Multidimensional Fluorescence Imaging and Super-resolution Exploiting Ultrafast Laser and Supercontinuum Technology EGIDIJUS AUKSORIUS Department of Physics Imperial College London Thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy of the University of London December, 2008 Skiriu tėvams 3 Abstract This thesis centres on the development of multidimensional fluorescence imaging tools, with a particular emphasis on fluorescence lifetime imaging (FLIM) microscopy for application to biological research. The key aspects of this thesis are the development and application of tunable supercontinuum excitation sources based on supercontinuum generation in microstructured optical fibres and the development of stimulated emission depletion (STED) microscope capable of fluorescence lifetime imaging beyond the diffraction limit. The utility of FLIM for biological research is illustrated by examples of experimental studies of the molecular structure of sarcomeres in muscle fibres and of signalling at the immune synapse. The application of microstructured optical fibre to provide tunable supercontinuum excitation source for a range of FLIM microscopes is presented, including wide-field, Nipkow disk confocal and hyper-spectral line scanning FLIM microscopes. For the latter, a detailed description is provided of the supercontinuum source and semi-confocal line-scanning microscope configuration that realised multidimensional fluorescence imaging, resolving fluorescence images with respect to excitation and emission wavelength, fluorescence lifetime and three spatial dimensions. This included the first biological application of a fibre laser-pumped supercontinuum exploiting a tapered microstructured optical fibre that was able to generate a spectrally broad output extending to ~ 350 nm in the ultraviolet. The application of supercontinuum generation to the first super-resolved FLIM microscope is then described. This novel microscope exploited the concept of STED with a femtosecond mode-locked Ti:Sapphire laser providing a tunable excitation beam by pumping a microstructured optical fibre for supercontinuum generation and directly providing the (longer wavelength) STED beam. This STED microscope was implemented in a commercial scanning confocal microscope to provide compatibility with standard biological imaging, and exploited digital holography using a spatial light modulator (SLM) to provide the appropriate phase manipulation for shaping the STED beam profile and to compensate for aberrations. The STED microscope was shown to be capable of recording superresolution images in both the lateral and axial planes, according to the settings of the SLM. 4 Acknowledgments I would like to express my thanks to all those that helped me during my research and thesis writing. I am indebted to my supervisors, Paul French and Mark Neil for all the help, supervision and trusting me with this challenging but very interesting project. Thanks to Paul for his guidance, drive and enthusiasm and also for his support and patients when things did not go according to the plan or materialise ‘before Christmas’. Thanks to Mark for his skilful advice on all things regarding microscopy and spatial light modulators. I would also like to thank Chris Dunsby who taught me everything in the lab from laser physics to microscopy. Thanks to Peter Lanigan who introduced me to all the particularities of the Leica SP2 + B&H microscope system and to James McGinty who has been ordering numerous items when I urgently needed them and for being useful with the whereabouts of things in the Lab. Further thanks to Bosanta Boruah and Dylan Owen who contributed more directly to the ‘physics’ results in this thesis. I would also like to acknowledge help from Roy Taylor and Sergei Popov for their advice on fibre laser and supercontinuum generation and in particular thanks to Andrei Rulkov for his numerous efforts to keep my fibre laser running and to John Travers for all things supercontinuum. Also thanks to Gordon Kennedy for helping me with the ‘big’ lasers on the STED setup. Everyone in the optics workshop was always helpful and some of the setups would have not existed without their help, therefore thanks to Martin Kehoe, Simon Johnson, Martin Dowman, James Stone and Paul Brown. Thanks again to Chris and Valerie for proofreading this thesis. Special thanks to Vincent for being a great mate both in the office and out. I am grateful to all other present and past member in the group whose help was sometimes so important: Dan, Jose, Richard, Raul, Bebhinn, Delisa, Damien, Pieter, Khadija, Ian, Cliff, Ewan, David, Sunil, Hugh, Stephane, Tom, Anca, Ali and anyone else I may have forgotten. I also enjoyed your company in the H-bar and other ‘great’ places in South Ken! Many thanks to my friends whom I met here in London that made my life much more interesting outside the lab, especially thanks those from Lithuanian community, Ceilidh and Jonkelis dancing clubs. In particular, thanks to Agnė, Sebastian, Natalia and Anne for their friendship over those years. Also, I am grateful to Michael for his help and support in the final stages of the writing-up. And finally, but not least, thanks to my whole family and friends back in Lithuania who haven’t seen me much during this time but always supported and believed in me. 5 Acronyms described in: 2 D Two-Dimensional 3 D Three-Dimensional B&H 3.3.2 Becker and Hickl GmbH (company) CCD 3.2.1 Charge Coupled Device CW Continuous Wave EEM 4.3.3 Excitation Emission Matrix FLIM 3 Fluorescence Lifetime Imaging FRET 2.2.8 Fluorescence Resonance Energy Transfer FWHM Full Width Half Maximum GOI 3.3.1 Gated Optical Intensifier GVD 4.2.2 Group Velocity Dispersion I5M 2.4.3 Incoherent Interference Illumination Image Interference Micr. IRF 3.2.3 Instrument Response Function MOF 4.2.2 Microstructured Optical Fibre NA 2.3.1 Numerical Aperture OTF 2.3.1 Optical Transfer Function PMT 3.2.1 Photo-Multiplier Tube PSF 2.3.1 Point Spread Function SLM 5.2.7 Spatial Light Modulator SMF 4.2.2 Single Mode Fibre STED 2.6.1 Stimulated Emission Depletion τ 2.2.7 Fluorescence lifetime TCSPC 3.2.3 Time Correlated Single Photon Counting Ti:Sapphire Titanium Sapphire Contents 6 Contents Abstract ...................................................................................................................................... 3 Acknowledgments ..................................................................................................................... 4 Acronyms ................................................................................................................................... 5 Contents ..................................................................................................................................... 6 1. Introduction ........................................................................................................................ 9 2. Fluorescence Microscopy and Super-resolution ........................................................... 13 2.1 Introduction ................................................................................................................. 13 2.2 Fluorescence ................................................................................................................ 14 2.2.1 Jablonski diagram ................................................................................................. 14 2.2.2 Franck-Condon principle ...................................................................................... 14 2.2.3 Vibrational relaxation and Kasha’s rule ............................................................... 16 2.2.4 Fluorescence emission .......................................................................................... 16 2.2.5 Phosphorescence ................................................................................................... 17 2.2.6 Fluorescence quantum yield ................................................................................. 19 2.2.7 Fluorescence lifetime ............................................................................................ 19 2.2.8 Fluorescence energy resonance transfer (FRET) ................................................. 20 2.3 Fluorescence microscopy ............................................................................................ 21 2.3.1 Optical microscopy ............................................................................................... 21 2.3.2 Other contrast enhancing microscopy techniques ................................................ 24 2.3.3 Wide-field fluorescence microscopy .................................................................... 25 2.3.4 Multidimensional fluorescence microscopy ......................................................... 29 2.4 Fluorescence microscopy beyond the diffraction limit ................................................ 30 2.4.1 Wide-field fluorescence deconvolution microscopy ............................................ 30 2.4.2 Confocal microscopy ............................................................................................ 31 2.4.3 Other axial resolution improvement techniques ................................................... 35 2.4.4 Structured illumination for lateral resolution improvement ................................. 38 2.5 Non-linear optical microscopy .................................................................................... 40 2.5.1 Multiphoton microscopy .....................................................................................