Optimized Photoswitchable Fluorophores for Superresolution Microscopy
Superresolution Microscopy Research in the Gibbs Lab
Why do we need Superresolution Microscopy?
Through rapid advances in genomics and proteomics, comprehensive lists of components that comprise a living cell have been complied. However, current understanding of cellular component organization required for necessary function is rudimentary at best, leaving a critical knowledge gap. This is due to a combination of the complexity of biological processes, the destructive assays conventionally used to quantify intracellular components, and lack of appropriate measurement tools to simultaneously probe and accurately localize molecules and their complexes at the nanometer scale.
Overview of Superresolution Microscopy and biological imaging techniques
The advent of superresolution fluorescence microscopy (SRM) has significantly advanced biological imaging in <10 years (Rust MJ, et al., 2006, and Betzig E, et al., 2006). Optimization of SRM has the potential to enable the quantitation necessary to understand the spatial orientation and organization of cellular components, facilitating molecule-specific visualization at resolution >10x that of conventional light microscopy. Current single molecule localization SRM technology, termed photoactivated localization microscopy (PALM) or stochastic optical reconstruction microscopy (STORM), relies heavily on fluorophore performance for image resolution where photoactivatable fluorescent proteins and photoswitchable small molecules are required for imaging. To date, a set of optimized fluorophores for SRM has not been designed or developed and commercially available fluorescent proteins and small molecule fluorophores are used. This limits the performance of SRM to a two-dimensional imaging tool that can only image up to 4-colors in a single sample with minimal quantitation available.
The Gibbs Lab is developing optimized photoswitchable fluorophores for SRM
The Gibbs Lab is developing optimized small molecule fluorophores that will enable SRM imaging in any spectral range and two-photon photocaged photoswitchable fluorophores that will enable three-dimensional SRM imaging capabilities as well as the potential for stoichiometrically quantitave SRM. A combinatorial solid phase synthetic approach is being utilized to synthesize libraries of fluorophores derivatives modeled after four fluorophore scaffolds known to photoswitch, including xanthene, BODIPY, oxazine/acridine, and cyanine. We have developed a platform to quantify our novel fluorophore photoswitching properties, which aids in the selection of optimal fluorophores for SRM in all spectral ranges. We are currently investigating the utility of SRM imaging with our photoswitchable fluorophores to map cell signaling architecture in breast cancer, visualize the immune complexity in triple negative breast cancer, quantify human breast cancer heterogeneity and quantitatively label cellular proteins.