Total internal reflection fluorescence correlation spectroscopy (TIR-FCS) with low background and high count-rate per molecule

We designed a fluorescence correlation spectroscopy (FCS) system for measurements on surfaces. The system consists of an objective-type total internal reflection fluorescence (TIRF) microscopy setup, adapted to measure FCS. Here, the fluorescence exciting evanescent wave is generated by epi-illumination through the periphery of a high NA oil-immersion objective. The main advantages with respect to conventional FCS systems are an improvement in terms of counts per molecule (cpm) and a high signal to background ratio. This is demonstrated by investigating diffusion as well as binding and release of single molecules on a glass surface. Furthermore, the size and shape of the molecule detection efficiency (MDE) function was calculated, using a wave-vectorial approach and taking into account the influence of the dielectric interface on the emission properties of fluorophores. (c) 2005 Optical Society of America.

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

Multiple scattering in wide-field optical coherence tomography

Boris Karamata

Optical Coherence Tomography (OCT), a well-established imaging method based on low-coherence interferometry, provides cross-sectional images of the internal structure of biological samples with a resolution in the micrometer range. OCT was successfully applied on various tissues such as for instance the retina, the skin or a tooth. In highly scattering tissues like the skin, probing depth is limited to approximately 2mm, mainly due to insufficient rejection of multiply scattered light. Presently, the contribution of multiple scattering in OCT is not fully understood. Therefore, there is a strong and urgent need to develop models allowing a reliable evaluation of the system's limitations as well as the improvement of the imaging capabilities. It is generally believed that a relevant model should account for loss of correlation between the reference and the sample field due to multiple scattering. We developed a new comprehensive model of OCT. Our preliminary study revealed that the reference and sample fields are actually fully correlated. This important result allowed us to model the OCT signal as a sum of stationary random phasors and treated it as a statistical signal. The mean of this signal can be calculated thanks to classical results of statistical optics and to a Monte Carlo simulation. Unlike other existing models, our model accounts for the source autocorrelation function. The model proved to be in excellent agreement with a whole range of experimental data gathered in a comprehensive study of cross-talk in wide-field OCT. Moreover, our results put in question the applicability of widely used models of OCT based on the "extended Huygens-Fresnel principle", which assume a partial correlation between interfering fields due to multiple scattering. The construction of conventional OCT images is based on lateral scanning of a beam focused within the sample. To increase image acquisition speed and eliminate the need for lateral scanning, wide-field OCT was recently developed. Our experimental and theoretical investigations of the potential and limitations of wide-field OCT revealed the crucial role played by the spatial coherence of the light source. Spatially coherent illumination generates considerable coherent optical cross-talk, which prevents shot-noise-limited detection and diffraction-limited imaging in scattering samples. The dependence on several parameters of the optical system and of the sample properties was investigated in a comprehensive study. Cross-talk increases with the wide-field diameter, numerical aperture, source coherence length, and sample optical density; and strongly depends on sample anisotropy. We showed that spatially incoherent illumination realized with a thermal light source permits cross-talk suppression in wide-field OCT, i.e. rejection of multiply scattered light to a level comparable to that of point scanning OCT. We performed a theoretical study which revealed that the power per spatial mode radiated by thermal light sources is too low to permit a high signal-to-noise ratio while maintaining a fast acquisition speed. Therefore, wide-field OCT realized with either spatially coherent or spatially incoherent illumination suffers from inherent fundamental limitation. This led us to investigate the possibility of exploiting a spatially incoherent light source brighter than a thermal light source. We came to the conclusion that such a "pseudothermal" light source can potentially lead to wide-field OCT systems devoid of cross-talk and an image acquisition speed higher than that of a point scanning OCT system. However, the attractive properties of pseudothermal light sources could be gained at the expense of the simplicity and the economical advantages offered by thermal light sources. Furthermore, fast acquisition speed also relies on a performing "smart pixel detector array". Presently, such detectors do not have sufficient sensitivity and their frequency readout is too low as shown in our feasibility study. We performed a theoretical investigation of the potential of thermal light sources in terms of axial resolution and power per mode. The former revealed that the maximum power per mode is radiated at a wavelength higher than the spectral peak of a blackbody radiator. This led to the important practical conclusion that, at 6000 K, the maximum power is collected in the therapeutic spectral window in OCT (600 - 1300 nm), while at 3000 K this peak is shifted out of the therapeutic window leading to significant power losses. More generally, our work provides a design tool for choosing the optimal thermal light source for a given therapeutic window in terms of signal-to-noise ratio. Currently available sources at 6000 K consist of high pressure gas arc lamps providing a spectrum endowed with spectral lines deleterious for OCT. By suppressing a portion of the spectrum devoid of spectral lines of a mercury arc lamp, we obtained amongst the highest axial resolution so far reported in OCT. Furthermore, the importance of the speckle statistics in OCT incited us to clarify the origin of a difference between two theoretical results reported in the literature. Indeed, two calculations of the amplitude distribution of speckles in OCT, each of them based on a different mathematical formulation, yield different results. We showed that a modification of an initial assumption in one of the formulation leads to equivalent results. In conclusion, this thesis provides a deeper understanding of the potential and limitations of widefield OCT, leading to important design rules. Moreover, it presents a new comprehensive model of OCT putting in question other widely used models.

EPFL2004

Model-Based Estimation of Three-Dimensional Stiffness Parameters in Photonic-Force Microscopy

Elena Bertseva, Andrzej Kulik, Amardeep Singh, Philippe Thévenaz, Michaël Unser

We propose a system to characterize the 3-D diffusion properties of the probing bead trapped by a photonic-force microscope. We follow a model-based approach, where the model of the dynamics of the bead is given by the Langevin equation. Our procedure combines software and analog hardware to measure the corresponding stiffness matrix. We are able to estimate all its elements in real time, including off-diagonal terms. To achieve our goal, we have built a simple analog computer that performs a continuous preprocessing of the data, which can be subsequently digitized at a much lower rate than is otherwise required. We also provide an effective numerical algorithm for compensating the correlation bias introduced by a quadrant photodiode detector in the microscope. We validate our approach using simulated data and show that our bias-compensation scheme effectively improves the accuracy of the system. Moreover, we perform experiments with the real system and demonstrate real-time capabilities. Finally, we suggest a simple adjunction that would allow one to determine the mass matrix as well.