Methodology for quantitative characterization of fluorophore photoswitching to predict superresolution microscopy image quality

Amy M. Bittel, Andrew Nickerson, Isaac S. Saldivar, Nick J. Dolman, Xiaolin Nan, Summer Gibbs

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

Single-molecule localization microscopy (SMLM) image quality and resolution strongly depend on the photoswitching properties of fluorophores used for sample labeling. Development of fluorophores with optimized photoswitching will considerably improve SMLM spatial and spectral resolution. Currently, evaluating fluorophore photoswitching requires protein-conjugation before assessment mandating specific fluorophore functionality, which is a major hurdle for systematic characterization. Herein, we validated polyvinyl alcohol (PVA) as a single-molecule environment to efficiently quantify the photoswitching properties of fluorophores and identified photoswitching properties predictive of quality SMLM images. We demonstrated that the same fluorophore photoswitching properties measured in PVA films and using antibody adsorption, a protein-conjugation environment analogous to labeled cells, were significantly correlated to microtubule width and continuity, surrogate measures of SMLM image quality. Defining PVA as a fluorophore photoswitching screening platform will facilitate SMLM fluorophore development and optimal image buffer assessment through facile and accurate photoswitching property characterization, which translates to SMLM fluorophore imaging performance.

Original languageEnglish (US)
Article number29687
JournalScientific Reports
Volume6
DOIs
StatePublished - Jul 14 2016

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Fluorophores
Image quality
Microscopic examination
Molecules
Polyvinyl Alcohol
Spectral resolution
Image resolution
Labeling
Buffers
Screening
Proteins
Imaging techniques
Adsorption
Antibodies

ASJC Scopus subject areas

  • General

Cite this

Methodology for quantitative characterization of fluorophore photoswitching to predict superresolution microscopy image quality. / Bittel, Amy M.; Nickerson, Andrew; Saldivar, Isaac S.; Dolman, Nick J.; Nan, Xiaolin; Gibbs, Summer.

In: Scientific Reports, Vol. 6, 29687, 14.07.2016.

Research output: Contribution to journalArticle

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