Development of wide-field TCSPC fluorescence microscopy for cell membrane studies

From the earliest invention of the camera, humans have been seeking to observe processes that are too fast or too complex for the human eye to follow. The first time-lapse images of a running horse, taken by Eadweard Muybridge in the 19th century, allowed us to understand its motion, freezing a moment in time so that we can examine minute details. It showed that a horse's feet all leave the ground when galloping, a controversial question hotly debated at the time. Importantly, the time lapse images were a full-frame view - a key concept which we will also employ in the instrument to be developed here. Today, in cellular biology, our understanding of cellular function continues to evolve as we observe complex dynamic processes played out under a microscope. The optical microscope is a non-invasive, non-destructive and non-ionising tool which can be used to study living cells and tissues. No other method can study molecules in living cells with anything remotely approaching its combination of spatial resolution, selectivity, sensitivity and dynamics. Modern sensitive and sophisticated electronic cameras can capture dynamic processes at high speed, revealing intricate details of these processes. Indeed, detector development is a very important aspect of progress in the field of microscopy. The aim of our project is to develop extremely sensitive and fast full-frame view cameras which will allow us to observe molecules and proteins in their natural habitat, the cell, without disturbing them - in a way the 21st century equivalent of Muybridge's galloping horse.

We are interested in molecules that play a role in inflammation, which is the body's response to some kind of harm or injury. These molecules are called proteins, and they are many different ones in our cells. We specialise in finding out about a protein called the coxsackie virus adenovirus receptor (CAR). We want to know how they move around in time, bump into each other and stick together. So we have labelled them with a fluorescent label to observe them under a microscope. The special cameras we are going to develop will be able to see them with a very high resolution, and also very quickly and very precisely, by measuring the polarization of the fluorescence emitted by its label. They will allow us to observe the moment a cell responds to a chemical stimulus at the level of single proteins. This will help us to understand how inflammation occurs, on a molecular basis - which, at the moment, is still unknown. Imaging living cells is the best available approach to study this kind of biological question, and others, and, ultimately, the knowledge and insight gained by doing this work will enable us to design and develop drugs against inflammation, for the benefit of all of humankind.

FLIM is a key technique to image the interaction of proteins, and is independent of fluorophore concentration, which is hard to control in cells. Time-correlated single photon counting (TCSPC) FLIM has the highest sensitivity of all FLIM approaches, but while scanning TCSPC FLIM is routinely implemented, some microscopy methods are performed without beam scanning, employing wide-field camera-based detection instead, e.g. time-lapse and TIRF microscopy. There is a technology gap for wide-field TCSPC FLIM, and this proposal will fill that gap: advanced 192x256 pixel SPAD array cameras with on-pixel time-to-digital converters - the most advanced TCSPC imaging detectors to date - will be adapted for wide-field TCSPC-based FLIM and TIRF microscopy, and bespoke data management solutions for this application implemented. They provide the high level of sensitivity, specificity and speed required - without beam scanning - to elucidate the control of receptor self-association at the membrane of living cells and how this is regulated by inflammatory insults.

Regulation of epithelial cell junction integrity is vital to many processes including embryonic development, tissue homeostasis, wound healing and inflammation. One transmembrane receptor playing a role in these processes is the coxsackie virus adenovirus receptor (CAR). The crystal structure of the CAR D1 domain has shown that D1 is able to form homodimers in solution, but how CAR dimerisation is regulated in intact cells remains unknown. Understanding how, where and when CAR dimerises is essential to dissecting its role in controlling epithelial cell adhesion, but hampered by the limitations of currently available techniques such as standard biochemical or immunofluorescence analysis which compromises the cell and does not provide spatial or temporal information. Current TIRF microscopes lack the versatility, time-resolution and photon throughput capabilities to address this issue.

In addition to the academic beneficiaries, commercial private sector beneficiaries may include STMicroelectronics who have developed the first high volume products based on SPAD sensors ("flightsense") for time-of-flight ranging. A recent spinoff company (PhotonForce) from the University of Edinburgh is commercialising SPAD image sensors in scientific/medical applications and is a likely licensee of IP generated in the project. The SPAD arrays can be used for range finding in mobile phones to switch off the display when the device is held to the ear, thus contributing to saving energy - an important mass market development (see http://www.st.com/content/st_com/en/about/media-center/press-item.html/stmicroelectronics-proximity-sensor-solves-smartphone-hang-ups.html). Our development and refinement of photon arrival timing techniques in this proposal may be able to further optimise this approach. Moreover, our novel fluorescence and photon arrival time detection technology will lead to SPAD array cameras optimised specifically for time-resolved fluorescence microscopy, which could be manufactured by PhotonForce. The proposed project would thus facilitate their entry into the life sciences fluorescence microscopy market. We will also organise a workshop for the academic community and industry in the final year of the project. Moreover, we will invite the industrial collaborators STMicroelectronics and PhotonForce to join the project review meetings every 4 months either in person or via skype. This would allow them to develop their applications alongside the main thrust of the project ensuring that beneficiaries are well represented even at the genesis of the project. In the longer term, when the SPAD array technology developed in this proposal is taken up by the biophotonics research community.

Beyond the field of fluorescence microscopy and inflammation, general photon time-of-flight measurement techniques such as photon-counting light detection and ranging (lidar) and photon counting optical tomography would benefit significantly from TCSPC detection. In lidar, single photon sensitivity and large number of pixels would allow rapid detection of reflected laser pulses, speeding up the process of mapping an archaeological site, for example, or industrial processes such as lidar-based non-contact inspection of car bodies or aircraft wings for fractures. In photon counting optical tomography, image acquisition could be sped up by orders of magnitude, as currently only 10s of detectors are used to map the specimen, now 10s of 1000s could, in principle, be used.

The single photon sensitive SPAD array cameras with picosecond resolution will allow us to observe the moment a cell responds to a chemical stimulus, and the location of that stimulus, at the level of single proteins. This will help us to understand how inflammation occurs, on a molecular basis. The technology we will develop will dramatically improve our understanding of dynamic events within cells offering insight into drug interactions in diverse applications throughout the life sciences - an area of great interest for the pharmaceutical industry. Their aim is to establish the efficacy of a new drug early in its development and on a molecular basis, reducing the reliance on lengthy clinical trials. The pharmaceutical industry saves money by adopting this approach, and patients benefit from an earlier availability of a new drug.

The public will also benefit from outreach activities, for example SPAD cameras were demonstrated at the Royal Society Summer Science Exhibition 2014, and the PI frequently talks at events such as the Pint of Science festival and the Crick's Science Museum Lates event "The Future of Biomedical Discovery", attracting 7000 visitors. He also oversaw the design and creation of the fluorescence exhibit in the Physics stand at the Big Bang fair in London's ExCel exhibition centre in 2011, an event attracting more than 29,000 visitors over 3 days.