Superresolution microscopy with novel BODIPY-based fluorophores

PLoS One. 2018 Oct 26;13(10):e0206104. doi: 10.1371/journal.pone.0206104. eCollection 2018.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Boron Compounds / chemistry*
  • Buffers
  • Carbocyanines / chemistry
  • Color
  • Fluorescent Dyes / chemistry*
  • Humans
  • Microscopy, Fluorescence / methods
  • Microtubules / ultrastructure
  • Photochemistry
  • Single Molecule Imaging / methods*
  • Tumor Cells, Cultured

Substances

  • 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene
  • Boron Compounds
  • Buffers
  • Carbocyanines
  • Fluorescent Dyes

Grants and funding

This work was supported by a Collaborative Research Agreement from Thermo Fisher Scientific (S.L.G) and a Damon Runyon-Rachleff Innovator Award from the Damon Runyon Cancer Research Foundation (S.L.G and X.N.). The author Dr. Nick J. Dolman is an employee of Thermo Fisher Scientific and we also received funding from Thermo Fisher Scientific. Dr. Dolman’s contribution to this paper include initial brainstorming on project directions as well as proof reading of the final manuscript. Therefore, 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."