Single molecule detection on surfaces

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.

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 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

Super-resolution fluorescence microscopy based on physical models

François Aguet

This thesis introduces a collection of physics-based methods for super-resolution in optical microscopy. The core of these methods constitutes a framework for 3-D localization of single fluorescent molecules. Localization is formulated as a parameter estimation problem relying on a physically accurate model of the system's point spread function (PSF). In a similar approach, methods for fitting PSF models to experimental observations and for extended-depth-of-field imaging are proposed. Imaging of individual fluorophores within densely labeled samples has become possible with the discovery of dyes that can be photo-activated or switched between fluorescent and dark states. A fluorophore can be localized from its image with nanometer-scale accuracy, through fitting with an appropriate image function. This concept forms the basis of fluorescence localization microscopy (FLM) techniques such as photo-activated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), which rely on Gaussian fitting to perform the localization. Whereas the image generated by a single fluorophore corresponds to a section of the microscope's point spread function, only the in-focus section of the latter is well approximated by a Gaussian. Consequently, applications of FLM have for the most part been limited to 2-D imaging of thin specimen layers. In the first section of the thesis, it is shown that localization can be extended to 3-D without loss in accuracy by relying on a physically accurate image formation model in place of a Gaussian approximation. A key aspect of physically realistic models lies in their incorporation of aberrations that arise either as a consequence of mismatched refractive indices between the layers of the sample setup, or as an effect of experimental settings that deviate from the design conditions of the system. Under typical experimental conditions, these aberrations change as a function of sample depth, inducing axial shift-variance in the PSF. This property is exploited in a maximum-likelihood framework for 3-D localization of single fluorophores. Due to the shift-variance of the PSF, the axial position of a fluorophore is uniquely encoded in its diffraction pattern, and can be estimated from a single acquisition with nanometer-scale accuracy. Fluroescent molecules that remain fixed during image acquisition produce a diffraction pattern that is highly characteristic of the orientation of the fluorophore's underlying electromagnetic dipole. The localization is thus extended to incorporate the estimation of this 3-D orientation. It is shown that image formation for dipoles can be represented through a combination of six functions, based on which a 3-D steerable filter for orientation estimation and localization is derived. Experimental results demonstrate the feasibility of joint position and orientation estimation with accuracies of the order of one nanometer and one degree, respectively. Theoretical limits on localization accuracy for these methods are established in a statistical analysis based on Cramér-Rao bounds. A noise model for fluorescence microscopy based on a shifted Poisson distribution is proposed. In these localization methods, the aberration parameters of the PSF are assumed to be known. However, state-of-the-art PSF models depend on a large number of parameters that are generally difficult to determine experimentally with sufficient accuracy, which may limit their use in localization and deconvolution applications. A fitting algorithm analogous to localization is proposed; it is based on a simplified PSF model and shown to accurately reproduce the behavior of shift-variant experimental PSFs. Finally, it is shown that these algorithms can be adapted to more complex experiments, such as imaging of thick samples in brightfield microscopy. An extended-depth-of-field algorithm for the fusion of in-focus information from frames acquired at different focal positions is presented; it relies on a spline representation of the sample surface and as a result yields a continuous and super-resolved topography of the specimen.

EPFL2009