Superresolution microscopy with novel BODIPY-based fluorophores.
Multicolor single-molecule localization microscopy (SMLM) expands our understanding of subcellular details and enables the study of biomolecular interactions through precise visualization of multiple molecules in a single sample with resolution of ~10-20 nm. Probe selection is vital to multicolor SM...
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2018-01-01
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doaj-ec9277f00a84498697e63e246ff716b22020-11-25T01:19:51ZengPublic Library of Science (PLoS)PLoS ONE1932-62032018-01-011310e020610410.1371/journal.pone.0206104Superresolution microscopy with novel BODIPY-based fluorophores.Amy M BittelIsaac S SaldivarNick J DolmanXiaolin NanSummer L GibbsMulticolor single-molecule localization microscopy (SMLM) expands our understanding of subcellular details and enables the study of biomolecular interactions through precise visualization of multiple molecules in a single sample with resolution of ~10-20 nm. Probe selection is vital to multicolor SMLM, as the fluorophores must not only exhibit minimal spectral crosstalk, but also be compatible with the same photochemical conditions that promote fluorophore photoswitching. While there are numerous commercially available photoswitchable fluorophores that are optimally excited in the standard Cy3 channel, they are restricted to short Stokes shifts (<30 nm), limiting the number of colors that can be resolved in a single sample. Furthermore, while imaging buffers have been thoroughly examined for commonly used fluorophore scaffolds including cyanine, rhodamine, and oxazine, optimal conditions have not been found for the BODIPY scaffold, precluding its routine use for multicolor SMLM. Herein, we screened common imaging buffer conditions including seven redox reagents with five additives, resulting in 35 overall imaging buffer conditions to identify compatible combinations for BODIPY-based fluorophores. We then demonstrated that novel, photoswitchable BODIPY-based fluorophores with varied length Stokes shifts provide additional color options for SMLM using a combination of BODIPY-based and commercially available photoswitchable fluorophores.http://europepmc.org/articles/PMC6203453?pdf=render |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Amy M Bittel Isaac S Saldivar Nick J Dolman Xiaolin Nan Summer L Gibbs |
spellingShingle |
Amy M Bittel Isaac S Saldivar Nick J Dolman Xiaolin Nan Summer L Gibbs Superresolution microscopy with novel BODIPY-based fluorophores. PLoS ONE |
author_facet |
Amy M Bittel Isaac S Saldivar Nick J Dolman Xiaolin Nan Summer L Gibbs |
author_sort |
Amy M Bittel |
title |
Superresolution microscopy with novel BODIPY-based fluorophores. |
title_short |
Superresolution microscopy with novel BODIPY-based fluorophores. |
title_full |
Superresolution microscopy with novel BODIPY-based fluorophores. |
title_fullStr |
Superresolution microscopy with novel BODIPY-based fluorophores. |
title_full_unstemmed |
Superresolution microscopy with novel BODIPY-based fluorophores. |
title_sort |
superresolution microscopy with novel bodipy-based fluorophores. |
publisher |
Public Library of Science (PLoS) |
series |
PLoS ONE |
issn |
1932-6203 |
publishDate |
2018-01-01 |
description |
Multicolor single-molecule localization microscopy (SMLM) expands our understanding of subcellular details and enables the study of biomolecular interactions through precise visualization of multiple molecules in a single sample with resolution of ~10-20 nm. Probe selection is vital to multicolor SMLM, as the fluorophores must not only exhibit minimal spectral crosstalk, but also be compatible with the same photochemical conditions that promote fluorophore photoswitching. While there are numerous commercially available photoswitchable fluorophores that are optimally excited in the standard Cy3 channel, they are restricted to short Stokes shifts (<30 nm), limiting the number of colors that can be resolved in a single sample. Furthermore, while imaging buffers have been thoroughly examined for commonly used fluorophore scaffolds including cyanine, rhodamine, and oxazine, optimal conditions have not been found for the BODIPY scaffold, precluding its routine use for multicolor SMLM. Herein, we screened common imaging buffer conditions including seven redox reagents with five additives, resulting in 35 overall imaging buffer conditions to identify compatible combinations for BODIPY-based fluorophores. We then demonstrated that novel, photoswitchable BODIPY-based fluorophores with varied length Stokes shifts provide additional color options for SMLM using a combination of BODIPY-based and commercially available photoswitchable fluorophores. |
url |
http://europepmc.org/articles/PMC6203453?pdf=render |
work_keys_str_mv |
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1725136974408318976 |