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Proceedings Paper

Effect of labeling density and time post labeling on quality of antibody-based super resolution microscopy images
Author(s): Amy M. Bittel; Isaac Saldivar; Nicholas Dolman; Andrew K. Nickerson; Li-Jung Lin; Xiaolin Nan; Summer L. Gibbs
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Paper Abstract

Super resolution microscopy (SRM) has overcome the historic spatial resolution limit of light microscopy, enabling fluorescence visualization of intracellular structures and multi-protein complexes at the nanometer scale. Using single-molecule localization microscopy, the precise location of a stochastically activated population of photoswitchable fluorophores is determined during the collection of many images to form a single image with resolution of ~10-20 nm, an order of magnitude improvement over conventional microscopy. One of the key factors in achieving such resolution with single-molecule SRM is the ability to accurately locate each fluorophore while it emits photons. Image quality is also related to appropriate labeling density of the entity of interest within the sample. While ease of detection improves as entities are labeled with more fluorophores and have increased fluorescence signal, there is potential to reduce localization precision, and hence resolution, with an increased number of fluorophores that are on at the same time in the same relative vicinity. In the current work, fixed microtubules were antibody labeled using secondary antibodies prepared with a range of Alexa Fluor 647 conjugation ratios to compare image quality of microtubules to the fluorophore labeling density. It was found that image quality changed with both the fluorophore labeling density and time between completion of labeling and performance of imaging study, with certain fluorophore to protein ratios giving optimal imaging results.

Paper Details

Date Published: 9 March 2015
PDF: 5 pages
Proc. SPIE 9331, Single Molecule Spectroscopy and Superresolution Imaging VIII, 93310M (9 March 2015); doi: 10.1117/12.2083209
Show Author Affiliations
Amy M. Bittel, Oregon Health & Science Univ. (United States)
Isaac Saldivar, Oregon Health & Science Univ. (United States)
Nicholas Dolman, Thermo Fisher Scientific (United States)
Andrew K. Nickerson, Oregon Health & Science Univ. (United States)
Li-Jung Lin, Oregon Health & Science Univ. (United States)
Xiaolin Nan, Oregon Health & Science Univ. (United States)
Summer L. Gibbs, Oregon Health & Science Univ. (United States)

Published in SPIE Proceedings Vol. 9331:
Single Molecule Spectroscopy and Superresolution Imaging VIII
Jörg Enderlein; Ingo Gregor; Zygmunt Karol Gryczynski; Rainer Erdmann; Felix Koberling, Editor(s)

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