New implementations of phase-contrast imaging

Phase-contrast imaging is a method of imaging widely used in biomedical research and applications. It is a label-free method that exploits intrinsic differences in the refractive index of different tissues to differentiate between biological structures under analysis. The basic principle of phase-contrast imaging has inspired a lot of implementations that are suited for different applications. This thesis explores multiple novel implementations of phase-contrast imaging in the following order. 1, We combined scanning Oblique Back-illumination Microscope (sOBM) and confocal microscope to produce phase and fluorescence contrast images in an endomicroscopy configuration. This dual-modality design provides co-registered, complementary labeled and unlabeled contrast of the sample. We further miniaturized the probe by dispensing the two optical fibers in our old design. And we presented proof of principle demonstrations with ex-vivo mouse colon tissue. 2, Then we explored sOBM-based phase and amplitude contrast imaging under different wavelengths. Hyperspectral imaging is achieved by multiplexing a wide-range supercontinuum laser with a Michaelson interferometer (similar to Fourier transform spectroscopy). It features simultaneous acquisition of hyperspectral phase and amplitude images with arbitrarily thick scattering biological samples. Proof-of-principle demonstrations are presented with chorioallantoic membrane of a chick embryo, illustrating the possibility of high-resolution hemodynamics imaging in thick tissue. 3, We focused on increasing the throughput of flow cytometry with principle of phase-contrast imaging and compressive sensing. By utilizing the linearity of scattered patterns under partially coherent illumination, our cytometer can detect multiple objects in the same field of view. By utilizing an optimized matched filter on pupil plane, it also provides increased information capacity of each measurement without sacrificing speed. We demonstrated a throughput of over 10,000 particles/s with accuracy over 91% in our results. 4, A fourth part, which describes the principle and preliminary results of a computational fluorescence endomicroscope is also included. It uses a numerical method to achieve sectioning effect and renders a pseudo-3D image stack with a single shot. The results are compared with true-3D image stack acquired with a confocal microscope

Microscopy and Analysis

Microscopes represent tools of the utmost importance for a wide range of disciplines. Without them, it would have been impossible to stand where we stand today in terms of understanding the structure and functions of organelles and cells, tissue composition and metabolism, or the causes behind various pathologies and their progression. Our knowledge on basic and advanced materials is also intimately intertwined to the realm of microscopy, and progress in key fields of micro- and nanotechnologies critically depends on high-resolution imaging systems. This volume includes a series of chapters that address highly significant scientific subjects from diverse areas of microscopy and analysis. Authoritative voices in their fields present in this volume their work or review recent trends, concepts, and applications, in a manner that is accessible to a broad readership audience from both within and outside their specialist area

Confocal Microscopy: a Powerful Tool for Biological Research

Conventional light microscopy allows the observation of living as well as fixed cells and tissues to generate two-dimensional images. The out-of-focus information often obscures the ultrastructural details, especially in thick specimens with overlapping structures. The earliest available light microscopy visualized the objects in hydrated state in two-dimensions during their temporal development. The emergence of electron microscopy (EM) provided superb resolution of ultrastructural details, but it was applicable only for objects in the dehydrated state and thereby potentially introducing handling artifacts. The usefulness of optical methods, however, has been limited by the poor depth discrimination. Often, the fluorescence and reflectance images are severely degraded by the scattered- or emitted-light from tissue structures outside the plane of focus. These limitations have been partially overcome by video image processing and deconvolution. Laser scanning confocal microscopy (LSCM) overcomes the above difficulties and produces improved light microscopic images of fixed as well as living cells and tissues. In terms of resolution of the image, the confocal microscope occupies a position in between the light and electron microscopes. Confocal microscope generates information from a well-defined optical section rather than from the entire specimen, thereby eliminating the out-of-focus glare and increasing the contrast, clarity and detection sensitivity. Optical sectioning is noninvasive and less time consuming compared to reconstruction algorithms to give 3-D images. Optical sectioning is achieved not only in the xy plane (perpendicular to the optical axis of the microscope) but also vertically in the xz or yz plane (parallel to the optical axis). With vertical sectioning, cells are scanned laterally (x or y axis) as well as in depth (z axis). Stacks of optical sections taken at successive focal plane (known as z series) are then reconstructed to generate a 3-D version of the specimen. The 3-D image can be directly visualized where each data point represents the quantity of specific contrast parameter used at a certain point in space. The image processing can be additionally used to enhance the confocal images. It is widely used in the fluorescence mode for different specimens and in bright field reflection mode for objects of different forms

4Pi live cell imaging

One distinctive advantage of light microscopy over electron microscopy is it ability to selectively image sub-cellular details three-dimensionally in living cells. Live cell imaging is the domain of light microscopy. In this thesis a live cell imaging setup relying on 4Pi fluorescence microscopy is presented. To speed up the imaging process and record cellular processes of the order of a few seconds a new method of multifoci-generation via the stacking of birefringent crystals is implemented. The axial optical sectioning ability of the system is ~120 nm and the recording of an average-sized 3D data stack is accomplished in less than 10 seconds. The microscope’s applicability to live cell imaging is verified by recording both mitochondrial fusion and fission events in live yeast and the movement of PML bodies in live human U2OS cells. To push the limits of applicability of the system further, second harmonic generation is tested as an alternative label-free coherent contrast mechanism and binary amplitude filters are successfully applied. Second harmonic generation turns out to be an imaging technique confined to specific applications. With the implementation of amplitude filters 4Pi microscopy of aqueous tissue becomes convenient and broadly applicable

Identification of morphological biosignatures in martian analogue field specimens using in situ planetary instrumentation

We have investigated how morphological biosignatures (i.e., features related to life) might be identified with an array of viable instruments within the framework of robotic planetary surface operations at Mars. This is the first time such an integrated lab-based study has been conducted that incorporates space-qualified instrumentation designed for combined in situ imaging, analysis, and geotechnics (sampling). Specimens were selected on the basis of feature morphology, scale, and analogy to Mars rocks. Two types of morphological criteria were considered: potential signatures of extinct life (fossilized microbial filaments) and of extant life (crypto-chasmoendolithic microorganisms). The materials originated from a variety of topical martian analogue localities on Earth, including impact craters, high-latitude deserts, and hydrothermal deposits. Our in situ payload included a stereo camera, microscope, Mössbauer spectrometer, and sampling device (all space-qualified units from Beagle 2), and an array of commercial instruments, including a multi-spectral imager, an X-ray spectrometer (calibrated to the Beagle 2 instrument), a micro-Raman spectrometer, and a bespoke (custom-designed) X-ray diffractometer. All experiments were conducted within the engineering constraints of in situ operations to generate realistic data and address the practical challenges of measurement

In-vivo Raman spectroscopy: from basics to applications

For more than two decades, Raman spectroscopy has found widespread use in biological and medical applications. The instrumentation and the statistical evaluation procedures have matured, enabling the lengthy transition from ex-vivo demonstration to in-vivo examinations. This transition goes hand-in-hand with many technological developments and tightly bound requirements for a successful implementation in a clinical environment, which are often difficult to assess for novice scientists in the field. This review outlines the required instrumentation and instrumentation parameters, designs, and developments of fiber optic probes for the in-vivo applications in a clinical setting. It aims at providing an overview of contemporary technology and clinical trials and attempts to identify future developments necessary to bring the emerging technology to the clinical end users. A comprehensive overview of in-vivo applications of fiber optic Raman probes to characterize different tissue and disease types is also given

Electrophysiological and cellular analysis of filamin-C mutations causing cardiomyopathy using human iPSC-derived cardiomyocytes

Background: Arrhythmogenic Cardiomyopathy (AC) is a genetic cardiac disease resulting from different mutations within proteins constituting the intercalated disc, including desmosomal and nondesmosomal proteins. Recent studies have revealed that mutations in filamin-C (FLNC) may lead to AC. The arrhythmogenesis and electrophysiological effects of FLNC-related AC are incompletely understood. Therefore, the aim of this study is to assess the potential electrophysiological consequences of FLNC loss as occurs in AC in human induced pluripotent stem cell-derived cardiomyocytes (hiPSCCMs). Specifically, I aimed to characterise abnormal electrical activity and the expression and function of key proteins in cardiac electrical activity such as gap junction protein connexin 43 (Cx43).// Methods: hiPSC-CMs were differentiated and observed by immunofluorescence microscopy. Small interfering RNA (siRNA) transfection was utilised to knockdown the expression of FLNC in hiPSC-CMs. Protein analysis was performed using western blotting to confirm the knockdown efficiency. Electrophysiological properties were recorded using a multielectrode array and manual patch clamping. Optical recording of membrane potential and calcium activity from hiPSC-CMs were also carried out using parameter sensitive dyes.// Results: Silencing of FLNC led to markedly decreased immunofluorescence signals of FLNC, Cx43, desmoplakin, and junctional plakoglobin. No significant reductions were noted in the immunofluorescence signals of voltage-gated sodium channel (Nav1.5) and plakophilin-2 compared with control hiPSC-CMs. Western blotting showed the reduction of FLNC and Cx43 expression following silencing of FLNC. Knockdown of FLNC resulted in disturbances to the recorded action and field potential signals of hiPSC-CMs and arrhythmic likeevents. Transfected hiPSC-CMs with siRNA-FLNC were associated with prolongation of calcium transient durations, optical action potential duration, and action potentials measured with patch clamping.// Conclusion: The current findings indicated that loss of FLNC resulted in a complex arrhythmogenic phenotype in hiPSC-CM

Methods and instrumentation for raman characterization of bladder cancer tumor

High incidence and recurrence rates make bladder cancer the most common malignant tumor in the urinary system. Cystoscopy is the gold standard test used for diagnosis, nevertheless small flat tumors might be missed, and the procedure still represents discomfort to patients and high recurrence can result from of urethral injuries. During cystoscopy, suspicious tumors are detected through white light endoscopy and resected tissue is further examined by histopathology. after resection, the pathologist provides information on the differentiation of the cells and the penetration depth of the tumor in the tissue, known as grading and staging of tumor, respectively. During cystoscopy, information on tumor grading and morphological depth characterization can assist onsite diagnosis and significantly reduce the amount of unnecessarily resected tissue. Recently, new developments in optical imaging and spectroscopic approaches have been demonstrated to improve the results of standard techniques by providing real-time detection of macroscopic and microscopic biomedical information. Different applications to detect anomalies in tissues and cells based on the chemical composition and structure at the microscopic level have been successfully tested. There is, nevertheless, the need to cope with the demands for clinical translation. This doctoral thesis presents the investigations, clinical studies and approaches applied to filling the main open research questions when applying Raman spectroscopy as a diagnostic tool for bladder cancer tumor grading and general Raman spectroscopy-based oncological clinical studies