Re-engineering of enzymes for use in super-resolution microscopy

Until recently, fluorescence microscopy in biological research has been limited in resolution (~ 250 nm) by the physical properties of light and diffraction. However, with the advent of super-resolution microscopy, this barrier has been broken, permitting the observation and quantitation of cellular processes down to the single molecule level. The strength of fluorescence microscopy is the ability to specifically label cellular components and most often to simultaneously label two or more components with fluorophores of differing spectral properties. For the detection of endogenous proteins in a cellular environment, antibodies conjugated to organic fluorescent dyes are almost ubiquitously used. However, when employed in super-resolution techniques such as direct stochastic optical reconstruction microscopy (dSTORM) the multi-valent nature of antibodies and their large size limits the quantitative analysis of this type of microscopy. Other approaches using nanobodies and aptamers are beginning to be employed but their targets are currently limited. What if enzymes could be re-engineered for use as detection reagents in super-resolution microscopy? This talk will discuss the utility of this approach using modified botulinum neurotoxins, the most toxic substance known, to specifically label specific cellular molecules.