Light microscopy has made several key advancements since the invention of the first compound light microscope in the late 16th century. Since then we have moved through several iterations from enhanced magnification, to improved resolution (diffraction limited), targeted fluorescence, high signal to noise confocal and most recently super resolution imaging. As recently as 2014 the Nobel Prize in Chemistry was awarded for 'the development of super-resolved fluorescence microscopy'. In 2015 we launched a revolutionary super resolved fluorescent microscope which not only broke the traditional limits of resolution (Abbe's limit) but also allowed for very fast and gentle imaging, therefore making it the most advanced tool for super resolved 'Live Cell Imaging.' However, this microscope was still limited to what is commonly referred to as the SIM/ISM limit of resolution for a light microscope, which is approximated as Resolution = (0.61\*λ)/(2\*NA). So for an emission λ of 525nm and an objective with Numerical Aperture (NA) of 1.49, the best achievable resolution would be 107nm. Whilst this does offer a window into some of the smallest interactions which take place within cell biology, the increase in the amount of information that can be gained from resolving down at the nanoscopy scale (≤50nm) is exponential. Currently the only tools available for fluorescence based nanoscopy (SMLM, STED, CLEM) all require the biological specimen to be fixed and therefore do not allow you to see the huge amount of sub cellular interactions which occur during critical biological events such as cell division, protein to protein interaction, cell interactions with viruses and bacteria, cell contraction, etc... What we are proposing to develop as part of this project is the worlds first fluorescent based nanoscope which enables live cell imaging. It should also be noted that the developed instrument would not only be suitable for the scientific research field; advanced light microscopes are no longer confined to scientific laboratories and are being used for a variety of clinical and industrial (pharmaceutical) applications, from medical screening to drug development. Such a high speed, live cell imaging nanoscope would open up new possibilities for screening applications in medical screening such as for the diagnosis of platelet granule disorder, high content screening (HCS) for drug development such as in the development of mRNA vaccines and gene sequencing for high density in-situ sequencing to spatially resolve RNA/DNA/Proteins for development of personalised medicines.

One of the key tools of bio-medical research are light microscopes and to be more specific fluorescent microscopes. A fluorescent microscope allows the scientist to view tagged parts of a Cell, Virus, etc... at a molecular level so it's behaviour can be monitored. Such fluorescent microscopes are driving modern bio-medical research to aid in our understanding of illness, disease and infection and in turn allow the development of improved treatments and possible cures. However, the performance of these light microscopes has been hampered by the previous limit of optical resolution which was defined by a German Physicist in the 19th Century, Ernst Abbe, and as such carries his name, Abbe's Law. Abbe's Law determined that the maximum achievable resolution of a light microscope is given by the wavelength of the light being used to view it (in many cases around 550nm) divided by twice the numerical aperture of the lens used for imaging (in modern microscopes the Maximum NA = 1.4). This limited the resolution at which light microscope could observe biological systems and interactions to >200nm, as scientists developed an improved understanding of biology this was becoming a frustrating bottle neck for furthering research, until that is the development of super-resolved or Nanoscopy techniques and applications. This new emerging field was highlighted by the 2016 Nobel Price in Chemistry which was awarded for the development of super-resolved fluorescent microscopy. However, whilst these new techniques did allow light microscope to resolve as low as 20nm the techniques were quite complex and could not always be used for every tool within the field of light microscopy which the scientist may have wished to use. One such tool is Total Internal Reflection Fluorescence (TIRF) Microscopy. TIRF is currently used in Cell biology to view events which happen at the surface of cells, such events can play a major roll in the behaviour of the cell and scientists who research cancer (for example) are trying to understand the movement and spread of cancer cells by looking at events at the cellular surface using TIRF microscopes. The instrument we propose to develop within this project will double the spatial resolution of a regular TIRF microscope enabling features <100nm to be imaged. This will offer the global science community an imaging system which can do true super-resolved live cell imaging with TIRF (SR-TIRF) allowing further advancements within bio-medical research.

Bio-imaging has to meet many requirements, not least the ability to image a living cell or organism without damaging it to an extent that any results attained from imaging it become unusable. At the same time to view some of the most critical biological process' requires an ability to see things which are less than 100nm which is 1/2000th the diameter of a human hair. The processes within which such microscopic biological features take place can happen within 1mS, or 1/1000th of a second. To be able to produce a Bio-imaging technique which can view biological processes of 1/1000th of a second with the detail of 1/2000th the diameter of a human hair and yet enable the living cell or organism to carry on behaving as it would in the body would truly be a development in Bio-imaging which could literally change how we look at living cells and organisms. It is this which VisiTech are proposing to develop a prototype of within this SMART grant application.

An ongoing challenge of imaging microscopy is the limitations of achievable optical resolution. Improved optical resolution allows smaller features to be resolved which in turn allows researchers to better understand such processes as cell division, movement of mitochondria and colloidal dynamics. The techniques to improve optical resolution beyond the current perceived limitations are called super resolution. Existing systems which can offer this super resolution are limited by the poor achievable temporal resolution and high cost. The poor temporal resolution of these systems limits them to taking a “snap shot” of a sample in super resolution but the dynamics cannot be studied in super resolution. This is hugely limited in applications such as those mentioned above. Also these existing super resolution techniques are very expensive (£1M) putting them beyond the budget of many scientists worldwide. The aim of this project is to develop a new technique for improving the spatial resolution of confocal imaging systems without impacting on the temporal resolution at a price which is targeted at being one quarter of the price of existing systems. We have developed a concept that combines our multi beam confocal imaging system with super resolution providing the world’s first multi beam super resolution system offering super resolution at a high enough temporal resolution to allow the study of dynamics in super resolution. A further objective of the project is to transfer our design into a working prototype ensuring that we develop clear records of each stage of manufacture so we can transfer the knowledge gained through prototype development into a product that we can manufacture at our target price and in turn market worldwide. Over 85% of the materials we procure to build our confocal imaging systems are taken from local, UK companies so any increase in turn over resulting from our new technology will have a direct impact on the local economy.