED) microscopy method with three-dimensional superresolution, and its application to osteoclast bone resorption study. In order to improve axial resolution in standard STED system by tomography, two axial projections were obtained by imaging a sample at two different angles; one conventionally from below and another from the side. The second observation was acquired via a metal-coated silicon mirror, positioned above the region of interest by a custom-built micro-positioner. The acquired two sets of 3D stacks were computationally registered and fused, with our own in-house-developed software, to produce a 3D tomogram with three-dimensional super-resolution. With the presented tomographic super-resolution method we optically investigated actin cytoskeleton through thin and smooth bone layer, particularly at ruffled boarders (RB), which are directly associated with active bone resorption in osteoclasts. Tomographic STED microscopy at RB of osteoclast, cultured on thin bone layer, demonstrated axial resolution of approx. 210 nm, revealing fine axial structures of actin cytoskeleton at RB. Further investigation of the cytoskeleton at RB in relation with associated proteins would provide understanding in the protein roles during the bone resorption.

Citation Download Citation

Takahiro Deguchi, Sami V. Koho, Tuomas Näreoja, Juha Peltonen, and Pekka Hänninen "Tomographic STED microscopy to study bone resorption", Proc. SPIE 9330, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XXII, 93301M (9 March 2015); https://doi.org/10.1117/12.2079157

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