Benjamin Rappaz
Obtaining high resolution 3D quantitative images is critical for elucidating the relationship between cell dynamics and physiological or pathological processes. However the feasibility of such cellular dynamic studies is limited by the resolution of the 3D images, the required acquisition time and the degree of invasiveness of the currently available imaging techniques as well as their limitation to provide quantitative information. Digital Holographic Microscopy (DHM) is an optical imaging technique developed to meet most of these requirements to accurately explore cell dynamics. In short, DHM allows to quantitatively measure the wavefront transmitted through a specimen and magnified by a microscope objective. A hologram, which contains the whole information of the transmitted wavefront, is formed by the interference of the wave coming from the object with a reference wave, and recorded with a camera. The hologram reconstruction process, based on the electromagnetic wave propagation theory, permits to numerically process the hologram in order to extract both amplitude and phase information. This process allows to obtain an exact replica of the wave diffracted or transmitted by the observed specimen. Thanks to its interferometric nature, DHM yields phase images with a nanometric sensitivity along the optical axis of the microscope, revealing extremely detailed minute information about the cell morphology and internal structure, as well as cellular micromovements in the nanometer range. So far, DHM has proven its efficiency on numerous fields in material sciences. This thesis presents some technical developments achieved with DHM and extends the applications of this technique to life sciences in general and cellular structure and dynamics in particular. The quantitative phase images generated by DHM represent an endogenous contrast signal which contains dual information about both the cell morphology and the refractive index (RI), which is essentially related to the intracellular (protein) content. The first part of this thesis (chapter 2) presents two different approaches to separately extract these two contributions and obtain an independent measurement of both the cell morphology and RI, thus providing an interpretation of the phase signal which is more readily exploitable and more relevant for cell biologists. The RI is a function of the dry mass, thus the phase signal variation in time can be related to biomass production and provides a direct interpretation of the phase signal. In section 3.1 this relation is exploited to study the dry mass production during the cell cycle of fission yeast. The decoupling procedure presented in section 2.1 allows to measure the thickness and RI of cells. In section 3.2, we use this decoupling procedure to measure the Mean Corpuscular Volume (MCV) and RI of red blood cells. These measurements are also compared to those obtained by other techniques. In addition, as far as red blood cell are concerned, the RI can be related to the Mean Corpuscular Hemoglobin Content (MCHC). The MCV, RI and MCHC are important clinical parameters that can be non-invasively and instantly measured with DHM. The membrane of red cells presents a flickering at a nanometric scale which can reflect their metabolic state, related to ATP content. Due to the technical difficulty to measure this flickering, it has been difficult to investigate the origin of these fluctuations. DHM, thanks to its nanometric and microsecond sensitivity, allows to monitor these Cell Membrane Fluctuations (CMF). In section 3.3 DHM is used to measure CMF of healthy and metabolically-impaired red blood cells. The last section of this thesis (section 3.4) assesses how DHM can quantitatively monitor minute cell morphological and optical changes induced by agonist-mediated activation of specific membrane receptors. This is illustrated by tracking the phase signal variations during excitotoxic activation of neurons by the neurotransmitter glutamate.
EPFL2008
