Effect of labeling density and time post labeling on quality of antibody-based super resolution microscopy images

Amy M. Bittel, Isaac Saldivar, Nicholas Dolman, Andrew K. Nickerson, Li Jung Lin, Xiaolin Nan, Summer L. Gibbs

Research output: Chapter in Book/Report/Conference proceedingConference contribution


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.

Original languageEnglish (US)
Title of host publicationSingle Molecule Spectroscopy and Superresolution Imaging VIII
EditorsIngo Gregor, Zygmunt Karol Gryczynski, Felix Koberling, Jorg Enderlein, Rainer Erdmann
ISBN (Electronic)9781628414219
StatePublished - 2015
EventSingle Molecule Spectroscopy and Superresolution Imaging VIII - San Francisco, United States
Duration: Feb 7 2015Feb 8 2015

Publication series

NameProgress in Biomedical Optics and Imaging - Proceedings of SPIE
ISSN (Print)1605-7422


OtherSingle Molecule Spectroscopy and Superresolution Imaging VIII
Country/TerritoryUnited States
CitySan Francisco


  • photoswitching
  • single molecule localization microscopy
  • small molecule fluorophore
  • super resolution microscopy

ASJC Scopus subject areas

  • Electronic, Optical and Magnetic Materials
  • Biomaterials
  • Atomic and Molecular Physics, and Optics
  • Radiology Nuclear Medicine and imaging


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