Developing and validating a new tool for simultaneous multi-channel wide-field imaging

Imaging biological systems provides a direct insight into how they work. As more systems are being studied there is an increasing necessity to label and visualise more components. However, despite there now being a large variety of colours with which to label proteins our ability to simultaneously visualise them is limited. There are two major methods to image in multiple colours, wide-field and confocal. The former approach involves taking a picture of the whole sample at once, but is limited to only four colours per image. The latter approach involves taking pictures in one location before moving to another position to take a second image and so on until the whole sample is catalogued. Clearly this confocal approach means that the whole sample cannot be imaged simultaneously, however it does permit many colours to be imaged. In this proposal we are developing a new method to image six-colours simultaneously in wide-field, with the hope that this could be further extended in the future to many more colours. To achieve this, we will build a simple device that attaches onto a standard microscope, this will allow two commercially available 3-colour splitters to be attached and then two cameras. These cameras need to talk to each other so that they take images at the same time, we will build this interface using software or hardware.

The six-colour imager developed here will then be tested using a biological system. In our lab we study excerpts from the bacterial nucleotide excision DNA repair (NER) process using a three colour imaging device. A six-colour imager will enable us to study simultaneously all of the NER proteins that act together to repair DNA damage. Our approach is to use DNA tightropes, which are nanowires of DNA suspended between microscopic platforms on a microscope coverslip. To these tightropes we will add all of the proteins involved in NER but each type will be labelled with a different coloured tag. We will then study how DNA is repaired one molecular complex at a time. As a test for the new technology developed here this model system is ideal because it requires multiple colour imaging and the highest level of detection sensitivity.

We anticipate that this work will pave the way for the study of many different biological systems with multiple components, not only at the single molecule level but also at the cellular level as a consequence of wide-field imaging. This is a hugely important tool that needs to be developed and tested.

This proposal seeks to address a key problem in our ability to study more complex biological systems, namely that we are limited to imaging up to four colours simultaneously in wide-field.
Presently a wide variety of fluorophores ranging from fluorescent proteins to quantum dots are available for protein detection. Despite this spectrum of colours, our ability to detect them simultaneously is limited in wide-field to just four. This is due to limited spectral separation of fluorophores and approaches to visualise more colours simultaneously require spot-scanning of the sample. Here we propose to solve this issue through the design of a primary image splitter that will spectrally separate two regions of the image. Each spectral region will be sent to a commercial three colour splitter and then onto a camera. Recent advances in CMOS technology have enabled large but sensitive camera chips to be used that overcome problems of visual field size. However to ensure accurate acquisition timing the cameras will require tethering to one another. This system is unique in its hardware design.
This multi-spectral divider will then be rigorously tested against a difficult biological problem at the single molecule level. Working at this level requires the highest levels of image quality and photon throughput. We will use bacterial nucleotide excision DNA repair as a test system due to our background in this field. Initial tests for sensitivity will be performed on the two protein complex UvrBC. Subsequently, we will attempt to study differentially Qdot labelled UvrA, UvrB, UvrC, UvrD, Pol1 and DNA damage all in the same assay simultaneously at frames rates >20 Hz. The results from this experiment will be transformative because we can extract kinetic and mechanistic information from a single experiment.
This proposal aims to develop a new tool with the potential to substantially advance our ability to study complex biological systems. This tool has enormous longer term value.

We are developing a new technology to enable scientists to directly image more complex biological systems in real time. Therefore there will be beneficiaries across a number of fields that use imaging technologies, whether within the biosciences or beyond. The multi-spectral divider will be simple and can be fit onto any commercial microscope to ensure that other scientists can implement this technology easily into their own systems.

Imaging is widely used to understand biological processes. This can be at the level of whole organisms through to single molecules. The impact of our new technology will be felt across all of these fields. Understanding complex systems relies on the development of technologies that can multiplex in real time. Biosciences are moving towards a more holistic view, where proteins interact with each other in a dynamic and complex fashion. By having tools to enable direct investigation of such interactions with the capability of super-localisation this goal can be more easily realised. Therefore, investigators studying whole organism physiology, optogenetics, cell biology, single molecule biophysics will all immediately benefit. The proposed techniques could also be applied beyond basic research to drug discovery, healthcare, food security, electronic engineering and materials science. By working with an imaging-technology manufacturer we have the vehicle to immediately fill the demand for this new technology. In collaboration with our lab, Cairn Research Ltd. will produce this device, this establishes a clear route to commercialise this multi-spectral divider for wider community access.

Real time imaging is used across disciplines ranging from number plate detection to airport security, having the tools present to image multi-spectrally in real-time will find beneficiaries in these areas. To ensure that the wider public is aware of our work we will publicise our findings through the University of Kent's Press Office looking for publication in local and national technical and non-technical journals.

The staff involved in this research will receive specific project related training across the range of disciplines, offering an excellent opportunity to learn cutting edge skills. In addition they will learn transferable skills from conference networking, giving seminars, and writing reports.