Extended focus optical coherence microscopy and fluorescence lifetime imaging

Fourier domain Optical coherence microscopy (FDOCM) offers excellent sensitivity and high axial resolution to image the structure of biological tissue. The depth information is extracted in parallel and allows very high volume acquisition rates. The present system uses a diffractionless beam, produced with an axicon lens, to achieve high lateral resolution all while maintaining an extended depth of field (xf). The xfOCM signal reveals the spatial distribution of changes of the refractive index in the sample that scatter the incident light. To identify and validate the functionality of the observed structures can proof difficult. In this work the xfOCM setup was interfaced with a fluorescent lifetime imaging (FLIM) system, working in the Fourier domain and measuring the phase offset between the modulated excitation signal and the returned fluorescence intensity. Both the fluorescence amplitude and lifetime are retrieved. The amplitude contains important information due to the selective labeling of the tissue. The lifetime is very sensitive to the surrounding environment and varies for different fluorophores, adding further contrast. The xfOCM tomograms and FLIM images are acquired in parallel. A complementary view of the sample is obtained that helps to understand and interpret the xfOCM signal. The lifetime measurement provides further contrast to perform functional imaging on biological samples such as the rat hair follicle.

Extended focus Fourier domain optical coherence microscopy and fluorescence lifetime imaging

Adrian Bachmann, Cédric Blatter, Theo Lasser, Rainer Leitgeb, Martin Villiger

Fourier domain Optical coherence microscopy (FDOCM) offers excellent sensitivity and high axial resolution to image the structure of biological tissue. The depth information is extracted in parallel and allows very high volume acquisition rates. An illumination scheme employing an axicon lens provides extended depth of field (xf) in combination with high lateral resolution. The xf-OCM signal reveals the spatial distribution of changes of the refractive index in the sample with high contrast at a near isotropic resolution of ~2um. However identification and differentiation between different tissues may be difficult with this purely structural sample information at hand. Combining the xf-OCM setup with fluorescence lifetime imaging (FLIM) offers a powerful method to obtain functional contrast with molecular specificity. The FLIM- system works in the Fourier domain measuring the phase offset between the modulated excitation signal and the returned fluorescence intensity. Both the fluorescence amplitude and lifetime are retrieved. Previously labeled structures can be identified and help interpreting the xf- OCM signal. The fluorescence lifetime provides additional functional information. It varies between different fluorophores and can be very sensitive to environmental changes. The xf-OCM tomograms and FLIM images are acquired in parallel and provide a complementary view of the sample. This combined system was applied to the imaging of biological tissue such as rat hair follicle and mouse pancreas.

SPIE2008

Endoscopic low coherence interferometry applied to tri-dimensional contouring of upper airways

Yves Delacrétaz

This thesis presents a novel approach for optical tri-dimensional contouring, requiring only one acquisition to extract a contour depth. It is based on an interferometric setup, using a reduced coherence light source, and is fully adaptable to endoscopic procedure commonly in used in ear-nose-throat (ENT) medicine. It has been demonstrated that the method can be successfully applied to imaging of porcine upper airways. Although this method uses interferometry to obtain depth range measurement, the phase signal is not directly evaluated, rather the location of the coherent superposition of two waves is extracted, and thus provides a robust technique even in harsh environment. The key feature is to extract the coherent area on the image by locating intentionally induced fringes created by off-axis superposition of a smooth reference wave and an object wave coming from a rough or diffusing sample through an optical system with limited aperture. A model for the propagation of the light through the optical system has been developed. It takes into account the broadened emission spectrum of the light source, as well as the statistical speckle effect induced by the illumination of a rough surface with partially coherent light. Simulated results have been carefully compared with measurements, both in the spatial domain and in the Fourier domain (spatial frequencies response), which has permitted to better understand the processes involved in the creation of the interference fringes, and improve the design of the device. Different filters have been investigated in order to extract the depth information from acquired interferograms. By using Gabor filterbanks, which do not need any a priori knowledge of the signal, selectivity of the extracted signal has been enhanced by a factor of 1.3 compared to a simple local Fourier spectrum evaluation, and time of the extraction procedure divided by 6, even when compared to fast iterative calculation in the Fourier spectrum. Moreover, by imposing the frequency and/or orientation of the signal to detect, which can be known by a simple calibration procedure, it has been proved that the selectivity can be further enhanced by a factor of 2.5, keeping the calculation time as short or even shorter. A compact prototype has been designed and built, with a specific multiple fibers arrangement for illumination. The system is separated in two parts: the first one is directly attached to the rigid endoscope, and contains imaging as well as recombining object/reference wave optics, and the detector. It fits into a box with overall dimensions of 115 × 83 × 94 mm. The second part contains the laser source, the scanning arm and the injection optics for the fibered system. It is designed to be left on a small cart. This device can potentially be brought to the bedside, and used during routine endoscopic check-up. Associated with the device, a complete framework has been elaborated in order to simplify the acquisition procedure as well as the signal processing. This results in a fully automated environment taking in charge hardware control, data storage and signal processing, up to the tri-dimensional scene rendering.

EPFL2009

Etude par microscopie électronique à transmission à haute résolution de l'élargissement thermique des parois de domaines ferroélectriques dans un système de premier ordre

The macroscopic properties of ferroelectric materials depend directly on the domain configuration and the structure of the domain boundaries. For this reason, their study is of great scientific and technological interest. Several models have been developed to describe the properties of domain walls (structure, thickness, stress at the interface, mobility, …). However, few quantitative experimental observations of ferroelectric domain walls at an atomic scale have been reported, and even less results exist at higher temperatures. The ferroelectric domain-wall thickness is an important parameter whose behavior as a function of temperature is directly related to the order of the phase transition. In a second-order system, the temperature dependence of the domain-wall thickness follows a power law L ~ (Tc - T)-ω where ω is the critical exponent characterizing the divergence of L near the critical temperature Tc The predictions concerning the numerical value of are different depending on the theoretical model considered. Nevertheless, the majority of ferroelectric materials are known to undergo a first-order transition. In these systems the domain-wall thickness increases when the transition temperature is approached, but without diverging as for a second-order phase transition. The major objective of this work was to measure this broadening predicted by the theory for the first time at the approach of the phase transition temperature. We have shown that high resolution transmission electron microscopy is powerful technique for the study of ferroelectric domain walls not only at room temperature but also at higher temperatures. This method allows a direct and local observation of interfaces at an atomic scale which is a considerable advantage over diffraction techniques (X-ray, neutron, electron) and most of the other electron microscopy methods. However, the requirements of this method concerning the performance of the optical system of the microscope, the quality of the specimen and its stability in the column are very high, and today high resolution microscopy at high temperature remains a major challenge. The domain-wall thickness is obtained from the measurement of the crystal lattice distortion near the interface using two different numerical techniques of image analysis. The first method is based on the accurate determination of the center of bright peaks which appear on high resolution images for a judicious choice of the experimental parameters (delocalization and crystal thickness) and which represent the crystal lattice nodes. The second technique consists in selecting a circular region of the power spectrum around a reflection peak, centering the Fourier space on this reflection and performing the inverse Fourier transform. The information about the local displacement of atomic planes corresponding to the selected reflection is extracted form the phase component of the obtained complex image. Both techniques have been successfully applied to the measurement of the thickness of the 90° ferroelectric domain walls in PbTiO3 single crystals. This perovskite presents a first order phase transition at Tc = 492.2°C. which corresponds to the transformation of the crystal from the high-temperature paraelectric cubic phase to the low-temperature ferroelectric tetragonal phase. For the first time, the thermal broadening of domain walls has been measured quantitatively using HRTEM. The results are compared with the predictions made by phenomenological theories. We find that the domain wall thickness increases continuously from 0.7 nm at room temperature up to 5.5 nm near the transition temperature. Our results are consistent with those obtained by other authors at room temperature and those of a very recent study performed at higher temperatures using weak-beam microscopy. The measured domain-wall broadening is greater than the one predicted by a three-dimensional Ginzburg-Landau model where the polarization which is the primary order parameter is coupled to the elastic strain which plays the role of secondary order parameter. Our results are better described by a one-dimensional Ginzburg-Landau model without secondary order parameter. In this case the gradient coupling coefficient κ of the Ginzburg-Landau free energy is found to be 1.3 · 10-10 m3F-1. The methods developed for the measurement of the domain-wall thickness can by applied not only to other first-order ferroelectric crystals but also to ferroelectric systems presenting a second-order phase transition thus allowing the determination of the critical exponent ω. Characteristics only present in 2-D systems could also be detected by studying very thin specimens. Moreover, the same techniques can be adapted to the study of domain walls in purely ferroelastic materials.

EPFL2000

Endoscopic low coherence interferometry applied to tri-dimensional contouring of upper airways

Yves Delacrétaz

EPFL2009

Etude par microscopie électronique à transmission à haute résolution de l'élargissement thermique des parois de domaines ferroélectriques dans un système de premier ordre

EPFL2000