Imaging intracellular protein dynamics by spinning disk confocal microscopy
Stehbens, Samantha, Pemble, Hayley, Murrow, Lyndsay, & Wittmann, Torsten (2012) Imaging intracellular protein dynamics by spinning disk confocal microscopy. Methods in Enzymology, 504, pp. 293-313.
The palette of fluorescent proteins (FPs) has grown exponentially over the past decade, and as a result, live imaging of cells expressing fluorescently tagged proteins is becoming more and more mainstream. Spinning disk confocal (SDC) microscopy is a high-speed optical sectioning technique and a method of choice to observe and analyze intracellular FP dynamics at high spatial and temporal resolution. In an SDC system, a rapidly rotating pinhole disk generates thousands of points of light that scan the specimen simultaneously, which allows direct capture of the confocal image with low-noise scientific grade-cooled charge-coupled device cameras, and can achieve frame rates of up to 1000 frames per second. In this chapter, we describe important components of a state-of-the-art spinning disk system optimized for live cell microscopy and provide a rationale for specific design choices. We also give guidelines of how other imaging techniques such as total internal reflection microscopy or spatially controlled photoactivation can be coupled with SDC imaging and provide a short protocol on how to generate cell lines stably expressing fluorescently tagged proteins by lentivirus-mediated transduction.
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|Item Type:||Journal Article|
|Keywords:||spinning disk confocal microscopy, live cell imaging, mammalian cell culture, lentivirus, flourescence microscopy, flourescent protiens|
|Subjects:||Australian and New Zealand Standard Research Classification > TECHNOLOGY (100000) > MEDICAL BIOTECHNOLOGY (100400)|
|Divisions:||Current > Schools > School of Biomedical Sciences
Current > QUT Faculties and Divisions > Faculty of Health
Current > Institutes > Institute of Health and Biomedical Innovation
|Deposited On:||01 Dec 2014 23:36|
|Last Modified:||03 Dec 2014 01:49|
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