Superresolved, 3D, multi-fluorophore tracking of live-cell dynamics

Recent work on adaptive optics technologies, funded by the Science and Technology Facilities Council, has demonstrated a method that allows simultaneous imaging of many depth layers, and ultra-precise measurement of fast-changes movements, in the biological microscopy, including live-cell applications. This 'small grant' research proposal will develop software and design tools that will allow any biology microscopy groups to use this novel imaging technique on their own microscopes. We will distribute the software and design tools to biologists who wish to use it to further their research.

This multi-disciplinary proposal will address a 'technology gap' in 'bioimaging and functional analysis', by making quantitative, high-resolution, 3D, time-resolved, information available from a new class of image data in fluorescence microscopy. We will develop new software tools for the capture and analysis of images to facilitate studies of fast, dynamic and 3D processes in live-cell biology, made accessible by recent optical and algorithmic developments.
The imaging technique involved uses a simple and inexpensive (~£5-6000) microscope attachment that provides between 2 and 9 different specimen planes simultaneously in focus on a single camera.
Spatial resolution better than 10nm has been shown to be routinely available in test measurements and from a single camera frame. The in-focus planes can be designed to cover the full depth of a cell (i.e. ~10 microns).
The technique uses a combination of the microscope attachment described above, with wavefront-sensing techniques derived from the exploitation of 'adaptive optics' in astronomy and a Maximum Likelihood algorithm, for superresolved determination of the depth of particles within the specimen. This will be combined with advanced tracking algorithms previously developed within our group.
We will develop and distribute software that will allow microscopy groups both to design a microscope attachment suitable their particular application and to analyse the data that that imaging system provides.

The principal academic beneficiaries are microscopists wishing to track fluorescent particles (e.g. vesicles) and fast-dynamic processes with high spatial resolution in live-cell microscopy and for whom the programme will make these technologies easily and openly accessible. In particular, by providing data on the dynamics associated with organelles in both normal and dysfunctional cells this study can make contributions to the understanding of important processes such as exocytosis, endocytosis, the dynamics of cytoskeletal structure and cargo transport.

Benefit will also available to microscopists working on fixed cells where a z-stack is required, but there are problems associated with bleaching of fluorophores.

For biologists working in these areas it is intended that the results from this programme, together will all optical designs, will become openly available shortly after the programme ends and the systems are as bug-free and user-friendly as is practical. For UK biologists we anticipate that dissemination of the results and system through our web site and through the contacts that we have established with biologists involved as investigators will occur rapidly and even during the programme (for those willing to work with systems still under development - indeed such interactions can strengthen our work in the later stages). For non-UK biologists we wish also to make all software and hardware freely available to those who wish to have such information, and will ensure open access at the earliest possible point. We see providing support to companies who in turn provide equipment to microscopists as a valuable method to assist wider dissemination.

Wider benefit from this work will be found in many fluid-flow areas, such as the fluid-flow in heat exchangers, the analysis of mixing in fuel systems and bioreactors, flow in food-drying towers, flow through porous media (in the context of models of such flow based on transparent constructions), applications in contexts such as the oil industry for monitoring the distribution and motion of and oil mixtures in sea-water pumped through wells in the later stages of oil extraction from wells in remote locations (esp the sea bed). In the context of fuel mixing, even minor fuel efficiencies achieved through improved understanding of these issues can lead to very-substantial savings and thus contribute to the reduction in emissions of both CO2 and particulate matter.

In addition, because the techniques considered here are clearly capable of achieving high-precision metrology measurements, the longer-term, prospects of surface-shape measurement may provide a useful tool for manufacturing.