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.
Highlights
Utilized fluorophores largely consist of commercially available probes that have been demonstrated to photoswitch or be photoactivatable under certain imaging conditions[10,18,19]
We examined polyvinyl alcohol (PVA) film as a method for facile characterization of fluorophores developed for Single-molecule localization microscopy (SMLM) or optimization of imaging buffer systems, where the photoswitching properties of fluorophores measured using both PVA film and the previously utilized antibody adsorption method were correlated with SMLM image quality (Figs 1 and 2)
We found microtubule width was statistically correlated with total photon output using both the antibody absorption and PVA film fluorophore isolation methods, while duty cycle was significantly correlated to microtubule continuity using both fluorophore isolation methods (Table 2 and Fig. 3)
Summary
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. While fluorophore photoswitching performance evaluated via protein adsorption has been shown to qualitatively correlate to cell imaging applications[18], it has not been established how the more advantageous polymer film isolation method compares, nor how photoswitching properties quantitatively compare to SMLM image quality. We compared the polymer film and the protein adsorption single-molecule systems for their ability to predict in vitro SMLM image quality within cells through measurement of photoswitching properties. We demonstrated PVA films were efficacious and robust in evaluating fluorophores for SMLM imaging applications and provide the necessary screening system for SMLM fluorophore development
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