Single Molecule Fluorescence Spectroscopy and Imaging: Advanced Methods and Applications in Life Sciences
by Arindam Ghosh
Date of Examination:2020-12-01
Date of issue:2021-06-03
Advisor:Prof. Dr. Jörg Enderlein
Referee:Prof. Dr. Jörg Enderlein
Referee:Prof. Dr. Fred Wouters
Files in this item
Format:PDFDescription:Doctoral thesis entitled "Single Molecule Fluorescence Spectroscopy and Imaging: Advanced Methods and Applications in Life Sciences"
EnglishThe visualization of biological structures down to the molecular length scale has been recently made possible by the development of super-resolution fluorescence microscopy. These techniques now routinely resolve biological structures down to a few nanometers. Various super-resolution techniques have been developed, the most successful being Stimulated Depletion Emission (STED) microscopy and Single Molecule Localization Microscopy (SMLM). In what follows, I will focus on the latter class of techniques which is based on the fact that a single molecule image allows for localizing the molecule with a much higher accuracy than the diffraction limit of resolution of the used microscope. However, a big challenge of SMLM is to achieve a similar super-resolution along the optical axis of a microscope. For this purpose, metal-induced energy transfer (MIET) imaging was recently introduced as an elegant method for axially localizing fluorophores with nanometer precision. The underlying principle of MIET is based on an electromagnetic near-field-mediated energy transfer from an excited fluorescent emitter (donor) to a thin planar metal film (acceptor). This energy transfer leads to a distance-dependent modulation of an emitter’s fluorescence lifetime (quenching), that can be easily measured with conventional fluorescence lifetime measurement techniques. The power of MIET is that it works with any fluorophore, and it only requires a conventional fluorescence lifetime imaging (FLIM) microscope. In this thesis, I present a powerful modification and further development of MIET, that is called graphene-induced energy transfer (GIET). GIET replaces the metal film of MIET with a single sheet of graphene which reduces the quenching range by one order of magnitude, leading to a tenfold improvement in axial resolution. This enables the localization of fluorophores with sub-nanometer accuracy. We demonstrate the potential of GIET by quantifying inter-leaflet distances in supported lipid bilayers (SLBs) and discuss the potential of the technique particularly in membrane biophysics applications. The second line of this thesis is devoted to the complementary topic of fast molecular dynamics. While super-resolution microscopy succeeds in resolving structural details with nanometer resolution, it is too slow for temporally resolving the fast dynamics of the observed molecules. For this purpose, spectroscopic techniques such as single molecule fluorescence spectroscopy (SMFS) have become an important tool that can resolve molecular dynamics down to timescales of nanoseconds. In my thesis, I focus on fluorescence lifetime correlation spectroscopy (FLCS), an advanced variant of fluorescence correlation spectroscopy (FCS). Using FLCS, I could disentangle two emission states in an autofluorescent protein that have otherwise highly overlapping spectra, and I could quantify the microsecond switching rates between these two states. As compared to other existing methods, FLCS offers the unique advantage of probing such fast switching kinetics with nanosecond temporal resolution under equilibrium conditions at room temperature, making it the method of choice for similar studies of complex luminescent emitters. Finally, I will also present another study where I utilized advanced FCS for studying protein self-assembly. In summary, my thesis presents several advanced methods in SMLM and SMFS which significantly enhance the spatial and temporal resolution at the single molecule level. I believe that the presented methods will find a wide range of applications in the life sciences.
Keywords: Single molecule fluorescence spectroscopy (SMFS); Super-resolution fluorescence microscopy; Metal-induced energy transfer (MIET); Graphene-induced energy transfer (GIET); Fluorescence lifetime correlation spectroscopy (FLCS); Dual color fluorescence cross-correlation spectroscopy (FCCS)