title: Methodology for Quantitative Characterization of Fluorophore Photoswitching to Predict Superresolution Microscopy Image Quality.
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.
authors.affiliation: Biomedical Engineering Department, Oregon Health &Science University, Portland, OR 97201, USA.
authors.forename: Amy M
authors.initials: AM
authors.lastname: Bittel
authors.affiliation: Biomedical Engineering Department, Oregon Health &Science University, Portland, OR 97201, USA.
authors.forename: Andrew
authors.initials: A
authors.lastname: Nickerson
authors.affiliation: Biomedical Engineering Department, Oregon Health &Science University, Portland, OR 97201, USA.
authors.forename: Isaac S
authors.initials: IS
authors.lastname: Saldivar
authors.affiliation: Thermo Fisher Scientific, Eugene, OR 97402, USA.
authors.forename: Nick J
authors.initials: NJ
authors.lastname: Dolman
authors.affiliation: Biomedical Engineering Department, Oregon Health &Science University, Portland, OR 97201, USA.|Knight Cancer Institute, Oregon Health &Science University, Portland, OR 97201, USA.|OHSU Center for Spatial Systems Biomedicine, Oregon Health &Science University, Portland, OR 97201, USA.
authors.forename: Xiaolin
authors.initials: X
authors.lastname: Nan
authors.affiliation: Biomedical Engineering Department, Oregon Health &Science University, Portland, OR 97201, USA.|Knight Cancer Institute, Oregon Health &Science University, Portland, OR 97201, USA.|OHSU Center for Spatial Systems Biomedicine, Oregon Health &Science University, Portland, OR 97201, USA.
authors.forename: Summer L
authors.initials: SL
authors.lastname: Gibbs