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 Gibbs

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

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.

Original languageEnglish (US)
Title of host publicationSingle Molecule Spectroscopy and Superresolution Imaging VIII
PublisherSPIE
Volume9331
ISBN (Print)9781628414219
DOIs
StatePublished - 2015
EventSingle Molecule Spectroscopy and Superresolution Imaging VIII - San Francisco, United States
Duration: Feb 7 2015Feb 8 2015

Other

OtherSingle Molecule Spectroscopy and Superresolution Imaging VIII
CountryUnited States
CitySan Francisco
Period2/7/152/8/15

Fingerprint

Fluorophores
antibodies
Antibodies
Labeling
marking
Microscopy
Microscopic examination
microscopy
Microtubules
Image quality
Photons
Fluorescence Microscopy
Proteins
proteins
Fluorescence
fluorescence
Light
conjugation
Imaging techniques
Molecules

Keywords

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

ASJC Scopus subject areas

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

Cite this

Bittel, A. M., Saldivar, I., Dolman, N., Nickerson, A. K., Lin, L. J., Nan, X., & Gibbs, S. (2015). Effect of labeling density and time post labeling on quality of antibody-based super resolution microscopy images. In Single Molecule Spectroscopy and Superresolution Imaging VIII (Vol. 9331). [93310M] SPIE. https://doi.org/10.1117/12.2083209

Effect of labeling density and time post labeling on quality of antibody-based super resolution microscopy images. / Bittel, Amy M.; Saldivar, Isaac; Dolman, Nicholas; Nickerson, Andrew K.; Lin, Li Jung; Nan, Xiaolin; Gibbs, Summer.

Single Molecule Spectroscopy and Superresolution Imaging VIII. Vol. 9331 SPIE, 2015. 93310M.

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

Bittel, AM, Saldivar, I, Dolman, N, Nickerson, AK, Lin, LJ, Nan, X & Gibbs, S 2015, Effect of labeling density and time post labeling on quality of antibody-based super resolution microscopy images. in Single Molecule Spectroscopy and Superresolution Imaging VIII. vol. 9331, 93310M, SPIE, Single Molecule Spectroscopy and Superresolution Imaging VIII, San Francisco, United States, 2/7/15. https://doi.org/10.1117/12.2083209
Bittel AM, Saldivar I, Dolman N, Nickerson AK, Lin LJ, Nan X et al. Effect of labeling density and time post labeling on quality of antibody-based super resolution microscopy images. In Single Molecule Spectroscopy and Superresolution Imaging VIII. Vol. 9331. SPIE. 2015. 93310M https://doi.org/10.1117/12.2083209
Bittel, Amy M. ; Saldivar, Isaac ; Dolman, Nicholas ; Nickerson, Andrew K. ; Lin, Li Jung ; Nan, Xiaolin ; Gibbs, Summer. / Effect of labeling density and time post labeling on quality of antibody-based super resolution microscopy images. Single Molecule Spectroscopy and Superresolution Imaging VIII. Vol. 9331 SPIE, 2015.
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