The 2020 Winkler Award Lecture: Richard Lincoln - Lighting and Enlightening - the Chemistry of the Cell by Rational Design of Fluorogenic Dyes

Abstract:

Cutting-edge, super-resolution fluorescence microscopy, combined with the rational design of fluorogenic probes provides a unique opportunity to study chemical processes inside living cellular systems, in a minimally invasive manner with unprecedented spatio-temporal resolution. To begin, I give an overview of the work conducted during my doctoral research, which focused on the development and application of fluorogenic probes and fluorescence microscopy methodologies for rationalizing the chemistry and biology of lipid oxidation at the cellular level. This work extends the concepts of fluorogenic probe design to developing activatable photosensitizers, as a means to exacerbate and ultimately probe the role of lipid oxidation in biology. The work focused on three major goals: 1) to improve on current methods of developing fluorogenic probes through a greater understanding of the fundamental photochemistry and photophysics underlying the sensing mechanisms (Lincoln et al. 2014, Lincoln et al. 2015); 2) to develop novel fluorogenic probes and imaging methodologies for studying the chemistry of lipid oxidation products in cell signaling (Lincoln et al. 2017); and 3) to exploit our understanding of the mechanisms of lipid oxidation to develop novel chemically-activated photosensitizers as tools to probe lipid oxidation in live cell systems (Lincoln et al. 2017, Lincoln et al. 2019).

Following this, I will address new opportunities in super-resolution microscopy stemming from MINFLUX, a recently reported methodology by the Stefan Hell lab (Balzarotti et al. 2017). Unlike stochastic-based super-resolution techniques (e.g. PALM/STORM) which rely on a large number of emitted photons to calculate the centroid position of single fluorescence molecules, MINFLUX probes the molecular position using a local intensity minimum of excitation light. Thus, MINFLUX requires only a modest photon budget to achieve a high localization precision. This enables not only single-nanometer localization precision of fluorescent emitters, but also the rapid tracking of single-fluorescent emitters in living systems (Eilers et al. 2018). Most recently, the concept has been further extended towards three-dimensional multicolor imaging of living samples (Gwosch et al. 2020). Critical to this technology are the fluorophores and labelling strategies employed. In particular, the desired photochemical and photophysical properties: including controllable mechanisms of photoactivation, consistent photon outputs (i.e. the absence of non-fluorescent “dark” states), and high biocompatibility, being of particular significance. With the implementation of MINFLUX, the limit of optical resolution is being fast approached. Now we turn our attention to new challenges in the field, notably in the design and synthesis of small and novel fluorogenic probes to aid in achieving this ultimate resolution.

Bio:

Richard Lincoln obtained his B.Sc. (Hons.) in Chemistry in 2012 from Acadia University, where he worked with Prof. Sherri McFarland studying the photochemistry of ruthenium organometallic complexes. During his undergraduate, he travelled to McGill University on the Reactive Intermediate Student Exchange (RISE) to work with Prof. Gonzalo Cosa studying the photophysics of BODIPY dyes. After graduating from Acadia, he returned to the research group of Prof. Cosa to pursue his Ph.D. in chemistry. His research focused on developing fluorescence probes to study the chemistry of lipid oxidation. Richard is currently a postdoctoral researcher at the Max Planck Institute for Medical Research in Heidelberg in the research group of Prof. Stefan Hell. He is currently developing novel fluorophores for MINFLUX nanoscopy.