This thesis is a collection of experimental attempts to enhance photoluminescence of fluorescent molecules and quantum dots with single gold nanorods (GNRs) and relevant applications. Special... Show moreThis thesis is a collection of experimental attempts to enhance photoluminescence of fluorescent molecules and quantum dots with single gold nanorods (GNRs) and relevant applications. Special attention is focused on the interactions between single emitters and GNRs. The idea is to increase the emission of weak emitters by the excellent optical properties of GNRs so that weak light emitters will then be bright enough to be detected and studied individually. We can thus generalize single-molecule fluorescence spectroscopy to weakly emitting species which are currently undetectable by conventional single-molecule spectroscopy. The research is important for extending the scope of single-molecule spectroscopy, which is a powerful technique for understanding the dynamic behaviors at the nanometer scale in biological systems and other materials. Show less
Single-molecule fluorescence was invented in the 1990s and has quickly developed into an indispensable technique in the biomedical sciences and condensed-matter research. It has revolutionized... Show moreSingle-molecule fluorescence was invented in the 1990s and has quickly developed into an indispensable technique in the biomedical sciences and condensed-matter research. It has revolutionized the fields of molecular biology, imaging (super-resolution), and catalysis, to name a few. In this thesis, we will apply fluorescence enhancement by single gold nanorods to extend single-molecule studies to chromophores with low fluorescence quantum yields and to high concentrations of probe molecules. Following single-molecule trajectories, we will explore variations in the electron-transfer rates of the metalloprotein azurin both from molecule to molecule and for the same molecule as a function of time. Evidence for conformational substates will be discussed based on dynamic heterogeneity. Show less
Super-Resolution Microscopy is an optical fluorescence technique. In this thesis we focus on single molecule super-resolution, where the position of single molecules is determined. Typically these... Show moreSuper-Resolution Microscopy is an optical fluorescence technique. In this thesis we focus on single molecule super-resolution, where the position of single molecules is determined. Typically these molecules can be localized with a 10 to 30nm precision. This technique is applied in four different studies. To determine the spatial distribution of Ras-protein in live cells, Ripley's analysis is used on localization data to quantify size an diffusion parameters of nanodomains. Particle image correlation spectroscopy (PICS) is a second order spatial distribution analysis to determine diffusion properties of single molecule populations. A mathematical framework to correct the data when applied on 3D diffusion is presented in the second chapter. In the fourth chapter the uptake of alpha-synuclein aggregates by the cells is observed using super-resolution microscopy. Their partial degradation was followed and showed the importance of the lysosome-dependent mechanism for protecting cells from exposure to potentially toxic a-synuclein. In the fifth chapter we correlate the number of vinculin proteins in a focal adhesion protein complex, to the local force generated by the cell via this complex. A method was developed to determine the local stoichiometry of molecules by their correlated distances as obtained from SMLM. Show less
The investigation of electron-transfer (ET) processes, as well as redox reactions is important to understand a whole series of biochemical processes. Single-molecule techniques are a precious tool... Show moreThe investigation of electron-transfer (ET) processes, as well as redox reactions is important to understand a whole series of biochemical processes. Single-molecule techniques are a precious tool whose diffusion and technical evolution made them available for the study of biologically relevant reactions. In this thesis an approach to the study of electron-transfer and redox reaction by means of single-molecule techniques is presented. Two proteins were investigated: azurin, from Pseudomonas aeruginosa, which contains a type-1 copper center, and the blue nitrite reductase (bNiR), from Alcaligenes xylosoxidans, which contains a type-1 and a type-2 copper center. By means of a resonant energy transfer (FRET) __ based approach it was possible to use a fluorescent label attached to the surface of the proteins to obtain information about their redox state. By using fluorescence correlation spectroscopy in solution it was possible to study ET processes between azurin and the label and between two covalently linked azurin monomers. Nitrite reductase was immobilized in an agarose matrix and single enzyme molecules were investigated by using scanning confocal microscopy: new details about the internal ET between the two copper centers of bNiR were revealed and new light was shed on the catalytic cycle of the enzyme. Show less
A human consists of billions of cells. All these cells need to know in which organ they are located and what their position inside the organ is. One way to obtain this information is via morphogens... Show moreA human consists of billions of cells. All these cells need to know in which organ they are located and what their position inside the organ is. One way to obtain this information is via morphogens, small particles providing positional information. We quantitatively studied the transport of the morphogen Decapentaplegic (Dpp) in the __wing imaginal disc__ (the precursor of the wing) of fruit fly larvae. Certain cells in this disc produce Dpp, while others receive it and determine their position according to the Dpp concentration. To study Dpp transport we first developed a microscope able to follow single molecules in three dimensions in living tissue with high spatial and temporal accuracy. With this microscope we then studied the subcellular processes governing intracellular Dpp transport. We determined how long Dpp resides in different types of endosomes (a cellular compartment involved in transport). We also found that the movement of endosomes is too small to facilitate Dpp transport. Furthermore we found differences in the in- and outflow of Dpp in endosomes. This work is one of the first to quantitatively study intracellular morphogen transport. It provides new insights into growth and development of organisms. Show less
This thesis centers around a novel fluorescence based method that allows to monitor the activity of redox enzymes and of electron (ET) or oxygen transfer proteins. It takes advantage of the fact... Show moreThis thesis centers around a novel fluorescence based method that allows to monitor the activity of redox enzymes and of electron (ET) or oxygen transfer proteins. It takes advantage of the fact that the absorption spectrum of the protein__s active site varies upon changing its redox state. This change can be translated into a change in the fluorescence intensity of a label that is covalently linked to the protein on the basis of F_rster Resonance Energy Transfer (FRET). With our method we could show that different redox proteins and enzymes can be studied down to the single molecule level. This exciting finding opens the door to the study of various redox enzymes and to monitor specific substances such as for example nitrite. Depending on the function of the enzyme under investigation a wide range of substrates can be monitored. Another example is the development of an oxygen sensor by employing proteins that are capable of binding oxygen. The findings presented in this thesis might be significant for applications in oxygen sensing and, more generally, in the fast growing field of biosensing Show less
