Development of a super-resolving STED FLIM microscope for biological applications

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

Abstract

We aim to develop a fluorescence microscope to image molecular processes with significantly improved spatial resolution in order to study the mechanisms of disease at a molecular level. By using fluorescent molecules ('fluorophores') to label proteins associated with signalling, e.g. at the interface ('immune synapse') between white blood cells and suspect cells being interrogated by the immune system, it is possible learn about the spatial and temporal organisation of specific proteins and their interactions. Unfortunately the resolution of fluorescence microscopes is limited by diffraction to ~300 nm and the molecules of interest are much smaller. One way to probe their interactions is to use spectroscopic techniques because the properties of fluorescence emission can vary according to the local environment of the fluorophore and can also be used to distinguish different molecular species. By labelling different proteins with fluorophores emitting at different wavelengths and then comparing the different 'colour' images, one can obtain information about co-localisation, from which interaction can be inferred - albeit limited by the spatial resolution. More can be learned from the fluorescence lifetime - the time over which a fluorescence signal decays - which can be sensitive to its local physical or chemical environment and to the presence of other molecules. Fluorescence lifetime imaging (FLIM) can be used to map changes in molecular environment and to detect protein-protein interactions by exploiting Förster Resonant Energy transfer (FRET), where the emission of one fluorophore-labelled protein is quenched by direct energy transfer to nearby suitable fluorophores that can be labelling a second molecule. This energy transfer, which can only occur if the fluorophores are within ~10 nm, also reduces the fluorescence lifetime and so FLIM provides a means to map when and where pairs of proteins interact - to a precision limited by the spatial resolution of the microscope. In a study of inter-cell signalling, we used FLIM-FRET to image the phosphorylation (i.e. the 'activation') of a key molecule (the KIR receptor) involved in determining the response of a white blood cell when interrogating a suspect cell. Unexpectedly, we observed that the phosphorylation of the KIR receptor occurred in small microclusters - but the confocal microscope did not have sufficient spatial resolution to do more than detect their presence. This is an exemplar application for which we are developing a new microscope capable of resolving structures below the diffraction limit. Others include the colocalisation and segregation of signalling molecules, the assembly of signalling complexes and the use of FLIM to elucidate the state and distribution of actomyosin cross bridges in muscle fibres. We aim to build a super-resolving fluorescence microscope using the technique pioneered by S. Hell called stimulated emission depletion (STED). A regular confocal microscope scans a focussed excitation beam across a sample and detects the resulting fluorescence to acquire an image, the resolution of which depends on the size of the focussed spot on the sample. In STED, the sample is scanned by two collinear beams: the first excites fluorescence in the usual way but the second 'STED' beam suppresses it by depleting the excited state population through stimulated emission. This second beam has a 'doughnut' profile with a hole in the middle such that the outside of the excitation spot is 'switched off' while the centre remains, thereby realising a smaller effective spot and resolution beyond the diffraction limit. Having demonstrated a STED prototype microscope incorporating FLIM and adaptive compensation of aberrations in the microscope, we now aim to build a system suitable for use by biologists to study the organisation of cell signalling and other molecules with resolution beyond the diffraction limit using techniques like FLIM and FRET.

Technical Summary

Microscopes offering resolution beyond the diffraction limit can probe biological structures and process on scales below ~300 nm. We have developed interdisciplinary research programmes to study cell signalling, many of which exploit FLIM to study molecular environments and interactions, including FLIM of membrane lipid microdomain, or 'lipid raft', activity, FLIM-FRET imaging of phosphorylation of signalling molecules and FLIM of actomyosin cross bridges in muscle sarcomeres. All these experiments were limited by the spatial resolution of our confocal microscope and our biology collaborators are keen to progress to experiments with superior resolution. As a first step, we demonstrated a novel STED FLIM microscope implemented in a standard laser scanning confocal microscope (LSCM) and utilising, for the first time, an ultrafast laser supercontinuum source to provide spectral versatility at relatively low cost. This novel instrument also included spatial light modulator technology to adaptively control the spatial profile of the depleting beam and to compensate for aberrations in the microscope. FLIM was implemented using time correlated single photon counting, making this the first super-resolved FLIM microscope, as well as the first STED microscope in the UK. Here we aim to develop a 'biology-friendly' instrument offering the full capabilities of a LSCM with the ability to 'zoom in' and obtain super-resolved (intensity and FLIM) STED images anywhere in the field of view. In our prototype STED microscope, we established that instabilities in the galvanometric scanners of our commercial LCSM limited our achievable resolution, as did vibrations from the pump laser. We will build a new, stable, instrument with stage and resonant scanning to be applied to studies of signalling molecule interactions in microclusters, the assembly of signalling complexes, kinetic segregation model studies, membrane nanotubes and actomyosin states in mammalian muscle sarcomeres.
 
Description We developed a novel STED microscope utilising a Ti:Sapphire laser to provide the depletion beam and also a tunable excitation beam from a supercontinuum generated using part of the Ti:Sapphire laser output. We also first demonstrated the use of a spatial light modulator (SLM) to generate the required "doughnut" depletion beam profile and to compensate for optical aberrations in the microscope. We also first demonstrated FLIM STED and how time-gated detection can be used to remove the early arriving non-super-resolved contribution to the signal.

This instrument has continued to be developed, building on the work done in this project, and was extended to provide super-resolution in 3-D and to compensate for aberrations in the instrument and sample using the spatial light modulator. It was applied to study biological and non-biological samples, including to provide super-resolved imaging of the immunological synapse (IS) between two cells with the IS in a vertical plane . Most recently we extended STED microscopy to our concept of easySLM STED, which provides robust operation by making it easier to maintain collinearity of excitation and depletion beams and enable correction of chromatic aberration that otherwise leads to walk-off between the excitation and depletion beams. This enables the STED field of view to be significantly extended.
Exploitation Route Following the end of the project, our STED instrumentation has been applied to cell biology and could be widely applied in this area. We also demonstrated its application to image colour centres in diamond and its application with nanoparticles that could provide a new labelling technique for biological samples.
Sectors Pharmaceuticals and Medical Biotechnology

URL http://dx.doi.org/10.1002/jbio.201300041
 
Description We developed this STED microscope to image cell biology, particularly at the immune synapse, and also applied it to nitrogen defects in diamond in a collaboration with De Beers, who sponsored an EPSRC CASE student. The technology and expertise gained in this project were later extended to a novel 3-D STED microscope configuration that has since been adopted by other researchers. Our approach to compensate for optical aberrations using a spatial light modulator has also been developed and further extended by others. Most recently we extended this work to the development of easySLM-STED, which provides robust operation with easy co-alignment of the exaction and depletion beams and facilitates the compensation of chromatic aberration to reduce walk-off between the scanning excitation and depletion beams and therefore enable STED microscopy over much larger fields of view. We are discussing this with a manufacturer of super-resolved microscopes.
First Year Of Impact 2011
Sector Education,Manufacturing, including Industrial Biotechology
Impact Types Economic

 
Description EPSRC CASE studentship with De Beers UK Ltd
Amount £22,279 (GBP)
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 10/2011 
End 03/2015
 
Description MRC MICA MR/K015834/1
Amount £2,466,513 (GBP)
Funding ID MR/K015834/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 02/2013 
End 02/2017
 
Description Nano-particle assisted super-resolution microscopy for live cell imaging
Amount £125,248 (GBP)
Organisation The Leverhulme Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 05/2014 
End 04/2016
 
Title Double passed Spatial Light Modulator arrangement for 3D-STED 
Description This method represents an elegant engineering solution for 3D-STED microscopy, simplifying alignment and also providing the means to perform adaptive imaging for improved image quality deep into aberrating samples such as live cells and tissue sections. 
Type Of Material Improvements to research infrastructure 
Provided To Others? No  
Impact Commercial instrumentation now uses a slightly modified version of this method. 
 
Title STED FLIM Microscope with adaptive optics 
Description We developed a novel configuration for STED microscopy utilising part of the output of a mode-locked femtosecond Ti:Sapphire laser to pump a supercontinuum in a microstructured fibre in order to produce tunable ultrashort pulse radiation for excitation of fluorescence. We used the remaining radiation from the Ti:Sapphire laser to provide the depletion beam, which we diffracted off a computer generated hologram on a spatial light modulator (SLM) to produce the desired "doughnut beam" profile. This provided a (relatively) low cost route to tunable excitation and depletion beams for STED microscopy. We also exploited our ability to control the spatial beam profile using the SLM to precompensate for optical aberrations in the microscope - and later in the sample. In addition we implemented time-correlated single photon counting detection (TCSPC) to provide fluorescence lifetime imaging (FLIM). We also used this time-resolution to select the later arriving photons since we discovered that the earliest arriving light did not contribute to the super-resovled STED image. 
Type Of Technology New/Improved Technique/Technology 
Year Produced 2010 
Impact This project produced the first demonstration of a STED microscope based on a mode-locked Ti:Sapphire laser pumping a supercontinuum for excitation and depleting with the Ti:Sapphire laser beam. It was also the first demonstration of STED FLIM, the first report of the variation in spatial resolution according to how the detected signal was analysed temporally and the first demonstration of the use of an SLM to compensate for aberrations in the system. All of these concepts have been built upon by others, including in commercial instruments. 
 
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 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 Multidimensional fluorescence imaging and metrology - for cell biology, high content analysis and clinical diagnosis 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Invited lectures at Biophotonics 2011 summer school, Ven, Sweden.
Year(s) Of Engagement Activity 2011
 
Description PicoQuant workshop 2013 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Invited oral presentation at the 3rd European Short Course on Time-Resolved Microscopy and Correlation Spectroscopy, Berlin, Germany. mainly aimed at post-graduate students

Pau French was asked to come back again to lecture on this training course
Year(s) Of Engagement Activity 2012
 
Description Plenary lecture ECBO 2013 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Plenary lecture, presented at the European Conference on Biomedical Optics, Munich 2013, entitled: "Fluorescence lifetime imaging and metrology for cell biology, high content analysis and clinical diagnosis"

Audience was > 500 including many attendees form industry since this conference was co located with Europe's largest laser trade fair.

Subsequently I received many enquiries about our work (including an request to give a webinar) and invitations to speak at further meetings.
Year(s) Of Engagement Activity 2013
URL http://www.osa.org/en-us/meetings/osa_meeting_archives/2013/european_conferences_on_biomedical_optic...