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PLoS One. 2013 Oct 9;8(10):e74604. doi: 10.1371/journal.pone.0074604. eCollection 2013.

Super-resolution imaging strategies for cell biologists using a spinning disk microscope.

Author information

1
Blizard Institute, Barts and the Royal London School of Medicine and Dentistry, Queen Mary University London, London, United Kingdom.

Erratum in

  • PLoS One. 2013;8(12). doi:10.1371/annotation/d96769fb-4e7d-4f47-98cf-443447c1471e.

Abstract

In this study we use a spinning disk confocal microscope (SD) to generate super-resolution images of multiple cellular features from any plane in the cell. We obtain super-resolution images by using stochastic intensity fluctuations of biological probes, combining Photoactivation Light-Microscopy (PALM)/Stochastic Optical Reconstruction Microscopy (STORM) methodologies. We compared different image analysis algorithms for processing super-resolution data to identify the most suitable for analysis of particular cell structures. SOFI was chosen for X and Y and was able to achieve a resolution of ca. 80 nm; however higher resolution was possible >30 nm, dependant on the super-resolution image analysis algorithm used. Our method uses low laser power and fluorescent probes which are available either commercially or through the scientific community, and therefore it is gentle enough for biological imaging. Through comparative studies with structured illumination microscopy (SIM) and widefield epifluorescence imaging we identified that our methodology was advantageous for imaging cellular structures which are not immediately at the cell-substrate interface, which include the nuclear architecture and mitochondria. We have shown that it was possible to obtain two coloured images, which highlights the potential this technique has for high-content screening, imaging of multiple epitopes and live cell imaging.

PMID:
24130668
PMCID:
PMC3793996
DOI:
10.1371/journal.pone.0074604
[Indexed for MEDLINE]
Free PMC Article

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