Optical spectral probing for epithelial tissue characterisation

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 (

Local in vivo optical measurements of neurometabolic and neurovascular coupling mechanisms in the rodent cortex

Elena Migacheva

This thesis introduces a novel approach for in vivo separation and quantification of spatio-temporal dynamics of optical coefficients (absorption, scattering), oxygen saturation (SO2) and haemoglobin concentrations within rat brain somatosensory cortex under baseline and neuronal stimulation conditions. The quantification of mentioned parameters became possible due to design of a novel optical setup for localized spatio-temporally-resolved (within ∼ 1 mm3 at 10 Hz) reflectance spectroscopy (STRReS) measurements at small source-detector separations. The possibility to automatically take into account the light propagation path lengths in tissue using Monte Carlo modeling, defines the novelty of this approach and its perspectives for brain research. This kind of corrections is currently limited or inaccurate with existing techniques like optical intrinsic signal imaging. In particular, the main aim of this thesis is to explore and better understand the local spatio-temporal characteristics of neurovascular and neurometabolic coupling mechanisms within in vivo rat cerebral cortex at initial and subsequent stages of neuronal stimulation with using STRReS technique. It has been proven theoretically (with Monte Carlo simulations) and experimentally (from in vivo rat brain cortex), that STRReS measurements are suitable from in vivo somatosensory cortex under neuronal stimulation conditions. As a part of this research, we have validated theoretically and experimentally the biological importance of new optical parameter ”gamma” (parameter of the phase function), which is nessesary to take into account for accurate optical coefficients extraction. On the other side, the STRReS technique was not found to be helpful to determine differential factors corrections for improvement of optical intrinsic signal imaging analysis due to differences in experiment’s geometries. Finally, it has been shown that temporal dynamics of reflectance is strongly influenced by light scattering variations within 500-900 nm range. Specifically, the shape of reflectance dynamics at 600 and 610 nm differs significantly from deoxy-haemoglobin concentration dynamics at initial stage of neural activation. For this reason, it is important always to take into account the variations of both components of reflectance signal: absorption and scattering. Additionally, an initial delay of 1 s was found in SO2 and haemoglobin concentrations dynamics. The interpretation of this delay, however, is strongly dependent on the origin of scattering signal. Our results are well correlated with ”metabolic” hypothesis of underlying neurovascular and neurometabolic coupling mechanisms, assuming that the scattering signal has a non-vascular origin at 500-599 nm. Nevertheless, the ”neurogenic” hypothesis is not completely excluded as far as the scattering signal has a vascular origin at 500-599 nm.

EPFL2012

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

Local in vivo optical measurements of neurometabolic and neurovascular coupling mechanisms in the rodent cortex

Elena Migacheva

EPFL2012

Optical microscopy for imaging through scattering media

Xin Yang

EPFL2014

Optical spectral probing for epithelial tissue characterisation

In Vivo Local Determination of Tissue Optical Properties: Applications to Human Brain

Local in vivo optical measurements of neurometabolic and neurovascular coupling mechanisms in the rodent cortex

Optical microscopy for imaging through scattering media

Local in vivo optical measurements of neurometabolic and neurovascular coupling mechanisms in the rodent cortex

In Vivo Local Determination of Tissue Optical Properties: Applications to Human Brain

Optical microscopy for imaging through scattering media