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

doi:10.1117/12.2083209

9 March 2015 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

Author Affiliations +

Proceedings Volume 9331, Single Molecule Spectroscopy and Superresolution Imaging VIII; 93310M (2015) https://doi.org/10.1117/12.2083209
Event: SPIE BiOS, 2015, San Francisco, California, United States

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.

Citation Download Citation

Amy M. Bittel, Isaac Saldivar, Nicholas Dolman, Andrew K. Nickerson, Li-Jung Lin, Xiaolin Nan, and Summer L. Gibbs "Effect of labeling density and time post labeling on quality of antibody-based super resolution microscopy images", Proc. SPIE 9331, Single Molecule Spectroscopy and Superresolution Imaging VIII, 93310M (9 March 2015); https://doi.org/10.1117/12.2083209

ACCESS THE FULL ARTICLE

PURCHASE THIS CONTENT

SUBSCRIBE TO DIGITAL LIBRARY

50 downloads per 1-year subscription

Members: $195

Non-members: $335 ADD TO CART

25 downloads per 1 - year subscription

Members: $145

Non-members: $250 ADD TO CART

PURCHASE SINGLE ARTICLE

Includes PDF, HTML & Video, when available

Members: $17.00

Non-members: $21.00 ADD TO CART

PROCEEDINGS
5 PAGES

DOWNLOAD PAPER SAVE TO MY LIBRARY

GET CITATION

RIGHTS & PERMISSIONS

Get copyright permission

KEYWORDS

Proteins

Image quality

Signal detection

Molecules

Luminescence

Microscopy

Super resolution microscopy

Show All Keywords

Keywords/Phrases

Search In:

Publication Years