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A simple, straightforward correlative live‐cell‐imaging‐structured‐illumination‐microscopy approach for studying organelle dynamics
Author(s) -
Sherman Shachar,
Nachmias Dikla,
Elia Natalie
Publication year - 2015
Publication title -
microscopy research and technique
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 118
eISSN - 1097-0029
pISSN - 1059-910X
DOI - 10.1002/jemt.22540
Subject(s) - organelle , live cell imaging , microscopy , golgi apparatus , super resolution microscopy , biological system , resolution (logic) , sted microscopy , fluorescence microscope , nanotechnology , biophysics , biology , cell , computer science , physics , microbiology and biotechnology , superresolution , materials science , fluorescence , optics , artificial intelligence , scanning confocal electron microscopy , image (mathematics) , genetics
Most cellular organelles are highly dynamic and continuously undergo membrane fission and fusion to mediate their function. Documenting organelle dynamics under physiological conditions, therefore, requires high temporal resolution of the recording system. Concurrently, these structures are relatively small and determining their substructural organization is often impossible using conventional microscopy. Structured Illumination Microscopy (SIM) is a super resolution technique providing a two‐fold increase in resolution. Importantly, SIM is versatile because it allows the use of any fluorescent dye or protein and, hence, is highly applicable for cell biology. However, similar to other SR techniques, the applicability of SIM to high‐speed live cell imaging is limited. Here we present an easy, straightforward methodology for coupling of high‐speed live cell recordings, using spinning disk (SD) microscopy, with SIM. Using this simple methodology, we are able to track individual mitochondrial membrane fission and fusion events in real time and to determine the network connectivity and substructural organization of the membrane at high resolution. Applying this methodology to other cellular organelles such as, ER, golgi, and cilia will no doubt contribute to our understanding of membrane dynamics in cells. Microsc. Res. Tech. 78:777–783, 2015 . © 2015 Wiley Periodicals, Inc.