About samples, giving examples: Optimized Single Molecule Localization Microscopy

Autor: Karoline Friedl, Angélique Jimenez, Christophe Leterrier
Přispěvatelé: Institut de neurophysiopathologie (INP), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)
Jazyk: angličtina
Rok vydání: 2019
Předmět:
Single molecule localization
Macromolecular Substances
Computer science
[SDV.NEU.NB]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology
STORM
Color
02 engineering and technology
General Biochemistry
Genetics and Molecular Biology
Cell Line
03 medical and health sciences
Imaging
Three-Dimensional
Chlorocebus aethiops
Microscopy
Image Processing
Computer-Assisted
Animals
Humans
Nanotechnology
Super-resolution microscopy
SMLM
Molecular Biology
Cytoskeleton
Fluorescent Dyes
030304 developmental biology
0303 health sciences
Sample fixation
030302 biochemistry & molecular biology
Resolution (electron density)
DNA
021001 nanoscience & nanotechnology
Immunohistochemistry
Single Molecule Imaging
Optical reconstruction
Microscopy
Fluorescence
Macromolecular Complexes
0210 nano-technology
Biological system
DNA-PAINT
Zdroj: Methods
Methods, 2019, ⟨10.1016/j.ymeth.2019.05.008⟩
Methods, Elsevier, 2019, ⟨10.1016/j.ymeth.2019.05.008⟩
ISSN: 1046-2023
1095-9130
DOI: 10.1101/568295
Popis: Super-resolution microscopy has profoundly transformed how we study the architecture of cells, revealing unknown structures and refining our view of cellular assemblies. Among the various techniques, the resolution of Single Molecule Localization Microscopy (SMLM) can reach the size of macromolecular complexes and offer key insights on their nanoscale arrangement in situ. SMLM is thus a demanding technique and taking advantage of its full potential requires specifically optimized procedures. Here we describe how we perform the successive steps of an SMLM workflow, focusing on single-color Stochastic Optical Reconstruction Microscopy (STORM) as well as multicolor DNA Points Accumulation for imaging in Nanoscale Topography (DNA-PAINT) of fixed samples. We provide detailed procedures for careful sample fixation and immunostaining of typical cellular structures: cytoskeleton, clathrin-coated pits, and organelles. We then offer guidelines for optimal imaging and processing of SMLM data in order to optimize reconstruction quality and avoid the generation of artifacts. We hope that the tips and tricks we discovered over the years and detail here will be useful for researchers looking to make the best possible SMLM images, a pre-requisite for meaningful biological discovery.
Databáze: OpenAIRE
Externí odkaz: