Coherent Imaging of Cellular Dynamics

Miguel Sison
EPFL, 2017
EPFL thesis

Abstract

Mitochondrial dynamics refers to the processes of fusion, fission, and transport that aid mitochondria in accomplishing their many roles; including ATP production, oxygen sensing, and homeostasis. Due to their involvement in numerous essential cellular activity, dysfunctional mitochondria have been implicated in a wide range of human diseases. Confocal microscopy using fluorophores for molecular specificity remains the gold standard of intracellular imaging. However, fluorescent labels can be toxic to the cell upon prolonged exposure and still suffer from photobleaching. These compromise the application of confocal fluorescence microscopy for true long lasting time-lapse imaging of living samples. Optical coherence microscopy (OCM) exploits the intrinsic variation in the scattering properties of the sample to achieve fast, label-free, and highly sensitive three-dimensional imaging. Unfortunately, being label-free means OCM lacks specificity and coherence based imaging techniques have no counterpart to fluorescent markers. The invention of the photothermal optical lock-in OCM (poli-OCM) brought about the possibility of specific OCM imaging using gold nanoparticles (AuNP) as photothermal bio-markers. The use of AuNPs as specific contrast agents has substantial advantages stemming from their well-established biocompatibility and photostability. Microscopic techniques that offer fast three-dimensional imaging over extended time durations would do well in revealing previously inaccessible knowledge on mitochondria. In this work, we quantify mitochondrial dynamics based on specific poli-OCM and surface functionalization of AuNPs. In realizing mitochondria specific poli-OCM imaging, it is necessary to functionalize AuNP with mitochondria targeting capabilities. We present copolymer surface coatings that provide the AuNPs with improved stability, solubility, and cellular uptake on top of mitochondria labeling. We further optimize the utilization of these AuNP labels for poli-OCM imaging. We also demonstrated poli-OCM imaging with differently structured gold nanolabels, which could lead to the realization of multimodal imaging using a single bio-marker. The two quantification techniques we developed were based on (1) temporal autocorrelation analysis combined with a classical diffusion model and (2) single particle tracking. Autocorrelation analysis is the foundation of fluorescence correlation spectroscopy (FCS); a technique extensively used for analyzing dynamic phenomena in chemistry and biophysics. We extend this analysis to three-dimensional poli-OCM imaging allowing us to map quantified mitochondrial diffusion parameters in three dimensions within the cell. We also investigate how the size of the mitochondria with respect to the point spread function (PSF) of the poli-OCM impacts the result of our autocorrelation analysis. Single particle tracking complements our temporal autocorrelation analysis since recent advances in localization and tracking algorithms have demonstrated precision better than the size of the PSF. Finally, we demonstrate the possibility of using mitochondria specific poli-OCM imaging with the quantification techniques we developed in studying the Cockayne syndrome (CS). CS is a very rare and fatal genetic disease that has been associated with mitochondrial dysfunction. To our knowledge, no study has been conducted focusing on quantifying the effect of CS on mitochondrial dynamics.

Official source

https://infoscience.epfl.ch/record/232682?ln=en

About this result

This page is automatically generated and may contain information that is not correct, complete, up-to-date, or relevant to your search query. The same applies to every other page on this website. Please make sure to verify the information with EPFL's official sources.

Related concepts

Related publications

Miguel Sison
EPFL, 2017
EPFL thesis

Abstract

Official source

https://infoscience.epfl.ch/record/232682?ln=en

About this result

Related concepts (20)

Related concepts

Related publications (33)

Related publications

Related publications (33)

Single molecule detection on surfaces

Marcel Leutenegger

Monitoring biological relevant reactions on the single molecule level based on fluorescence spectroscopy techniques has become one of the most promising approaches for understanding a variety of phenomena in biophysics, biochemistry and life science. By applying techniques of fluorescence spectroscopy to labeled biomolecules a manifold of important parameters becomes accessible. For example, molecular dynamics, energy transfer, and ligand-receptor reactions can be monitored at the molecular level. This huge application field was and still is a major drive for innovative optical methods as it opens the door for new quantitative insights of molecular interactions on a truly micro- and nano-scopic scale. This thesis contributes new single molecule detection (SMD) concepts, correlation analysis and optical correlation spectroscopy to study fluorophores or labeled biomolecules close to a surface. The search beyond the classical confocal volume towards improved confinement was a key objective. In a first approach, fluorescence correlation spectroscopy (FCS) using near field light sources to achieve highly confined observation volumes for detecting and measuring fluorophores up to micromolar concentration was investigated. In a second approach, FCS and fluorescence intensity distribution analysis (FIDA) based on dual-color total internal reflection fluorescence (TIRF) microscopy was conceived to achieve a common observation volume for dual-color fluorescence measurements. This resulted in two novel fluorescence fluctuation spectroscopy instruments providing observation volumes of less than 100al. The first instrument generates a near field observation volume around and inside nano-apertures in an opaque metal film. Back-illumination of such an aperture results in a highly confined excitation field at the distal aperture exit. This instrument was characterized with FCS and observation volumes as small as 30al were measured. The second instrument confines the observation volume with total internal reflection (TIR) at a glass-water interface. Today, the last-generation instrument provides a dual-color ps pulsed excitation and time-resolved detection for coincidence analysis and time-correlated single photon counting. It was characterized with FCS and FIDA and observation volumes of 70al to 100al were achieved. Moreover, the presence of the interface favors emission into the optically denser medium, such that nearly 60% of the emitted fluorescence can be collected. This very efficient light collection resulted in a two- to three-fold stronger fluorescence signal and led to a high signal to background ratio, which makes this instrument particularly suitable for SMD studies on surfaces. In parallel to these experimental investigations, a theoretical analysis of the total SMD process including an analysis of optical focus fields, molecule-interface interactions, as well as the collection and detection efficiency was performed. This analysis was used as a guideline for steady instrument improvements and for the understanding of the SMD process. Finally, SMD concepts were applied for a first investigation of in vitro expression of an odorant receptor and for monitoring the vectorial insertion into a solid-supported lipid membrane. These receptors were incorporated and immobilized in the lipid membrane. With increasing expression time, an increasing amount of receptors as well as an increasing aggregation was observed. The incorporation density and the receptor aggregation were investigated with TIRF microscopy and image correlation spectroscopy.

EPFL2007

Single molecule detection and fluorescence correlation spectroscopy on surfaces

Kai Hassler

In this thesis a new approach for single molecule detection and analysis is explored. This approach is based on the combination of two well established methods, fluorescence correlation spectroscopy (FCS) and total internal reflection fluorescence microscopy (TIRFM). In contrast to most existing fluorescence spectroscopy techniques, the subject of primary interest in FCS is not the fluorescence intensity itself but the random intensity fluctuation around the mean value. Intensity fluctuations are induced by thermal noise in a minute observation volume, which is in classical FCS the confocal volume of a confocal microscope. E.g. FCS is commonly utilized to investigate diffusion. In this case, diffusing fluorescent molecules entering or leaving the observation volume cause intensity fluctuations, which are analyzed by calculating the temporal autocorrelation of the observed signal. The autocorrelation is a measure for the self-similarity of a signal and contains information about the average fluctuation strength and duration. The confocal observation volume, i.e. the measurement volume that is actually seen by the detector is approximately given by the product of the optical transfer function with the fluorescence exciting intensity distribution of a focused laser beam. To achieve a high signal-to-background ratio a small observation volume is absolutely essential, first of all because the background from e.g. scattered light increases with the size of the observation volume. Second, a small volume assures for a small average number of fluorophores inside the observation volume and therefore for a high fluctuation amplitude i.e. FCS signal. This thesis proposes and discusses an alternative to confocal FCS specially conceived for measurements on surface-bound molecular systems, such as biological receptors or immobilized enzymes. In contrast to confocal FCS, fluorescence is excited within an evanescent field generated by total internal reflection (TIR) of a laser beam at the interface between a microscope coverslip and the sample. This is achieved by focusing the laser beam off-axis at the back focal plane of a high NA oil-immersion objective. The collimated beam that emerges from the objective is incident at an oblique angle at the coverslip-sample interface and totally internal reflected. In contrast to confocal FCS, the generated observation volume is completely confined to the surface and background fluorescence as well as scattered light from the bulk is efficiently suppressed. Our method, called objective-type TIR-FCS in the following, features an increased collection efficiency compared to existing techniques that combine evanescent wave excitation and FCS. Existing techniques use total internal reflection on the surface of a prism to generate an evanescent field. This leads to a configuration where the choice of objectives is limited to air or water-immersion objectives. In our system we use a high NA oil-immersion objective, specially conceived for TIR applications, which collects light efficiently. The collection efficiency is further enhanced by a naturally occurring change of the emission properties of fluorophores close to interfaces between dielectric media. The presence of the interface favors emission into the optically denser medium so that about 60% of the emitted light can be collected. These factors, together with a reduced observation volume lead to a very sensitive method with a high potential for applications in single molecule detection and analysis. The performance of the proposed method was experimentally shown for measurements on molecules subject to Brownian motion and binding to modified coverslips. In particular, it was experimentally shown that objective-type TIR-FCS features high signal-to-background ratio on a single molecule level. In this thesis, concise derivations of analytical expressions for autocorrelation functions for diffusion and most important, the case of ligands reversibly binding to a single and localized binding site are presented. The derived model allows for the quantitative determination of binding rates for a single receptor. We strongly believe that the application of these results in the context of investigations of receptor-ligand binding kinetics will allow for deeper understanding of cellular signaling. Moreover, this thesis discusses the applicability of the proposed method in enzymology. Enzymes, as most proteins are subject to continuous changes of their structure or conformation. These conformational changes are correlated with the function of the enzyme. In the discussed example the enzyme catalyzes an oxidation where the product is fluorescent but the substrate is not. The function of the enzyme i.e. the recurring product formation leads to observable intensity fluctuations. Since function and conformational states are correlated, conformational fluctuations can be investigated by means of FCS as was already shown for confocal FCS. A technique closely related to FCS is fluorescence lifetime spectroscopy (where lifetime refers to the mean lifetime of the electronic excited state). Whereas in FCS the relaxation after random deviations from thermal equilibrium is investigated, relaxation of excited fluorophores towards their electronic ground-state is investigated in lifetime spectroscopy. The technique is used for e.g. discrimination between fluorophores with different lifetimes. Lifetime spectroscopy combined with imaging is used in many domains of life-science, including microarray reading and medical diagnostics. In preliminary work we developed a novel approach to perform lifetime imaging that is based on a multiplexing technique. The proposed method requires no mechanical scanning stage and only a single-point detector. Furthermore, noise is reduced under certain circumstances if the signal is low. Characteristics of this technique as well as advantages and disadvantages are shortly discussed.

EPFL2006

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

Single molecule detection on surfaces

Marcel Leutenegger

EPFL2007

Single molecule detection and fluorescence correlation spectroscopy on surfaces

Kai Hassler

EPFL2006