Chapter 15
Practical Aspects of Super-Resolution Imaging
and Segmentation of Macromolecular Complexes
by dSTORM
Leonid Andronov, Jean-Luc Vonesch, and Bruno P. Klaholz
Abstract
Super-resolution fluorescence microscopy allows imaging macromolecular complexes down to the nanoscopic scale and thus is a great tool to combine and integrate cellular imaging in the native cellular
environment with structural analysis by X-ray crystallography or high-resolution cryo electron microscopy
or tomography. Here we describe practical aspects of SMLM imaging by dSTORM, from the initial sample
preparation using mounting media, antibodies and fluorescent markers, the experimental setup for data
acquisition including multi-color colocalization and 3D data acquisition, and finally tips and clues on
advanced data processing that includes image reconstruction and data segmentation using 2D or 3D
clustering methods. This approach opens the path toward multi-resolution integration in cellular structural
biology.
Key words Super-resolution microscopy, dSTORM, SMLM, Immunofluorescence, Fluorescence
microscopy
1 Introduction
Fluorescence microscopy is a key technique for the specific observation of proteins in their native cellular environment. Unfortunately, the resolution of fluorescence microscopy is limited by the
wavelength of light to around 200–300 nm, which limits the
studies to the scale of cellular organelles. The recently emerged
super-resolution fluorescence microscopy allows to overcome this
limit and improves the resolution by about one order of magnitude,
i.e., down to the nanoscopic scale of large macromolecular complexes. Super-resolution microscopy is therefore a powerful technique that bridges the gap between high-resolution
crystallographic and cryo electron microscopy (cryo-EM) structures of proteins and their functions in the cellular context [1].
Among the super-resolution techniques, single-molecule localization microscopy (SMLM), which includes techniques such as
Arnaud Poterszman (ed.), Multiprotein Complexes: Methods and Protocols, Methods in Molecular Biology, vol. 2247,
https://doi.org/10.1007/978-1-0716-1126-5_15, © Springer Science+Business Media, LLC, part of Springer Nature 2021
271
Practical Aspects of Super-Resolution Imaging
and Segmentation of Macromolecular Complexes
by dSTORM
Leonid Andronov, Jean-Luc Vonesch, and Bruno P. Klaholz
Abstract
Super-resolution fluorescence microscopy allows imaging macromolecular complexes down to the nanoscopic scale and thus is a great tool to combine and integrate cellular imaging in the native cellular
environment with structural analysis by X-ray crystallography or high-resolution cryo electron microscopy
or tomography. Here we describe practical aspects of SMLM imaging by dSTORM, from the initial sample
preparation using mounting media, antibodies and fluorescent markers, the experimental setup for data
acquisition including multi-color colocalization and 3D data acquisition, and finally tips and clues on
advanced data processing that includes image reconstruction and data segmentation using 2D or 3D
clustering methods. This approach opens the path toward multi-resolution integration in cellular structural
biology.
Key words Super-resolution microscopy, dSTORM, SMLM, Immunofluorescence, Fluorescence
microscopy
1 Introduction
Fluorescence microscopy is a key technique for the specific observation of proteins in their native cellular environment. Unfortunately, the resolution of fluorescence microscopy is limited by the
wavelength of light to around 200–300 nm, which limits the
studies to the scale of cellular organelles. The recently emerged
super-resolution fluorescence microscopy allows to overcome this
limit and improves the resolution by about one order of magnitude,
i.e., down to the nanoscopic scale of large macromolecular complexes. Super-resolution microscopy is therefore a powerful technique that bridges the gap between high-resolution
crystallographic and cryo electron microscopy (cryo-EM) structures of proteins and their functions in the cellular context [1].
Among the super-resolution techniques, single-molecule localization microscopy (SMLM), which includes techniques such as
Arnaud Poterszman (ed.), Multiprotein Complexes: Methods and Protocols, Methods in Molecular Biology, vol. 2247,
https://doi.org/10.1007/978-1-0716-1126-5_15, © Springer Science+Business Media, LLC, part of Springer Nature 2021
271