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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.
The organisation of molecules into dynamic cells, and collaboration of many of those cells over a billion years led to the evolution of human life. During the last century, biologists then began to unravel the marvels of cellular organisation with ever increasing detail. We now know, single cells not only comprise a defining DNA, but also a multitude of organelles. Mitochondria are such organelles, and produce cellular energy, at the heart of cellular metabolism. For this, they transcribe their own genome into mtRNA, and assemble respiratory chains. In recent decades, novel approaches, and technology enabled to shed new light on the spatial organisation of sub-cellular and mitochondrial processes. Thereby mtRNA was discovered to accumulate into small foci inside the mitochondrial matrix by fluorescence microscopy. Certain mitochondrial proteins also have also been found to conglomerate in these mitochondrial RNA granules (MRGs), but merely nothing is known about the structural, dynamic, and biophysical properties of MRGs, and their functional importance remains elusive. The development of superresolution microscopy has led to a revolution and revival of imaging methods for cell biology. This technology now enables investigation of sub-cellular biology at the nanometre scale, and to illuminate MRGs inside mitochondria.
In this thesis I investigate the organisational principles governing single cell and mitochondrial biology. First, I explore the application of superresolution microscopy to study the spatial organisation of nuclear DNA domains (TADs). I find high throughput STORM microscopy (htSTORM) allows to monitor effects of drug treatment on spatial rearrangement of a cancer associated TAD, together with established bulk-sample analysis. Next, I study the organisation of mitochondrial dynamics, by live-cell structured illumination microscopy (SIM). How different molecular pathways associated with mitochondrial proliferation or degradation are organised has long been a matter of debate. We discover a pattern for spatial organisation of mitochondrial fission, which distribute along the mitochondrial network in a bimodal manner. With this framework we distinguish different types of fissions, and show that distinct molecular features are associated with mitochondrial proliferation, or degradation through mitophagy. I find actin to be involved in proliferative midzone fission, whereas peripheral fission is associated with elevated reactive oxygen levels, and precedes mitophagy. By htSTORM, SIM and additional microscopy methods I then elucidate the biophysical organisation of intra-mitochondrial processes and MRGs. I show that the MRG ultrastructure consists of compacted RNA embedded within a dynamic protein cloud. Furthermore, MRGs associate with the inner mitochondrial membranes, and their distribution is governed by mitochondrial dynamics. MRGs can now be understood as nanoscopic, robust and fluid compartments, likely to influence intra-mitochondrial reaction kinetics. Finally, I also begin to unravel the spatial and biophysical organisation of mitochondrial transcription.
My results contribute to a holistic and dynamic picture of mitochondrial organisation. I highlight advantages of state-of-the-art microscopy to investigate spatial and temporal organisation of sub-cellular processes, and mark the onset of single organelle biology, in the context of other recent studies of sub-mitochondrial organisation.