TY - GEN
T1 - Effect of labeling density and time post labeling on quality of antibody-based super resolution microscopy images
AU - Bittel, Amy M.
AU - Saldivar, Isaac
AU - Dolman, Nicholas
AU - Nickerson, Andrew K.
AU - Lin, Li Jung
AU - Nan, Xiaolin
AU - Gibbs, Summer L.
N1 - Publisher Copyright:
© 2015 SPIE.
PY - 2015
Y1 - 2015
N2 - 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.
AB - 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.
KW - photoswitching
KW - single molecule localization microscopy
KW - small molecule fluorophore
KW - super resolution microscopy
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UR - http://www.scopus.com/inward/citedby.url?scp=84930470682&partnerID=8YFLogxK
U2 - 10.1117/12.2083209
DO - 10.1117/12.2083209
M3 - Conference contribution
AN - SCOPUS:84930470682
T3 - Progress in Biomedical Optics and Imaging - Proceedings of SPIE
BT - Single Molecule Spectroscopy and Superresolution Imaging VIII
A2 - Gregor, Ingo
A2 - Gryczynski, Zygmunt Karol
A2 - Koberling, Felix
A2 - Enderlein, Jorg
A2 - Erdmann, Rainer
PB - SPIE
T2 - Single Molecule Spectroscopy and Superresolution Imaging VIII
Y2 - 7 February 2015 through 8 February 2015
ER -