Accelerated Wavelet-Regularized Deconvolution for 3-D Fluorescence Microcopy

Modern deconvolution algorithms are often specified as minimization problems involving a non-quadratic regularization functional. When the latter is a wavelet-domain $ℓ _{ 1 }$ -norm that favors sparse solutions, the problem can be solved by a simple iterative shrinkage/thresholding algorithm (ISTA). This approach provides state-of-the-art results in 2-D, but is harder to deploy in 3-D because of its slow convergence. In this paper, we propose an acceleration scheme that turns wavelet-regularized deconvolution into a competitive solution for 3-D fluorescence microscopy. A significant speed-up is achieved though a synergistic combination of subband-adapted thresholds and sequential TwIST updates. We provide a theoretical justification of the procedure together with an experimental evaluation, including the application to real 3-D fluorescence data.

Structure and function of the nicotinic acetylcholine receptor

Jörg Grandl

The subject of this thesis is the study of the structure and function of the human muscle nicotinic acetylcholine receptor (nAChR), which is a typical member of the large class of ligand-gated ion channels. Appropriate techniques, like site-directed mutagenesis, patch-clamp and fluorescence microscopy, and combinations thereof, were applied to gain insight into selected aspects of the structure-function relationship of this receptor. Special emphasis was put on the investigation of single receptors, wherever this promised scientific benefit. Experiments with the aim to study structural rearrangements of nAChR upon ligand binding by fluorescence resonance energy transfer (FRET) were undertaken. Such intramolecular motions are expected to link ligand binding to channel gating and are therefore fundamental to understand the function of nAChR. Double-cysteine mutants were produced and receptors were labelled on living cells in order to measure changes in FRET efficiency due to ligand binding. A novel class of functionally modified ligands for the nAChR was characterized by patch-clamp technique. Derivatives that carry a thiol-reactive group were shown to be full and very potent agonists. On the bases of a structural model of the binding site, residues were selected, that are likely to be in contact with the bound ligand. Equivalent single-cysteine mutants were produced to covalently bind these agonists and thereby to gain information about the mechanisms of ligand binding and receptor activation. Ligands that were coupled to fluorophores turned out to be either partial or full agonists with nanomolar efficacies for the nAChR. Their use to study the diffusion of single nAChR on living HEK293-cells for different functional states is described in detail. The dependency of the lateral diffusion on the activation state (closed/opened/desensitized) was studied. Additionally these ligands served to demonstrate the feasibility to simultaneously combine patch-clamp and fluorescent binding experiments on a single-molecule level. Acetylcholine receptors carrying a single point-mutation, related to the congenital myasthenic syndrome, were found to stabilize an intermediate conductance state and were studied in detail by single-channel electrophysiology. The dependency of the gating behavior on different ligands, ligand concentration and transmembrane voltage was investigated. Mutants, carrying equivalent mutations on different subunits were produced to reveal the origin of this effect. Quantitative characterization was obtained by dwell-time and spectral noise-analysis, in order to gain insight on sub-millisecond fluctuations of the channel lining transmembrane regions of the nAChR and the gating mechanism in general.

EPFL2005

Lipid membranes for the fabrication of functional micro- and nano-structures

Gopakumar Gopalakrishnan

The central goal of this thesis work is to fabricate novel, functional fluorescent nanostructures in confined systems offered by phospholipid membranes, which are known to have highly ordered, thermotropic and lyotropic structures. In separate approaches, we have used three different lipid systems: multilamellar planar lipid membranes, unilamellar vesicular membranes as well as lipid monolayers for the development of functional fluorescent nano-, micro- and meso-scopic structures. Techniques like fluorescence microscopy, single particle imaging, electron microscopy, electron diffraction were used to achieve fundamental understanding of the resulting structures. Multilayer stacks of phospholipid membranes have been used as an effective template for the growth of high aspect ratio fluorescent nanowires. The room temperature synthesis was achieved in the confined nanometer-sized interlamellar water space of lipid multilayers where supersaturating CdCl2 concentrations were induced by acidification leading to controlled unidirectional growth of nanowires. The possibility to render the nanowires fluorescent by doping with CdS quantum dots (QDs) and the light waveguiding along hundreds of micrometers together with the possibility of lateral manipulation make these nanowires attractive candidates for future optoelectronic applications. Novel organic-inorganic functional nanocontainers have been designed and tested by making use of vesicle forming lipid bilayers in combination with semiconductor QDs. Hydrophobic QDs can be integrated into bilayers of lipid vesicles and such lipid/QD hybrid vesicles are capable to fuse with live cells, thereby stain the cell's plasma membrane selectively with fluorescent QDs and transfer the vesicle's cargo into the cell. Modification of the membrane of such hybrid vesicles on the other hand, made them capable to enter the cytoplasm of live cells. Additionally, these hybrid vesicles were found extremely useful for long-term model membrane imaging studies. The results described in this thesis imply that cell and lipid membranes can integrate any kind of hydrophobic nanoparticle whose size matches the membrane thickness, opening novel possibilities to manipulate them as individuals or in ensemble with wide-ranging applications for nanobiotechnology. In a further step, the ability of phospholipid molecules to exhibit lamellar to non-lamellar transition using external stimuli enabled a directed self-assembly of QDs into mesoscale fluorescent structures. An easy and versatile method for the surface modification of TOPO coated CdSe QDs using 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid (DPPA) have been achieved. DPPA predominantly form non-lamellar phases when dispersed in water, for example, upon addition of Ca2+ or at a pH below 6 are known to form hexagonal II phases. This particular property of DPPA has been exploited to form mesoscale self-assemblies of QD based structures both in solution and in confined systems. Potential applications include the detection or removal of Ca2+ ions in attoliter volumes, the construction of functional devices where QDs are reversibly organized in different forms as well as use of fluorescently labeled DPPA molecules for cellular studies using fluorescence microscopy.

EPFL2006

The Colored Revolution of Bioimaging

François Aguet, Michaël Unser, Cédric René Jean Vonesch

With the recent development of fluorescent probes and new high-resolution microscopes, biological imaging has entered a new era and is presently having a profound impact on the way research is being conducted in the life sciences. Biologists have come to depend more and more on imaging. They can now visualize subcellular components and processes in vivo, both structurally and functionally. Observations can be made in two or three dimensions, at different wavelengths (spectroscopy), possibly with time-lapse imaging to investigate cellular dynamics. The observation of many biological processes relies on the ability to identify and locate specific proteins within their cellular environment. Cells are mostly transparent in their natural state and the immense number of molecules that constitute them are optically indistinguishable from one another. This makes the identification of a particular protein a very complex task—akin to finding a needle in a haystack. However, if a bright marker were attached to the protein of interest, it could very precisely indicate its position. Much effort has gone into finding suitable markers for this purpose, but it is only over the course of the past decade, with the advent of fluorescent proteins, that this concept has been revolutionized. These biological markers have the crucial properties necessary for dynamic observations of living cells: they are essentially harmless to the organism, and can be attached to other proteins without impacting their function. Fluorescence microscopy was invented almost a century ago, when microscopists were experimenting with ultraviolet light to achieve higher resolutions. In the very beginning, observations were limited to specimens that naturally fluoresce. Rapidly, fluorescent dyes for staining tissues and cells were investigated. But it was not until the 1940s that fluorescence microscopy became popular, when Coons and Kaplan introduced a technique to label antibodies with a fluorescent dye to study antibody-antigen interactions, profoundly changing the field of immunohistochemistry. The discovery that really brought fluorescence microscopy to the forefront came in 1994, when M. Chalfie et al. succeeded in expressing a naturally fluorescent protein, the now-famous green fluorescent protein (GFP), in living organisms. This was a landmark evolution in the field, fostering a whole new class of tagging methods. While genetic engineering is at the origin of this new methodology, a number of innovations from the fields of physics, optics, and mechanical and electrical engineering have been combined to provide the necessary instrumentation. Impressive enhancements in classical microscopy have been achieved, and new imaging systems are actively being developed. A key element for the evolution of microscopy in general was the shift to digital imaging in the 1990s, with the availability of affordable high-sensitivity acquisition devices and powerful computer hardware. The capabilities of today's systems often lead to enormous data sets that, in most cases, require postprocessing for their interpretation. Signal processing methods for biological research are only at their prelude; the needs are considerable and most probably not even clearly formulated yet. It is thus predictable that signal processing will be one of the main challenges of fluorescence microscopy in the forthcoming years. The goal of this article is to provide an overview of the main aspects of modern fluorescence microscopy. We first cover the principles of fluorescence and highlight the key discoveries in the history of fluorescence microscopy. In subsequent sections, we present the optics of fluorescence microscopes and examine various types of detectors. Finally, we discuss the signal and image processing challenges in fluorescence microscopy and highlight some of the present developments and future trends in the field.

IEEE2006