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.

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.

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.

Single-molecule localization microscopy (SMLM) utilizes photoswitchable fluorophores to detect biological entities with 10-20 nm resolution. Multispectral superresolution microscopy (MSSRM) extends SMLM functionality by improving its spectral resolution up to 5 fold facilitating imaging of multicomponent cellular structures or signaling pathways. Current commercial fluorophores are not ideal for MSSRM as they are not designed to photoswitch and do not adequately cover the visible and far-red spectral regions required for MSSRM imaging. To obtain optimal MSSRM spatial and spectral resolution, fluorophores with narrow emission spectra and controllable photoswitching properties are necessary. Herein, a library of BODIPY-based fluorophores was synthesized and characterized to create optimal photoswitchable fluorophores for MSSRM. BODIPY was chosen as the core structure as it is photostable, has high quantum yield, and controllable photoswitching. The BODIPY core was modified through the addition of various aromatic moieties, resulting in a spectrally diverse library. Photoswitching properties were characterized using a novel polyvinyl alcohol (PVA) based film methodology to isolate single molecules. The PVA film methodology enabled photoswitching assessment without the need for protein conjugation, greatly improving screening efficiency of the BODIPY library. Additionally, image buffer conditions were optimized for the BODIPYbased fluorophores through systematic testing of oxygen scavenger systems, redox components, and additives. Through screening the photoswitching properties of BODIPY-based compounds in PVA films with optimized imaging buffer we identified novel fluorophores well suited for SMLM and MSSRM.View presentation recording on the SPIE Digital Library: http://dx.