Abstract
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.
Highlights
Single-molecule localization microscopy (SMLM) is one of several superresolution microcopy (SRM) techniques that enable fluorescence imaging below the diffraction limit of light (~250 nm) [1, 2]
Photoswitching properties were evaluated in 35 unique imaging buffer conditions, which consisted of seven redox conditions, each evaluated with five additives (Table 1) using a polyvinyl alcohol (PVA) single molecule isolation methodology [10]
Multicolor SMLM is a powerful tool that will enhance our understanding of complex biological processes at the nanoscale
Summary
Single-molecule localization microscopy (SMLM) is one of several superresolution microcopy (SRM) techniques that enable fluorescence imaging below the diffraction limit of light (~250 nm) [1, 2]. SMLM is able to achieve resolution on the order of ~10–20 nm through the use of photoswitchable fluorophores that stochastically switch between the fluorescent “on” state and the non-fluorescent “off” state [3]. Small molecules organic photoswitchable fluorophores are used with the SMLM technique most often termed stochastic optical reconstruction microscopy (STORM) [2, 4, 5], which has recently been expanded to multicolor techniques [6,7,8]. We agree with the statement that “The funder provided support in the form of supplies for the project, but did not have any additional role in study design, data collection and analysis, decision to publish or preparation of the manuscript."
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