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

Lead Research Organisation: Heriot-Watt University
Department Name: Sch of Engineering and Physical Science

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

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Yu H (2016) Estimation of Fluorescence Lifetimes Via Rotational Invariance Techniques. in IEEE transactions on bio-medical engineering

 
Description We have developed a computational method to correct an inherent optical aberration present in diffractive optics based multi-plane imaging systems. This approach greatly simplifies the use of multi-plane imaging for the biological sciences, and enables the technology to expand into practical applications.

As proposed in the grant we have developed fully comprehensive ImageJ software to use mutli-plane imaging systems that localizes particles in 3D using the above computational method and previously published depth localization methods.

The software is being tested by collaborators, and will be made open source once testing is complete.
Exploitation Route The programs will be made open source, available to the community and have been written such that different elements of the analysis can be used for multiple applications and methods. Preliminary work on toxicology and particle tracking in water have shown promise.

Methodology has been taken up by numerous other research groups in the UK.
Sectors Chemicals,Digital/Communication/Information Technologies (including Software),Environment,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description Facility Access to Central Laser Facility
Amount £1,000 (GBP)
Funding ID 16130044 
Organisation Science and Technologies Facilities Council (STFC) 
Sector Academic/University
Country United Kingdom
Start 05/2016 
End 05/2017
 
Description Facility Access to Central Laser Facility
Amount £1,000 (GBP)
Funding ID 16130035 
Organisation Science and Technologies Facilities Council (STFC) 
Sector Academic/University
Country United Kingdom
Start 05/2016 
End 03/2017
 
Description Facility Access to Central Laser Facility
Amount £1,000 (GBP)
Funding ID 15230040 
Organisation Science and Technologies Facilities Council (STFC) 
Sector Academic/University
Country United Kingdom
Start 01/2016 
End 04/2016
 
Description Facility Access to Central Laser Facility
Amount £1,000 (GBP)
Funding ID 16130041 
Organisation Science and Technologies Facilities Council (STFC) 
Sector Academic/University
Country United Kingdom
Start 05/2016 
End 05/2017
 
Description Standard Research Grant
Amount £973,622 (GBP)
Funding ID EP/R035156/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Academic/University
Country United Kingdom
Start 08/2018 
End 04/2021
 
Description ATC CDT 
Organisation UK Astronomy Technology Centre (ATC)
Country United Kingdom 
Sector Academic/University 
PI Contribution Provide optical support, expertise and application for developing adaptive optics systems for life science microscopy. Share and EngD student between sites.
Collaborator Contribution Provide expertise on adaptive optics for astronomy, and basis in which to translate it into life science microscopy, in particular for developing 4D imaging solutions.
Impact None to date, early stage project.
Start Year 2016
 
Description Francis Crick - Anderson Group 
Organisation Francis Crick Institute
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution We provide multi-plane optics and analytical software for super resolution 3D imaging solutions for cellular biology.
Collaborator Contribution Anderson group will apply the provided technology to various imaging modalities and investigate its viability for single molecule localisation challenges in real biological applications.
Impact Multi-disciplinary in that the developed technology will have uses throughout the life sciences at the Francis Crick.
Start Year 2013
 
Description ICFO 
Organisation ICFO - The Institute of Photonic Sciences
Country Spain 
Sector Charity/Non Profit 
PI Contribution Provided a multi-plane imaging system for trial to Dr, Melike Lakadamyali at the Institute of Photonic Sciences
Collaborator Contribution Offered an alternative 4D imaging system to explore single molecule fluorescence.
Impact Proof of concept trial.
Start Year 2016
 
Description IGMM - Bickmore Group 
Organisation University of Edinburgh
Department Institute of Genetics & Molecular Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution We supply experimental protocols, analytical solutions and supporting calibration standards for performing super resolution 3D imaging in cellular environments, with a particular emphasis on dual colour imaging challenges.
Collaborator Contribution IGMM provide exemplar biologically relevant data, notably that of chromatin folding in the nucleus of mammalian cells for the study of gene enhancers. A joint PhD student is shared between us at HWU and IGMM to work on this project.
Impact Multi-disciplinary between IGMM biologists and HWU physicists with cellular microscopy the common application. Two conference presentations to date (including dual poster presentatiosn at SULSA 2014 meeting in Edinburgh). EPSRC first grant was submitted, but rejected, based on this collaboration in 2014.
Start Year 2013
 
Description RAL - Webb 
Organisation Rutherford Appleton Laboratory
Country United Kingdom 
Sector Public 
PI Contribution We share and EngD PhD student and receive and provide in kind support for an MRC Advanced imaging grant to develop 3D multi-plane and adaptive optics technologies for single molecule localization in cellular systems. I co-supervise the student, meet with them regularly and form part of a highly active research partnership.
Collaborator Contribution RAL hosts the EngD student and leads the development of the work.
Impact None yet.
Start Year 2013
 
Title Grism optical system 
Description An adaptive optics Grism system for correcting chromatic aberations in a grating base multi-plane imaging platform. 
IP Reference GB2504188 
Protection Patent application published
Year Protection Granted 2014
Licensed No
Impact n/a
 
Title 3D Plug In 
Description An ImageJ plug-in to handle and analyse multi-plane imaging data for microscopy applications. Designed in particular for use with grating base imaging platforms, the software is compatible with any multi-plane imaging system. The software is separated into distinct parts, image segmentation, spot localisation, deconvolution and 3D localization. Each part has multiple possible applications. The software includes a novel deconvolution approach to account for chromatic distortion introduced in grating based imaging systems. 
Type Of Technology Software 
Year Produced 2016 
Open Source License? Yes  
Impact Software is being trialed by collaborative groups in Glasgow (Beatson) and RAL STFC. Both groups are applying this to novel imaging platforms for life sciences. 
 
Description Applied Photonics EngD conference (Edinburgh) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact ~50 EngD students and ~30 academics and others attended the Centre for Doctoral Training in Applied Photonics Annual Conference. An EngD student who worked on the research covered by this awarded gave a talk, assisting in the training of his fellow postgraduate students by contributing to the demonstration of the wide applications of photonics.
Year(s) Of Engagement Activity 2016
URL http://www.cdtphotonics.hw.ac.uk/sites/www.cdtphotonics.hw.ac.uk/files/CDTAP%20handbook%202016%20.pd...
 
Description Bang Goes the Borders Science Festival 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Presented a hands on public exhibition that demonstrated microscopy, fluorescence, cellular imaging and biological application.

Positive feedback from participants and organisers.
Year(s) Of Engagement Activity 2014
URL http://www.bgtb.org/
 
Description Focus on Microscopy 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Presentation at the 2015 conference in Gottinberg by Eric Pitkeathly, project postdoc.

n/a
Year(s) Of Engagement Activity 2015
 
Description IOP Teachers Talk 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Gave talk to high school physics teachers as part of IOP meeting on microscopy, and impact of physics on the life-sciences.
Year(s) Of Engagement Activity 2016
URL http://enlightenment.hw.ac.uk/
 
Description MMC2015 Conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Poster presentation on 3D imaging by Eric Pitkeathly, project postdoc

n/a
Year(s) Of Engagement Activity 2015
 
Description Midlothian Science Festival 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Presented a hands on public exhibition that demonstrated microscopy, fluorescence, cellular imaging and biological application. Public from age 5 to adults engaged with demonstrations, used microscopes, discussed science with group of expert PhD students and volunteers.

Positive feedback from participants and organisers. Meeting with representative from Wellcome trust sparked a discussion that has led to a large scale public outreach grant application.
Year(s) Of Engagement Activity 2014
URL http://midlothiansciencefestival.com/
 
Description SULSA Meeting 2014 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Scientific discussion

n/a
Year(s) Of Engagement Activity 2014
URL http://www.sulsa.ac.uk/sulsa-2014-research-symposium-optical-imaging-cells-single-molecules-organell...
 
Description School Talk - North Berwick 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Gave presentation to secondary school students on academic career. Received positive feedback from teachers and some questions from students.

School showed interest in returning for future talks.
Year(s) Of Engagement Activity 2014
 
Description Techfest 2014 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Presented a hands on public exhibition that demonstrated microscopy, fluorescence, cellular imaging and biological application. Public from age 5 to adults engaged with demonstrations, used microscopes, discussed science with group of expert PhD students and volunteers.

Also presented a ticketed public lecture on microscopy and fluorescence.

Positive feedback from participants and organisers. Major questions and discussions after public lecture from public audience.
Year(s) Of Engagement Activity 2014
URL http://www.techfestsetpoint.org.uk/tis/