Pushing Back the Limits of Optical Microscopy - Enhancing dSTORM Resolution through Controlled Fluorophore Switching

Sight allows humans to observe the world around us. The ability to see small objects, whether a minute insect, the iridescent scales on the wings of a butterfly or the individual dots on a new HD television, allows us to directly study tiny things which can have a big influences on human society. Despite the complexity of the human eye, it is limited to being able to perceive objects no bigger than 0.1 millimetres (or 1/10,000th of a metre). Thus study of objects smaller than 0.1 mm was impossible until the invention of high quality optics and the optical microscope, circa 1600. The optical microscopes of the time resulted in the pioneering discoveries of both red blood cells and micro-organisms (e.g. bacteria) by Antonie van Leeuwenhoeks. His discoveries resulted in a massive increase in the popularity and use optical microscopes by scientists. Subsequently the optical microscope has become a key piece of technology, revolutionising our understanding of the world and providing the foundations on which all modern medicine is built.
Despite the many design improvements, the optical microscope is still limited by the rules of physics, namely an important property of light known as the diffraction limit. As such, normal optical microscopes can only resolve objects larger than 0.0005 mm (or 1/2,000,000th of a metre) in size.
Our research proposal involves a technique called "super-resolution" optical microscopy. Super resolution microscopes circumvent the defraction limit barrier through a combination of dye molecules with special properties, lasers and computerised image processing. These systems can see objects down to 0.00002 mm (1/50,000,000th of a metre) approximately 25 times better than optical microscopy. Use of these systems will reveal previously unknown levels of detail, allowing the internal workings of the cell. to be seen in higher resolution then ever before. One can only imagine what these next generation microscopes could reveal about life and what discoveries they will provide in the future.
However super resolution optical microscopy is currently a highly specialist tool, used only by a few skilled research teams. We aim to improve the accessibility of super resolution optical microscopy, allowing it to become a standard tool for laboratories around the world. Though a greater understanding of the underlying science and the development of practically simple protocols we will help other research groups to apply super resolution optical microscopy to understanding a huge variety of biological systems.
We will use a super resolution technique known as STORM (STochastic Optical Reconstruction Microscopy) which promises to give the best resolution as well as being the simplest technique to perform. Our aims are to understand the science of STORM (currently poorly understood) and to use that knowledge to simplify both the imaging process and sample preparation, resulting in easy, reproducable, high resolution imaging.
Our chemistry team will build new molecules, designed as specialist dyes with just the right properties. These molecular dyes absorb the laser light from the STORM microscope and then release the light again in a way which allows each individual molecule to be observed. The light from these individual dye molecules is combined by a computer to build the high resolution picture of our target point by point. Until now, custom dyes have not been available for use in these systems and "normal" dyes have been used which do not provide the best images. Armed with our custom dye molecules the biology team will then work out the best way to prepare the biological samples (e.g. cells, microbes) to give the highest resolution and the best reproducibility. We will then pass our best dyes and best protocols onto other groups (commercial and public) to allow them to get the best possible high resolution images of their own targets.

(1). Our specific aim is to identify highly fluorescent molecules with controllable ON/OFF switching and low duty cycles suitable for dSTORM imaging of biological systems. Initial synthetic targets are cyanine dyes bearing N tethered thiol groups. The nature of the connecting tether and the reduction potential of the appended quencher will be varied systematically using rapid synthetic methods to optimise ON/OFF switching of the emissive state, levels of emission and stability during redox cycling. Lead compounds will be equipped with activated esters facilitating direct attachment to biological probes.
(2) To demonstrate improved STORM imaging resolution vs. commercial fluorophores and standard protocols, (100 nm - 20 nm) latex beads will be labelled and imaged to a sub-20 nm accuracy, in imaging buffer, using Nikon STORM equipment. Lead candidates and protocols will be used to label selected biological structures such as the cytoskeleton. Phalloidin and/or antibody tagged probes will be used to image the actin filaments and microtubules. In addition, candidate probes will be used to image, at nanometer resolution, bacterial and mitochondrial proteins. Validation will involve comparison with positional data collected using both conventional light and electron microscopy.
(3) Evaluation of our synthesized fluorophores as bio-imaging agents will be supported through quantitative studies to establish rates of ON/OFF switching and mechanisms for the switching action. These studies will include steady-state and time-resolved laser spectroscopy, laser flash photolysis to monitor intermediate species and in situ EPR spectroscopy to search for meta-stable free radicals. Environmental variables (pH, solvent, molecular oxygen, radical traps, etc.) will be used to help rationalise mechanisms and switching efficiencies. Particular attention will be given to understanding the reaction mechanism, limitations and improving the overall stability of the system.

The proposed research is intrinsically enabling and combines basic scientific progress, application and ultimately tool provision. As such, the research programme is designed not only to improve scientific understanding of photo-switching mechanisms but to apply that gained knowledge to the generation of real tools for the imaging community, applicable to a number of major challenges in the field of optical microscopy. As such, the immediate impact of this work will be to benefit the optical imaging community, this being an advancing cohort of specialist researchers, by providing access to sophisticated tools. At present, super-resolution microscopy is still the preserve of a few groups but our output should make this technique readily available to all. Immediate benefit will be realised by commercial suppliers of super-resolution equipment and associated consumables, in particular international companies (with UK presence) such as Nikon UK (manufactures of STORM microscopes) and Life Technologies (suppliers of molecular imaging agents). As our research will both advance and simplify STORM protocols, the uptake of the technology will accelerate increasing sales. Commercial development of our technological developments will be managed by partner companies (see supporting letters) and Newcastle University BDD.
In the midterm, uptake of our research outputs by the bio-imaging community will have disenable impact across biological research and will lead to a growth in the range of applications that would benefit from STORM technology. This is particularly evident in the event that a marked improvement results in both ultimate resolution and operational simplicity. As the resolution improves, more users will join the community creating new and exciting challenges. Part of our proposal seeks to improve data collection and analysis and such protocols will be useful for several ancillary areas of imaging. Spin-offs from our research could aid all kinds of imaging technology and provide patients with better understanding of the diagnostic tools. At a local level, on-going projects within mitochondrial and bacterial research groups at Newcastle would be prime beneficiaries from increased positional detail available with STORM imaging. The ready availability of our instrumentation through international collaborations would further extend this type of research.
The long-term impact of improved super-resolution microscopy will be far reaching, not least in terms of cost reduction. The most significant benefit will be felt in terms of the range of biological systems amenable to study by this technique. Better resolution will mean shorter exposure times such that transient structures can be analysed in real time. Improved contrast will allow smaller and smaller structures to be probed in detail. More stable dyes will facilitate those studies where long exposures are needed. We might foresee an improved understanding of the cellular processes involved in cell division and growth (with impact on the understanding of human growth, ageing and cancer) as well as further study on micro-organisms improving our knowledge of important human pathogenic bacteria. Knowledge of the underlying cellular mechanisms will lead to improvements in treatment and patient care. In-depth knowledge of the biology will underpin future advances in medicine and instrumentation, which in turn will benefit the high technology end of the business.
As super-resolution microscopy becomes a generic tool, we anticipate merging of this technology with existing protocols used in nano-science and information technology. The general public retains a fascination with the ever-growing ability to see inside the human body and to visualise cell functions at the molecular level. Such developments aid public awareness of the importance of imaging science and assist in the furtherance of policy makers' understanding of the importance of biology.