Dual-mode digital holographic and fluorescence microscopy for the study of morphological changes in cells under simulated microgravity

A dual mode microscope is developed to study morphological evolution of mouse myoblast cells under simulated microgravity in real time. Microscope operates in Digital Holographic Microscopy (DHM) and widefield epifluorescence microscopy modes in a time sequential basis. DHM provides information on real time cellular morphology. EGFP transfected actin filaments in mouse myoblast cells function as the reporter for the fluorescence microscopy mode. Experimental setup is fixed in the RPM to observe microgravity induced dynamic changes in live cells. Initial results revealed two different modifications. Disorganized structures become visible in the formed lamellipodias, and proteins accumulate in the perinuclear region.

Application of CMOS image sensors in optical sectioning and polarization microscopy

Jelena Mitic

In this thesis new imaging approaches for optical microscopy are proposed and studied. They are based on the use of dynamic structured illumination in combination with a demodulation detection concept employing CMOS image detectors. Two particular implementations are suggested: real-time optical sectioning microscopy and whole field quantitative polarization microscopy. Optical sectioning, i.e depth resolved imaging, allows observation of thin sections in volumetric samples. In its classical configuration the wide-field microscope does not allow optical sectioning of the investigated objects. In this thesis the optical sectioning in a wide-field microscope is achieved by illuminating the sample with a continuously moving single spatial frequency pattern, which temporarily modulates the signal obtained on the photodetector. Only the object parts that are in the focus have a good contrast and consequently generate stronger signal on the detector. The optically sectioned images are obtained employing a CMOS image detector, which demodulates the time-dependent component of the image. Two different systems for real-time acquisition of optically sectioned images are proposed. The first one is based on a specially designed smart-pixel-detector-array (SPDA), which allows performing the signal processing by an electronic circuit integrated to each pixel of the sensor. The second system utilizes a commercial CMOS image sensor. Here, the images are treated by a digital signal processor (DSP) integrated into the camera (iMVS-155). Both approaches provide real-time acquisition of optically sectioned images. The proof of principle for the new method is demonstrated by imaging artificial three-dimensional reflective samples. The optical sectioning imaging of biological samples in bright-field mode and in fluorescence mode is also demonstrated. The optical sectioning performances of the studied systems are similar to that of the confocal microscope. However the new method has some advantages over the classical confocal system: e.g. the difficulties with the alignment and the post-processing inherent to the confocal system are avoided. The theoretical framework presented in the thesis describes the image formation in structured illumination for both reflective and fluorescent samples. The influence of longitudinal chromatic aberration and spherical aberration caused by specimen induced index mismatch on depth discrimination property are studied. In extension to the work related to optically sectioned microscopy, the quantitative polarization microscopy imaging method is proposed as a new application for CMOS detectors. The polarization state of the illuminating light is dynamically changed and the demodulation detection approach is employed to extract the retardance and azimuth of the birefringent sample in real-time; for a whole field of view. Examples of polarization sensitive measurements for different samples are presented. The theoretical analysis is performed using the Jones formalism. Finally, the potentials and limitations of the CMOS detectors for applications in optical microscopy are discussed. Thanks to constant advancements in CMOS technology, the acquisition speed, resolution and sensitivity of new CMOS detectors generations have been improved. This will allow adapting the methods proposed in this thesis for investigations of fast dynamic process where high temporal and spatial resolution is required.

EPFL2004

Time-lapse high-resolution microscopy to study the morphogenesis of microorganisms

Mélanie Thérèse Marie Hannebelle

Mycobacterium tuberculosis, the etiological agent for the tuberculosis disease, is a bacterial pathogen thought to infect about a quarter of the global human population. It is the first cause of death among infectious diseases, and is most prevalent in low-income populations. Much remains unknown about how these bacteria grow, divide, and manage to persist in the host even during month-long antibiotic treatments. Bacteria are typically at the edge of the spatial resolution that conventional optical microscopy techniques can offer, and so new tools are required to study bacterial substructures or surface morphologies with nanometer resolution. We developed a platform for automated optical microscopy and atomic force microscopy (AFM) in order to study the morphogenesis of mycobacteria. What is the growth dynamics of mycobacteria? Opposite models coexist in the literature for the pole growth dynamics of mycobacteria: the unipolar model and the bipolar model. We show that the growth dynamics of mycobacteria follows a new end take off NETO model. There is a surprising similarity between the NETO growth dynamics of mycobacteria and of fission yeast, which hints at shared mechanisms for pole growth in evolutionarily distant pole growing organisms. How do pole-growing organisms maintain their shape through insertion of new cell wall material at the tip? To explore further pole growth morphogenesis, we developed a method for imaging with AFM the pole of a growing rod-shaped microorganism. We observed the formation of a stiff fibril mesh on the pole of fission yeast cells, and showed that this mesh is stretched as the cell grows and insert new cell wall material at the tip. How do mycobacteria divide into two sibling cells? Another essential aspect of morphogenesis of rod-shaped cell is the division of a mother cell into two sibling cells. Using AFM, we measured the relative contributions of mechanical forces and molecular mechanisms driving the division process in mycobacteria. We showed that there is a concentration of mechanical stress at the future division site, using a combination of COMSOL finite element modeling and AFM measurements while controlling the cell turgor pressure. The accumulation of mechanical stress, in combination of enzymatic activity, ultimately leads to mechanical fracture and separation of the sibling bacteria. Conventional optical microscopy techniques tend to have low resolution and high speed, while conventional AFMs have comparatively high resolution and low speed. We designed and built a 3D-printed structured illumination (SIM) module, the openSIM, as an add-on for fluorescence microscopes. It provides structured illumination to obtain SIM images with higher resolution. With the aim of improving the imaging speed of AFM, we implemented photothermal actuation in a tip-scanning AFM head, and quantified the design constraints for high-speed photothermal off-resonance tapping imaging. AFM is most often used to scan and acquire high-resolution images of a sample. Its nanometer precision make it a unique tool for other applications. We combined an AFM head with a volcano-shaped probe for electrogram measurements, and demonstrated simultaneous recording of extracellular field potentials and contraction of the cell. This new tool opens the way for future studies exploring the intriguing links between mechanobiology and electrophysiology, with single-cell resolution.

EPFL2020

Cellular dynamics explored with digital holographic microscopy

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