Dual-Activatable Cell Tracker for Controlled and Prolonged Single-Cell Labeling

Cell trackers are fluorescent chemical tools that facilitate imaging and tracking cells within live organisms. Despite their versatility, these dyes lack specificity, tend to leak outside of the cell, and stain neighboring cells. Here, we report a dual-activatable cell tracker for increased spatial and temporal staining control, especially for single-cell tracking. This probe overcomes the typical problems of current cell trackers: off-target staining, high background signal, and leakage from the intracellular medium. Staining with this dye is not cytotoxic, and it can be used in sensitive primary cells. Moreover, this dye is resistant to harsh fixation and permeabilization conditions and allows for multi-wavelength studies with confocal microscopy and fluorescence-activated cell sorting. Using this cell tracker, we performed in vivo homing experiments in mice with primary splenocytes and tracked a single cell in a heterogeneous, multicellular culture environment for over 20 h. These experiments, in addition to comparative proliferation studies with other cell trackers, demonstrated that the signal from this dye is retained in cells for over 72 h after photoactivation. We envision that this type of probes will facilitate the analysis of single-cell behavior and migration in cell culture and in vivo experiments.

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

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

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