Optical Coherence Correlation Spectroscopy

Questions such as biomolecular interactions, kinetics of enzymes, DNA conformational changes, cellular structures properties (among others) are addressed by characterizing the biological function of molecules. This function depends on their structure and their dynamic properties, and are usually studied with optical labels. A widely used technique for that purpose is fluorescence correlation spectroscopy (FCS) that adopts fluorophores as reporters of dynamic processes. However, photobleaching limits the observation time on the one hand, and saturation at high excitation intensities imposes constraints for the study of fast molecular processes on the other hand. Gold nanoparticles (NPs) are labels of particular interest to overcome these drawbacks due to their photostability, low cytotoxicity and their availability with biocompatible surface coatings. In addition, NPs have found widespread application in life sciences and medicine, such as labels for optical imaging, carriers for drug and gene delivery, targets for cancer cell imaging and phototherapy, and contrast agent for magnetic resonance imaging (MRI). These applications all require methods to characterize their behaviour in a biological environment. Medical use of NPs usually requires intravenous injection. Once NPs are introduced into the blood stream they become exposed to biomolecules in the plasma that form a so-called protein corona. Obviously, knowledge about this interaction is needed for a safe medical application of NPs. The uptake of NPs by cells depends mainly on this protein corona. It is thus very important to understand the structural and dynamic properties of the protein corona at the molecular level to judge the fate of NPs in the human body prior to clinical studies. We introduce Optical Coherence Correlation Spectroscopy (OCCS) which exploits the backscattered light of NPs illuminated by a broadband light source. Due to the measurement of a scattering signal, OCCS gives access to long time scales that are hardly accessible with FCS experiments. The signal acquisition is similar to Fourier-domain optical coherence microscopy (FDOCM) with a dark-field contrast. We present the theory of OCCS based on auto-correlation analysis. Numerical simulations were used to estimate the performance of our new spectroscopy method and to build an analytical model for the auto-correlation function for measuring the diffusion coefficient and concentration of NPs in solution. An application to the study of the protein corona on NPs is presented. The hydrodynamic radius of superparamagnetic iron oxide nanoparticles (SPIONs) with adsorbed bovine serum albumin (BSA) is measured under physiological conditions using OCCS.We also aimed at improving the detection of these NPs in highly scattering environments, for instance inside a cell. Photothermal optical lock-in OCCS (poli-OCCS) is proposed, which exploits the absorption properties of gold NPs to generate a specific signal due to the photothermal effect. Poli-OCCS is introduced and first proof-of-principle measurements with gold NPs are presented.

Optical microscopy for imaging through scattering media

Xin Yang

Optical microscopy has been widely used in the life sciences and biological research for several decades. It possesses many advantages over x-ray radiography, computed tomography, ultrasound imaging and magnetic resonance imaging, in terms of being low cost, high resolution, harmless and capable of multiple contrast mechanism, etc. However, the weakness of optical microscopy is the inability to perform deep tissue imaging. Owing to the short wavelength of optical waves, it experiences much more scattering than longer waves in inhomogeneous materials such as biological tissues. Scattering scrambles the wavefront and limits the penetration depth of optical microscopy to only hundreds of microns. We explore in this thesis, novel optical approaches to image through scattering media. There are two main categories of the state of art techniques: imaging with ballistic photons, which requires separation of ballistic photons from the sneak or multiple scattered ones; and imaging with scattered photons, which relies on manipulation of the scattered photons according to the specific scattering properties. We primarily focus on the second category in this thesis. Taking advantage of the reversibility of light, phase conjugation has a long history in aberration compensation. A deterministic wave is incident on a scattering medium and results in a random scattered field. If we record the full information of the scattered field (amplitude and phase) and generate a phase conjugated replica of it, the phase conjugated wave can follow the same path and reconstruct the original deterministic wave after passing back through the scattering medium. In this work, phase conjugation is achieved using digital method, called digital phase conjugation. The scattered field is recorded through digital holography, and a spatial light modulator is applied to construct the phase conjugated wave. Second harmonic radiation imaging probes are used as the original light source, rendering the generation of a focus behind the scattering medium. The memory effect, which indicates the correlation between speckles with different incident angles, is exploited to move the focus around and obtain three-dimensional scanning microscopy behind scattering medium. Speckle scanning microscopy is the second method demonstrated in this thesis for imaging through turbid media. In contrast to digital phase conjugation, which requires access from both sides of the scatterers to measure the scattered field, in speckle scanning microscopy, excitation and collection can be achieved from the same side. The specific scattering events are not taken into account, instead, the statistic property of the scatterers plays a major role. This technique makes full use of two facts: 1. The statistically averaged autocorrelation of speckle fields approaches a delta function; 2. When a speckle pattern scans across a fluorescence object, the collected fluorescence intensity is proportional to the convolution of the speckle field and the object. Linking these two facts with delicate calculation, we can eventually minimize the influence of the speckle, and reconstruct two-dimensional images of objects behind turbid medium. Encouraged by the success of two-dimensional speckle scanning microscopy, we investigate more advanced possibilities by simulation, including three-dimensional speckle scanning and reference based speckle scanning techniques. [...]

EPFL2014

Biomedical optical techniques for intracochlear cellular imaging

Marilisa Romito

Imaging the inner ear microanatomy is of great importance for the assessment of the state of the cells responsible for sound detection. Hearing disorders are mainly due to a malfunction of the cochlea, the bone containing the cells inside the inner ear. Under this situation, using suitable imaging techniques would be extremely helpful in detecting disease and diagnosing pathologies of the inner ear. However, cochlear small size and encasement in bone provide challenging obstacles, preventing visualization of intracochlear microanatomy using standard clinical imaging modalities. In this thesis, we explore optical techniques to image inner ear cells and we report an innovative method to observe intracochlear structures through the scattering cochlear bone. Firstly, we report different imaging techniques we used for intracochlear cellular investigation, starting with conventional optical microscopy. Two-photon excitation fluorescence (TPEF) microscopy is the most suitable technique for high quality images of the hair cells within the organ of Corti. Our results from extracted mouse organ of Corti show that we are able to distinguish between healthy and damaged sensory cells and nerve fibers, analyzing TPEF images from different mouse samples: young, old, exposed to noise and not exposed to noise. We then describe different noninvasive methods that have been used to image intact cochleae, such as Optical Coherence Tomography (OCT), X-ray Computed Tomography (X-Ray CT), micro-Computed Tomography (X-Ray µCT) and Optical Diffraction Tomography (ODT). These techniques allow the observation of the internal ear anatomy, despite the bone. We report tomographic volumetric reconstructions of mouse cochlea and we compare them in terms of achievable image resolution. µ-CT is the most appropriate techniques providing both penetration through bone and high-level resolution images down to 2µm. However, the high dose of radiation used in µCT studies prevent translation of these techniques to living humans. Due to the discussed challenges for imaging the hair cells through the highly scattering bone, the aim of my thesis is to develop a new method for intracochlear cellular imaging with minimum trauma. The last chapter represents the core of this PhD project. In this chapter, after considering the advantages and disadvantages of all the possible imaging techniques, we report our innovative technique for visualization of cochlear cells through the overlying scattering bone by combining femtosecond laser bone ablation and TPEF microscopy. The controlled ultrafast laser ablation reduces the optical scattering of the cochlear bone while the TPEF provides a contrast mechanism to resolve individual cells behind the bone. We implemented a simultaneous OCT with the laser ablation to enhance the precision of ablation and prevent inadvertent violation of the delicate cells hidden behind bone. An additional bright-field camera shows real-time images of the sample. We demonstrate that our approach improves the light focusing capability through the cochlear bone, allowing imaging of intracochlear structures in intact cochleae with high resolution. This extremely highly promising approach can be further developed for clinical ablation instruments and endoscopes that can reach the cochlea and produce images of cells within the inner ear to establish precise diagnosis, guide treatment and assess response to therapies, which is not yet possible in the clinic.

EPFL2019

Optical spectral probing for epithelial tissue characterisation

Philippe Thueler

In this thesis, a new method has been developed, which determines the optical coefficients spectra (absorption and scattering) of small volumes of tissue (1 mm3) by the measurement of spatially resolved reflectance between 480 and 950 nm. Firstly, an algorithm has been developed to determine the optical coefficients form reflectance measurements: It is based on the optical propagation model introduced by Bevilacqua [Bevilacqua 99 JOSA]. This model takes the first two moments of the phase function into account through the γ parameter, allowing to accurately simulate the propagation of photons close to the source by the Monte Carlo method. These simulations are then fitted to the reflectance to obtain the optical coefficients. To obtain any value of absorption, scattering or γ parameter, the simulations are interpolated by 3D B-splines. A complete robustness to noise study of the algorithm has been presented. A theoretical study of the γ parameter in tissue over broad wavelengths has been reported. Tissue was simulated with Mie theory for a fractal distribution of sphere diameters. For such a distribution, the γ spectrum is almost flat, instead of being wavy like in the case of mono-disperse sphere diameter. The correlation of the mean value of γ with the fractal power (or dimension) of the distribution was made and showed an analytical form of hyperbolic tangent. It allowed deepening the interpretation of the γ parameter as being an indicator of the relative number of small scatterers (much smaller than the wavelength, e.g. of Rayleigh type) in comparison of bigger ones (their size being comparable to the wavelength). The assessment of the set-up and the algorithm was made by measuring the reflectance of a solution containing 5 different diameters of polystyrene spheres simulating the scattering and Nigrosin colorant as absorber. It showed a good agreement between measurements and prediction. In order to built a better comprehension of absorption and scattering at the tissue scale, a first part of in vivo investigations has been made on an animal model. The skin of the mouse back was altered with Freund's adjuvant and TPA and the optical coefficients were measured on treated and control sites. The effect of alteration (epidermis and dermis thickening, oil vesicle appearing, inflammatory cells infiltration, etc.) was clearly correlated with optical coefficients variations. The scattering spectrum always showed the better contrast. The absorption spectrum, especially in the haemoglobin peak region (between 550 and 600 nm) also showed significant information, in terms of oxy and deoxy-haemoglobin concentration and saturation. Sensitivity and specificity of the "diagnosis" was evaluated. The study of stomach epithelium in vivo on 29 human subjects was made using a small optical fibre probe (

EPFL2001