Single Molecule Sensing | EPFL Graph Search

Living organisms consist of cells, the elementary components of which are proteins providing cellular structures and functionality. Nowadays, proteins of the size of one to a few tens of nanometers can be efficiently monitored by fluorescence microscopy and/or spectroscopy reaching the necessary single molecule sensitivity. However, for a more complete understanding of biological processes, the combined investigation of both cellular structures and function at the single molecule level is essential. This work extends the possibilities of existing single molecule investigation methods. The goal was to conceive a functional super-resolution imaging technique by joining the strong concepts from state-of-the-art microscopy and spectroscopy and was approached in three steps. As a first step, confocal fluorescence correlation spectroscopy (FCS) was applied in nanochannels to create sub-diffraction sampling volumes. The resulting enhanced sensitivity to the surface effects depending on ionic surface layers led to a modified protein diffusion measured with FCS. Triplet state lifetime and population analysis have confirmed the model developed for the protein diffusion as a function of the ionic concentration. In the second step, stochastic optical reconstruction microscopy (STORM) was implemented and enhanced by merging with a recent labeling technique called SNAP-tag. As a result, the labeling technique for bright and switchable organic fluorescent dyes attached by a short linker of only ≈ 2.5 nm to the target molecule enabled a localization precision down to ≈ 10 nm. To meet the stringent photo-physical requirements imposed by STORM, toxic imaging buffers based on thiols have to be used. It was found that these requirements could be relaxed by super-resolution optical fluctuation imaging (SOFI) making a step forward to live-cell super-resolution investigations, which introduced the third step. In the third step, SOFI was characterized providing the basis for combining functional and structural super-resolution imaging techniques. The comparison of STORM and SOFI revealed the strengths of each technique. Under ideal blinking conditions with ultra-stable long-lived dark states, STORM outperforms SOFI. However, if the emitters are not well isolated, SOFI achieves a higher effective resolution than STORM and even without mislocalization artifacts. This led onto a novel imaging technique derived from SOFI: Cumulant microscopy merges structural super-resolution with functional imaging. Simulations and measurements showed that photo-physical parameters can be extracted from the emitters serving as nano-sensors. In conclusion, concepts of FCS have been merged with an improved super-resolution technique leading to a novel functional super-resolution imaging technique, which is compatible with living cells.

Optical spectroscopy of the bladder washout fluid to optimize fluorescence cystoscopy with Hexvix

Blaise Lovisa, Carla Martoccia, Hubert van den Bergh, Georges Wagnières, Matthieu Zellweger

Fluorescence cystoscopy enhances detection of early bladder cancer. Water used to inflate the bladder during the procedure rapidly contains urine, which may contain fluorochromes. This frequently degrades fluorescence images. Samples of bladder washout fluid (BWF) or urine were collected (15 subjects). We studied their fluorescence properties and assessed changes induced by pH (4 to 9) and temperature (15 degrees C to 41 degrees C). A typical fluorescence spectrum of BWF features a main peak (excitation/emission: 320/420 nm, FWHM = 50/100 nm) and a weaker (5% to 20% of main peak intensity), secondary peak (excitation/emission: 455/525 nm, FWHM = 80/50 nm). Interpatient fluctuations of fluorescence intensity are observed. Fluorescence intensity decreases when temperature increases (max 30%) or pH values vary (max 25%). Neither approach is compatible with clinical settings. Fluorescence lifetime measurements suggest that 4-pyridoxic acid/riboflavin is the most likely molecule responsible for urine's main/secondary fluorescence peak. Our measurements give an insight into the spectroscopy of the detrimental background fluorescence. This should be included in the optical design of fluorescence cystoscopes. We estimate that restricting the excitation range from 370-430 nm to 395-415 nm would reduce the BWF background by a factor 2. (C) 2014 Society of PhotoOptical Instrumentation Engineers (SPIE)

Spie-Soc Photo-Optical Instrumentation Engineers2014

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

Biophysical applications of correlative scanning probe and super-resolution microscopy

Vytautas Navikas

Imaging live cells in their native environment is crucial for the understanding of complex biological phenomena. Modern optical microscopy methods such as fluorescence super-resolution microscopy are increasingly combined with complementary, label-free techniques to put the high-resolution fluorescence information into cellular context. Common high-resolution imaging approaches used in combination with fluorescence imaging such as electron microscopy and atomic force microscopy (AFM) were originally developed for solid-state material characterization and thus are not straightforwardly applicable to image live cells for sustained periods of time. In this thesis, I am focusing on advancing state-of-the-art correlative scanning ion-conductance microscopy (SICM) combined with novel super-resolution imaging techniques. The work presented in this thesis demonstrates various technological advancements for both imaging modalities as well as experimental proof-of-principle studies. The first part of the work focuses on a novel experimental combination of SICM and super-resolution optical fluctuation imaging for single-cell imaging (SOFI). To demonstrate the capabilities of our method we show correlative 3D cellular maps with SOFI implementation in both 2D and 3D with self-blinking dyes for two-color high-order SOFI imaging. We employ correlative SICM/SOFI microscopy for visualizing actin dynamics in live COS-7 cells with subdiffraction resolution. In order to increase the imaging depth for thick samples, a novel remote focusing modality based on an adaptive optics device was developed. Here, we use a deformable mirror to acquire multiple image planes of the labelled cells and we process the data using a single-molecule localization routine and/or computed spatiotemporal correlations, demonstrating subdiffraction resolution within a whole imaging volume. Second, we demonstrate the advantages of a new SICM probe manufacturing method allowing the batch production of glass nanopipettes used for SICM imaging and sensing. Finally, we show a new application of SICM tailored for single-molecule characterization based on electrical signatures while performing controlled translocations of surface-immobilized DNA molecules. The method is developed to be compatible with single-molecule fluorescence imaging and was applied for the detection of various analytes in a correlative manner. We believe that a synergy between fundamentally different imaging modalities is going to become necessary in an age of computational microscopy to ensure an unbiased data interpretation in the rapidly progressing field of biological research.