Focus enhanced single molecule super-resolution microscopy - correlative confocal and nanoscale imaging in thick tissues

Fluorescence microscopy is widely used as a sensitive tool to investigate the biology and biophysical properties of cells and tissues, including its use as a healthcare technology in diagnostic pathology of tissue samples. Larger structures in complex three-dimensional cells and tissues have been successfully investigated with confocal microscopy which produces sharp images by effectively rejecting out of focus light. However, many of the structures found within cellular organisms are much smaller than the wavelength of light and have therefore been difficult to observe with fluorescence microscopy. In this project we plan to combine principles from confocal microscopy with a new "super-resolution" microscopy method which overcomes the limits of conventional light microscopy and can resolve detail down to ~20 nm. To date, however, such high resolution has been difficult to achieve in thicker cell preparations (e.g. > 5 um), as many super-resolution methods rely on the precise localisation of individual molecules that emit light when excited in the fluorescence microscope. This "single molecule localisation microscopy" approach suffers from extensive background light that is generated in thick samples such as tissues and limits the achievable resolution.

In this project we propose an improvement to the imaging process that can be easily implemented and which combines ideas from conventional confocal microscopy with super-resolution imaging based on single molecule localisation while maximising the collection of light. Key to the new approach is the use of a digital micro device (DMD) array for patterned illumination of the sample. Adopting this new approach also allows the new device that we will build to implement standard confocal microscopy. It therefore allows us to combine both conventional 3D microscopy and our new effective super-resolution modes and obtain increased information from the samples. This means we can combine the high throughput of conventional confocal microscopy with local high resolution provided by super-resolution, in other words the best of both worlds for effective imaging in complex biological samples obtained for pathology testing.The utility of our approach is increased because we will adopt an improved confocal mode that has been recently demonstrated. Most importantly, we will be able to seamlessly switch between the various modes under sophisticated software control.

To improve the ability to provide extended 3D super-resolution data in thick tissue samples we will introduce a recently demonstrated technique into the workflow of our new approach. This technique, called DNA-PAINT, uses the modern understanding of DNA interactions to construct new markers for super-resolution imaging. DNA-PAINT provides a versatile way to combine many different marker types in the same sample and also introduces a convenient way to localise marker molecules as complementary DNA strands transiently bind to each other. In combination with our super-resolution improvements this will provide a way to record images throughout the depth of a thick sample and construct very high-resolution 3D volume images. The "confocal principle" in the new super-resolution approach is critical to avoid the background light that otherwise would greatly impair DNA-PAINT in tissue.

To demonstrate the impact of our new combined super-resolution, confocal and correlative microscopy modes we will conduct pilot studies that establish our new approach as a healthcare technology for diagnostic pathology in heart tissue, imaging in the brain and in cell "clumps" that resemble tumour tissue in their complex 3D arrangement.

The combination of new capabilities of our new microscope, its efficient implementation and sophisticated but intuitive software interface will make this a versatile new approach that will be highly relevant for academic and commercial users in many different fields.

A broad range of groups and stakeholders will benefit from the proposed project. These include:

- Commercial microscope manufacturers (for research, clinical and commercial markets)
- Clinical researchers and pathologists, i.e. users of fluorescence microscopy as a healthcare technology
- Biomedical scientists and other academic beneficiaries
- The pharmaceutical and wider biotechnology industry

In addition, we suggest that the wider public has an interest in simple, robust and affordable microscopy techniques to possibly allow interested lay groups as a mechanism of widening public engagement in the future to build their own microscopes, exploiting recent developments in 3D printing, computing and embedded devices as well as continuously decreasing component cost.

Ways in which these groups will benefit
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The technical developments that we will pursue will allow commercial manufacturers to increase the capability of their instruments and offer improved products that should create demand in their respective markets.

We anticipate increasing use of super-resolution methods in clinical research and diagnosis, particularly the use for advanced pathology diagnosis is a realistic possibility, currently only limited by both complexity and inability of current methods to deal with tissue samples easily. Our improvements in optical performance and ease of use in tissue slice samples, combined with a straightforward implementation, will make this much more practical. It is critical to demonstrate the applicability and versatility of our proposed methods and we therefore aim to conduct several applications directly related to the use by beneficiaries. This includes the study of samples from hearts in failure for improved diagnosis and two other biomedical applications of correlative confocal and focus enhanced super-resolution microscopy.

The types of benefit outlined above make a contribution to the nation's health and wealth and help to enhance quality of life and long term health when applied in the clinical setting.

We anticipate that towards the latter part of the proposed project, once the focus enhanced super-resolution modalities and the use of DNA PAINT technology with our new modality have been established, talks with commercial manufacturers will take place to address commercialisation of the technology (building on our track record with Zeiss, with support from Exeter RKT).
We have also formed a relationship with Badrilla Inc, a biotechnology company in the UK which specialises in immuno-marker technology, to trial the technology with their markers.

The research and technical staff working on this project will acquire advanced skills in applied optical design, computer control of modern imaging hardware and interdisciplinary skills in biophysics, cell biology and imaging applications. The research fellow in particular will acquire and refine a set of skills that should make them sought after for employment in applied computing, optical design, advanced application development and a variety of research and development roles.