Multidimensional fluorescence imaging of PIP2-derived intracellular signals in directional cell movement

Lead Research Organisation: Imperial College London
Department Name: Dept of Physics

Abstract

Directional cell migration is important during early development, inflammatory responses to infection, wound healing, and also during tumor invasion and metastasis. Since deregulation of this process has been linked to various pathological events, it has become an active area of research for therapies. Different lines of experimental evidence suggest that various types of migratory cells share a conserved set of signals involved in cell polarization and motility. Several classes of signalling molecules, including enzymes involved in turnover and modification of phosphoinositides and components of signalling networks controlling Rho GTP-ases, play key roles in these processes. Recent studies using advanced fluorescence microscopy suggest that understanding how intracellular signals control directional cell movement is critically dependent on their dynamic organization in time and space. Fluorescence microscopy entails 'labelling' proteins of interest with fluorescent molecules ('fluorophores') such as genetically expressed fluorescent proteins that can be used to tag specific proteins in living cells. Fluorophores are 'excited' by illumination at a wavelength that they absorb and the resulting emission (fluorescence) is recorded using an imaging detector. The technique called Förster Resonant Energy transfer (FRET) works by labelling two proteins with different fluorophores or incorporating different fluorophors into single protein that changes shape in response to specific signals. The property of the two fluorophores is chosen so that the excitation spectrum on one (the 'acceptor') overlaps with the emission spectrum of the other (the 'donor') and results in the transfer of energy from the excited donor to the acceptor only if they are in close proximity. This provides a basis to measure protein/protein interactions (not just co-localisation) and to use single protein probes as biosensors. One can image FRET by observing the donor or acceptor fluorescence intensity distributions but such intensity-based FRET is often unreliable because of background noise. More reliable techniques include mapping the ratio of acceptor to donor fluorescence and fluorescence lifetime imaging (FLIM) of the donor signal. In general, fluorescence lifetime is measured by exciting fluorophores with a short pulse of light and observing how long it takes the fluorescence signal to decay away as they relax back to their ground state. Using ultrafast camera technology, it is possible to image fluorescence decays across a sample and map the fluorescence lifetime. Because FRET provides an additional route for excited donor fluorophores to lose their energy, FLIM can map where FRET is occurring by observing the resulting reduction in donor fluorescence lifetime. In a recent BBSRC project we developed a novel high-speed FLIM microscope able to 'multiplex' FRET imaging of two probes (either for protein-protein interaction or biosensors). This permits us to simultaneously map the spatiotemporal properties of two different signalling events in live cells. Here we intend to extend the approach to multiplex more simultaneous signalling events and to use FRET to focus on some of the key components controlling the directional movement of cells, in particular those associated with intracellular signals derived from a membrane phosphoinositide, PIP2. Our goal is to correlate these with other intracellular signals in the same polarized, moving cell and to analyse dynamic aspects of their timing and localisation (e.g. front and back of polarized cell). This would provide new insights into the sequence of cell signalling interactions and some underlying molecular mechanisms important for the development of therapies for various pathological events resulting from deregulation of directional cell movement. The technical innovations of FRET methodology proposed here would find wide application in biology.

Technical Summary

Building on our ongoing collaboration, this multidisciplinary joint proposal aims to study cell signalling, in live cells and in vitro, using novel fluorescence imaging technology to measure FRET, particularly using biosensors. In our previous BBSRC funded project (BB/E003621/1) we successfully developed custom FRET constructs and robust new multiplexed FRET imaging technology to independently read out two signalling events simultaneously. This was complemented by the development of a multidimensional fluorometer for cuvette-based studies, including FRET, at Imperial (BB/E000495/1). We now wish to apply this new technology to study the signalling networks controlling directional cell movement. We plan to focus on intracellular signals derived from PIP2, which we have extensively studied at the ICR. Our multiplexed FRET methodologies give us a unique capability to tackle one of the main challenges, i.e. to correlate different dynamic signalling events. First, we will determine which candidate molecules (PLC isoforms) involved in intracellular signalling from PIP2 are critical to regulation of directional movement of fibroblasts towards platelet-derived growth factor. We will then correlate signalling events generated by PLC and PIP2-derived signals with activation of other components involved in cell movement, particularly Rho GTP-ases, and will test the links between them. For this we will make FRET constructs from known biosensors and refine and apply fluorescence instrumentation utilising spectral, polarisation and time-resolved measurements. Multiplexing FRET biosensors will permit us to study the timing and localisation (front and back of a polarized, moving cell) of specific signalling events under the same conditions, within the same cell. We will further determine the molecular mechanism of PLC activation by Rac, a molecule with an established role in cell movement, through in vitro FRET studies.

Planned Impact

The biological questions we propose to address have direct relevance to understanding various diseases including cancer, with clear impact on the development of more effective therapies, thereby enhancing the quality of life for patients and providing significant commercial opportunities. In general, the outcomes of this project will be directly relevant to scientists using fluorescence - in almost any field of science - since FLIM provides a robust tool for molecular imaging, contrasting different fluorophores and different fluorophore environments. Multiplexed FRET is especially useful to life scientists wishing to study cell signalling networks. This is a vital area for the understanding of disease and the development of more effective therapies. The specific results related to cell movement would directly impact tumour invasion and metastasis as targets for therapies. Longer term beneficiaries therefore include patients suffering from diseases, clinicians and the taxpayer, who would benefit from the reduced healthcare costs associated with more effective therapies. The FRET based biosensors that we will develop could be applied in a wide range of disease related fields including cancer. They could be used by scientists in universities, research institutes and industry. In the medium term, the biosensors and fluorescence-based technology could be applicable to many fluorescence assays and so would be applicable to drug discovery, e.g. using High Content Analysis and other approaches. The outcomes of this project could therefore be of significant commercial interest to pharmaceutical companies and the drug discovery industry in general - including, assay developers, instrumentation developers and drug screening companies. The pharmaceutical sector is important to the UK economy and the ability to study multiple events in cell signalling networks could enhance the success of screening campaigns and the efficiency of the drug discovery pipeline. Developing even one successful drug brings enormous financial benefits and the clinical benefits directly impact the quality of life for patients. If the efficiency of the drug discovery process can be improved, this will reduce the cost of drug discovery and therefore the cost of successful drugs. It could also reduce the number of animals required for testing drugs. Besides giant pharma, there are many smaller companies and SME's in the UK who innovate assay and instrumentation development for drug discovery. Access to a range of proven biosensors could create new opportunities for assay developers and the demonstrated success of multiplexed FRET would create new commercial opportunities for instrumentation manufacturers who could upgrade their microscopes and plate readers. Successful outcomes of this project would also drive the development of software tools since there are many challenges in the data analysis of multidimensional time lapse fluorescence imaging and this would create employment and commercial opportunities in the software sector. We anticipate that this project could immediately lead to new drug targets and the impact on assay developers and instrumentation manufacturers could be significant within 5 years. The researchers working on our project would develop valuable skills in molecular biology, imaging, software development, instrument design and data analysis. This could lead to employment in the pharmaceutical or other industrial sector, academic or clinical research or in consulting or policy development in the public or private sectors. For commercial exploitation, the ICR's dedicated department for technology transfer and exploitation will liaise with Imperial Innovations. We note that Matilda Katan has an established link with Cancer Research Technology Limited (CRT Ltd) with the aim to implement new therapies for direct patient benefit and Mark Neil is a cofounder of a successful microscope spin-out company (Aurox Ltd).

Publications

10 25 50
 
Description Directional cell migration is important during early development, inflammatory responses to infection, wound healing, and also during tumor invasion and metastasis. Deregulation of this process has been linked to various pathological events and has become an active area of research. Previous work suggests that various types of migratory cells share a conserved set of signals involved in cell polarization and motility. Several classes of signalling molecules, including enzymes involved in turnover and modification of phosphoinositides and components of signalling networks controlling small GTP-ases, play key roles in these processes. The Imperial College London group developed new quantitative fluorescence microscopy techniques during this project and applied these with the UCL team to study how intracellular signals, particularly signalling of enzymes in the PLC family, control directional cell movement. This entailed imaging the dynamic organization of signalling molecules and their interactions in space and time, including in live cells migrating towards a chemoattractant.
Fluorescence microscopy entails "labelling" proteins of interest with fluorescent molecules ("fluorophores") such as genetically expressed fluorescent proteins that can be used to tag specific proteins in living cells. Protein interactions have been widely studied by labelling specific kinds of protein with a different fluorophores and comparing the images acquired at the characteristic fluorophore emission wavelength. Unfortunately this "co-localisation" is limited by diffraction to a few 100 nm resolution while typical signalling proteins are much smaller (<~1 nm). It is possible, however, to detect and even measure protein separations <~10 nm using a technique called Förster Resonant Energy transfer (FRET), which entails labelling proteins with fluorophores for which the excitation spectrum of one (the "acceptor") overlaps with the emission spectrum of the other (the "donor") and observing the transfer of energy between them. While FRET can be imaged by observing the donor or acceptor fluorescence intensity distributions, this is often unreliable because of background noise and instrument/sample attenuation. More reliable techniques include mapping the ratio of acceptor to donor fluorescence intensity or fluorescence lifetime imaging (FLIM) of the donor signal. In general, fluorescence lifetime is measured by exciting fluorophores with a short pulse of light and observing how long it takes the fluorescence signal to decay away as they relax back to their ground state. Using ultrafast camera technology, it is possible to image fluorescence decays across a sample and map the fluorescence lifetime. Because FRET provides an additional route for excited donor fluorophores to lose their energy, FLIM can map where FRET is occurring by observing the resulting reduction in the donor fluorescence lifetime.
Building on a previous BBSRC project, we developed a novel high-speed FLIM microscope able to image FRET in live cells and to "multiplex" such readouts, e.g. to simultaneously map different signalling events using FRET-based and other biosensors. This included the development of novel hardware and software tools. In particular we developed a powerful software tool for characterizing complex fluorescence decay profiles that we have released as open source code for non-commercial public use.
We applied FRET to study key components controlling the directional movement of cells, in particular to intracellular signals derived from a key signalling molecule (membrane phosphoinositide, PIP2), reading out the spatiotemporal evolution of this signal in cells moving towards a stimulant (i.e. cells undergoing "chemotaxis"). Thus we determined that a specific enzyme (PLCe) is critically involved in directional movement of fibroblasts towards a chemoattractant and contributes to localisation and persistence of signalling in protrusions responding to chemotactic gradient. This was unexpected because another enzyme (PLC?) in the same family was believed to be the only relevant one for this process. We also studied signalling processes associated with the PLC family of enzymes in solution-based studies and applied intra-molecular FRET to study changes in the conformation of a PLC member (PLC?2) when binding Rac, another important signalling molecule and important regulator of cell motility. Intramolecular FRET entails labelling the same molecule with both donor and acceptor fluorophores and observing when the FRET signal (i.e. donor fluorescence lifetime) changes if and when the molecule changes its configuration.
These biological findings have implications for the development of therapies for various pathological events resulting from deregulation of directional cell movement. The advances in qualitative fluorescence microscopy (i.e. FRET and FLIM) should find wide application studying signalling molecular processes in biology.
Exploitation Route The new biology knowledge gained during this project will be utilized by ourselves and other researchers studying disease mechanisms with a view to developing new therapies to address pathological events resulting from deregulation of directional cell movement, which is important for cancer (e.g. tumour invasion and metastasis) and for tissue remodelling, regeneration and wound healing. We have published our key findings in peer-reviewed journals and we aim to build on these findings in future research projects
The improved FRET sensors developed during project should be of interest to other biologists wishing to study cell signalling and we will distribute the plasmids for these on request. These new FRET sensors have improved fluorescence decay properties for fluorescence lifetime imaging that is useful if they are to be multiplexed with other fluorescence readouts. A key future direction for us is to translate these techniques for studying protein interactions in cell signalling networks to increasingly higher-throughput formats for high-content biology assays that would be useful for accelerating drug discovery.
The new fluorescence microscopy instrumentation, acquisition and analysis experience gained by our postdoctoral research associates and 4 PhD students will be exploited by them in their future careers and help spread the instrumentation and analysis approaches developed during this project. The specific hardware and software that we have developed have been reported in journal publications and will be useful for future research by us and by laboratories interested in studying cell signalling. The FLIMfit fluorescence lifetime image analysis software that was developed during this project has been made publically available through the Open Microscopy Environment (http://www.openmicroscopy.org/site/products/partner/flimfit). This software includes global fitting tools to fit the complex fluorescence decay profiles often encountered when analysing FRET data derived from fluorescence proteins in biosensors and can also be widely applied to fluorescence lifetime imaging-based studies in general.
The biological knowledge gained on the roles of PLCepsilon and Rac activation of PLC in cell movement could be used by companies developing therapeutics for cancer and other diseases or processes associated with directed cell motion including tissue remodelling, regeneration and wound healing.
The new FLIM instrumentation and software developed during this project is being translated to extended live cell imaging studies and to automated high content analysis. This work is closely followed by our partners in industry (AstraZeneca, GSK, Pfizer and GE Healthcare) and we hope to work with them in the future to implement our new technology in the drug discovery process.
The FLIMfit software is available under an open-source license and will therefore provide a sophisticated tool for industrial research utilising fluorescence lifetime imaging, e.g. in drug discovery.
One specific route of direct follow up is related to new insights into importance of PLC enzymes in a disease context. It has recently been discovered that PLCgamma isoforms not only contribute to cancer (and some other diseases) as components of the aberrant signal transduction networks but also themselves incorporate mutations that trigger such aberrant signalling (reviewed in Koss et al. TIBS 2014). The data obtained and methodologies developed during this project established several potential signalling links and assays to assess them that can now be used to test the role of mutated PLCgamma variants in cell motility related to angiosarcoma. Furthermore, the renewed interest in drug discovery by targeting PLCgamma will utilise some of the downstream FRET readouts established in this project.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology,Other

 
Description We believe that our work has stimulated other researchers to build similar systems which has benefited the manufacturers of the key technology components that we use by increasing their sales, particularly the supercontinuum source and the time-gated image intensifier. We also note that there is considerable activity developing alternative technologies to realise rapid FLIM and FRET and we believe that some of this activity has been stimulated by our work
First Year Of Impact 2013
Sector Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Description BBSRC IPA BB/M006786/1
Amount £508,815 (GBP)
Funding ID BB/M006786/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 05/2015 
End 04/2017
 
Title TagRed-T-PLCdelta-PH/GFP-PLCdelta-PH FRET sensor 
Description TagRed-T-PLCdelta-PH/GFP-PLCdelta-PH FRET sensor for PLCdelta-PH aggregation following changes in PIP2 concentration 
Type Of Material Model of mechanisms or symptoms - in vitro 
Provided To Others? No  
Impact This FRET biosensor enables PIP2 signalling to be visualised in live cells 
 
Title TagRed-T/mPlum-PBD FLAIR FRET sensor for Rac1 activation 
Description TagRed-T/mPlum-PBD FLAIR FRET sensor for Rac1 activation, red FRET sensor can be multiplexed with CYP/YFP FRET sensors (issues of photobleaching and photoconversion) 
Type Of Material Model of mechanisms or symptoms - in vitro 
Provided To Others? No  
Impact In principle this FRET biosensor can be multiplexed with other biosensors utilising CFP/YFP derivative fluorophore pairs. However, the photo physical properties of the donor mean that it is challenging to use for FLIM 
 
Title mCherry-AKT-PH homoFRET sensor 
Description mCherry-AKT-PH homoFRET sensor for AKT aggregation following changes in PIP3 concentration, read out using FAIM; red FRET sensor can be multiplexed with other CYP/YFP FRET sensors 
Type Of Material Model of mechanisms or symptoms - in vitro 
Provided To Others? No  
Impact This FRET sensor was multiplexed with a calcium FRET sensor and the work was published in the International Journal of Molecular Sciences 
 
Title mTurquoise-Rac 1/Cypet-PBD FLAIR FRET sensor for Rac1 activation 
Description mTurquoise-Rac 1/Cypet-PBD FLAIR FRET sensor for Rac1 activation, contrast different lifetimes of mTurquoise and CyPet to identify cells transfected with both donor and acceptor; read out using time-resolved fluorescence anisotropy imaging (TR-FAIM) or lifetime 
Type Of Material Model of mechanisms or symptoms - in vitro 
Provided To Others? No  
Impact This is an improved FRET biosensor for Rac 
 
Title mTurquoise-Rac 1/Ypet-PBD FLAIR FRET sensor for Rac1 activation 
Description mTurquoise-Rac 1/Ypet-PBD FLAIR FRET sensor for Rac1 activation, optimised for FLIM by replacing CFP with (non-exponential) mTurquoise 
Type Of Material Model of mechanisms or symptoms - in vitro 
Provided To Others? No  
Impact This FRET biosensor for Rac was improved for fluorescence lifetime readouts by changing the donor to mTurquoiseFP 
 
Title mTurquoise/EYFP LIBRA FRET sensor for IP3 concentration 
Description mTurquoise/EYFP LIBRA FRET sensor for IP3 concentration, optimised for FLIM by replacing CFP with (non-exponential) mTurquoise 
Type Of Material Model of mechanisms or symptoms - in vitro 
Year Produced 2017 
Provided To Others? Yes  
Impact This improved FRET biosensor enables IP3 signalling to be visualised in live cells. We have provided it to the Netherlands Cancer Institute who are investigating PLC signaling by GPCRs. They believe our FRET sensor may provide a superior response to others they have tried. 
 
Description Kentech Instruments Ltd 
Organisation Kentech Instruments Ltd
Country United Kingdom 
Sector Private 
PI Contribution We have advised Kentech on improvements to be made to their gated optical image intensifiers and related components and have tested prototypes. We have demonstrated that their gated intensifiers can be integrated into a range of optical instruments, particularly for fluorescence lifetime imaging (FLIM) for biomedical applications, and this has led to increased sales for them. We collaborated with them on an EPSRC Healthcare Partnership project that built on earlier collaborations with Kentech and also on two MRC projects to develop FLIM instrumentation.
Collaborator Contribution Kentech Instruments Ltd advised us (with no charge) on the design of FLIM systems using gated optical image intensifiers and supplied us with a custom device for handheld FLIM that we designed in collaboration. They have sold us customised gated intensifier systems and refined the specifications of their products at our request.
Impact Joint publications Feedback to Kentech's product development
 
Title FLIMfit 
Description New open source fluorescence lifetime fitting tool with unprecedented functionality and speed ?FLIMfit? developed and released through Open Microscopy Environment http://www.openmicroscopy.org/site/products/partner/flimfit This software includes global fitting tools to fit the complex fluorescence decay profiles often encountered when analysing FRET data derived from fluorescence proteins in biosensors. 
Type Of Technology Software 
Year Produced 2017 
Open Source License? Yes  
Impact This FLIMfit software has been downloaded and is in use in many locations including the UK Europe, USA and Australia. It has been used by us for FLIM/FRET high content analysis and for optical projection tomography. It has been continuously refined and updated until the present (2017). 
URL http://www.openmicroscopy.org/site/products/partner/flimfit
 
Title openFLIM HCA: Automated multiwell plate FLIM microscope for optically sectioned assays in 3-D cell culture including time-lapse FLIM/FRET studies in live cells 
Description This automated multiwell plate FLIM microscope is controlled by open source (MIcroManager) software and the component lists are shared on our website to enable other research groups to replicate our instrumentation. FLIM data analysis, including global fitting, is provided by our open source software tool: FLIMfit, written in MATLAB. Exciation is provided by a super-continuum source or a frequency-doubled mode-locked Ti:Sapphire laser. FLIM detection is realised though time-gated imaging using a gated image intensifier. This instrument typically acquires a field of view every 10 seconds, including the time for sample movement and autofocus. 
Type Of Technology New/Improved Technique/Technology 
Year Produced 2016 
Impact This instrument has been applied to a number of applications including protein interactions of the kinetochore, metabolic changes in differentiating embryonic stem cells, bromodomain-histone binding and a genetically expressed FRET biosensor for EPAC,a Rap-1 guanine exchange factor that binds specifically to cAMP. 
URL http://www.imperial.ac.uk/photonics/research/biophotonics/instruments--software/openflim-hca/
 
Title openHCA-FLIM µManager Plugin 
Description This is the (MicroManager Plugin) control software for our openFLIM HCA instrument that provides automated multiwell plate FLIM, with the option to implement optical sectioning. 
Type Of Technology Software 
Year Produced 2016 
Impact This open source software is freely available and should enable other research groups to build their own FLIM HCA instrumentation. 
URL http://www.imperial.ac.uk/photonics/research/biophotonics/instruments--software/openflim-hca/
 
Description Biophotonics Summer School Hven 2015 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact I lectured on the Biophotonics Summer School organised in Hven, Sweden every two years. This is probably the highest level school int he field in terms of the lecturers and attracts students from all over the world. I presented the basic principles of fluorescence microscopy and our latest research in multidimensional fluorescence imaging including super-resolved microscopy, FLIM and optical tomography as well as clinical applications
Year(s) Of Engagement Activity 2015
URL http://www.biop.dk/biophotonics15/school/school.asp?page=main_school_lecturers
 
Description Crick retreat 2011 MDFI across scales 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other audiences
Results and Impact Invited talk, Francis Crick Institute retreat, Ashridge, 2011

no actual impacts realised to date
Year(s) Of Engagement Activity 2011
 
Description GSK Stevenage 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Industry visit and seminar.

Paul French gave an invited seminar on FLIM and its biomedical applications including results from this project.
Year(s) Of Engagement Activity 2012
 
Description MDFI for HCA workshop 2014 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact This workshop on multidimensional imaging for high content analysis was intended to showcase our MDFI technology, including FLIM and FRET, implemented in automated multiwell plate readers, endoscopes, microscopes and in vivo tomographic imaging of zebrafish. Our target audience was industry, including pharma and technology developers, as well as research students and academics from Imperial and other universities.
Discussions with industry led to new collaborations with SME to develop advanced imaging technology and continued interest from pharma who gave feedback on our technology and potential applications. This event included an internet presentation from Rainer Pepperkok at EMBL.
Year(s) Of Engagement Activity 2014
 
Description MDFI user-orientated workshop 2017 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact This one day user-orientated workshop entitled "Multidimensional fluorescence imaging technology: super-resolution, HCA and preclinical imaging" showcased the biophotonics instrumentation and applications of the technologies developed in the Photonics Group at Imperial College London. It attracted 80 participants including 11 from industry and 40 for other research organisation. The talks and posters were well received and this event led to further collaborations, including with industry.
Year(s) Of Engagement Activity 2017
 
Description MDFIM user-orientated workshop 2013 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Type Of Presentation Workshop Facilitator
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Multidimensional fluorescence imaging and metrology user-orientated workshop held at Physics Department, Imperial College London on 27/09/13.
~50 external visitors attended talks, posters and networking sessions. The aims were to identify new users of our research, to reinforce relationships with existing users, to identify new research partners, sponsors and opportunities. The event was also a showcase for our research progress and students and staff and a source of feedback to inform our ongoing technology development.

Three companies expressed interest in commercialising aspects of our work
Several external research partners learned about our capabilities and suggest new research collaborations
We received excellent feedback about quality of work presented and of presentations
Year(s) Of Engagement Activity 2013
URL http://www3.imperial.ac.uk/photonics/events/photworkshop
 
Description Multidimensional fluorescence imaging - for clinical diagnosis, cell biology and drug discovery 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact 1st Europhotonics Spring School, Barcelona, Spain.
Year(s) Of Engagement Activity 2012
 
Description PQ Short course MDFI 2008 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Invited talk, 6th European Short Course on Principles and Applications of Time-resolved fluorescence, PicoQuant GmbH, Berlin, Germany.
Year(s) Of Engagement Activity 2008