Design and development of BODIPY-based photoswitchable fluorophores to visualize cell signaling with multispectral super resolution microscopy

Amy M. Bittel, Andrew K. Nickerson, Li Jung Lin, Xiaolin Nan, Summer L. Gibbs

Research output: Chapter in Book/Report/Conference proceedingConference contribution

Abstract

Super resolution microscopy (SRM) has overcome the historic spatial resolution limit of light microscopy, enabling fluorescence visualization of cellular structures and multi-protein complexes at the nanometer scale. Using singlemolecule 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. However, the spectral resolution of current SRM techniques are limited by existing fluorophores with only up to four colors imaged simultaneously, limiting the number of intracellular components that can be studied in a single sample. In the current work, a library of novel BODIPY-based fluorophores was synthesized using a solid phase synthetic platform with the goal of creating a set of photoswitchable fluorophores that can be excited by 5 distinct laser lines but emit throughout the spectral range (450-850 nm) enabling multispectral super resolution microscopy (MSSRM). The photoswitching properties of all new fluorophores were quantified for the following key photoswitching characteristics: (1) the number of photons per on cycle (2) the number of on cycles (switching events), (3) the percentage of time the fluorophore spends in the fluorescent on and off states, and (4) the susceptibility of the fluorophore to photobleaching (time of last event). To ensure the accuracy of our photoswitching measurements, our methodology to detect and quantitate the photoswitching properties of individual fluorophore molecules was validated by comparing measured photoswitching properties of three commercial dyes to published results.1 We also identified two efficient methods to positionally isolate fluorophores on coverglass for screening of the BODIPY-based library.

Original languageEnglish (US)
Title of host publicationSingle Molecule Spectroscopy and Superresolution Imaging VII
PublisherSPIE
ISBN (Print)9780819498632
DOIs
StatePublished - Jan 1 2014
EventSingle Molecule Spectroscopy and Superresolution Imaging VII - San Francisco, CA, United States
Duration: Feb 1 2014Feb 2 2014

Publication series

NameProgress in Biomedical Optics and Imaging - Proceedings of SPIE
Volume8950
ISSN (Print)1605-7422

Other

OtherSingle Molecule Spectroscopy and Superresolution Imaging VII
CountryUnited States
CitySan Francisco, CA
Period2/1/142/2/14

Keywords

  • BODIPY
  • Fluorophore
  • Photoswitch
  • Polyacrylamide
  • Polyvinyl alcohol
  • Super resolution microscopy

ASJC Scopus subject areas

  • Electronic, Optical and Magnetic Materials
  • Biomaterials
  • Atomic and Molecular Physics, and Optics
  • Radiology Nuclear Medicine and imaging

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  • Cite this

    Bittel, A. M., Nickerson, A. K., Lin, L. J., Nan, X., & Gibbs, S. L. (2014). Design and development of BODIPY-based photoswitchable fluorophores to visualize cell signaling with multispectral super resolution microscopy. In Single Molecule Spectroscopy and Superresolution Imaging VII [89500S] (Progress in Biomedical Optics and Imaging - Proceedings of SPIE; Vol. 8950). SPIE. https://doi.org/10.1117/12.2040498