Optical microscopy is a cornerstone of biomedical research. Advances in optical techniques enable specific, high resolution, sterile, and biologically compatible imaging. In particular, beam shaping has been used to tailor microscopy techniques to enhance microscope performance. The aim of this Thesis is to investigate the use of novel beam shaping techniques in emerging optical microscopy methods, and to apply these methods in biomedicine. To overcome the challenges associated with high resolution imaging of large specimens, the use of Airy beams and related techniques are applied to light-sheet microscopy. This approach increases the field-of-view that can be imaged at high resolution by over an order of magnitude compared to standard Gaussian beam based light-sheet microscopy, has reduced phototoxicity, and can be implemented with a low-cost optical system. Advanced implementations show promise for imaging at depth within turbid tissue, in particular for neuroscience. Super-resolution microscopy techniques enhance the spatial resolution of optical methods. Structured illumination microscopy is investigated as an alternative for electron microscopy in disease diagnosis, capable of visualising pathologically relevant features of kidney disease. Separately, compact optical manipulation methods are developed with the aim of adding functionality to super-resolution techniques.

In the past two decades, Fluorescence microscopy has imparted tremendous impact in Biology and Imaging. Several super-resolution Fluorescence imaging techniques (e.g. PALM, STED, STORM, 4Pi and structured illumination) have enabled diff raction-unlimited imaging. But high resolution is limited to a depth of few tens of microns. Thus, deep tissue imaging and simultaneous volume imaging have become a highly sought after feature in Fluorescence microscopy. The research work in this thesis address these issues by using spatial filtering techniques to tailor the point spread function (PSF) which uniquely characterizes the optical sys-tem. The advantage of this approach lies in the fact that intricate details about the focal region can be computed and designed with the help of well established theory and experimentation. In particular, this technique was applied to both spherical and cylindrical lenses. The former was used to generate Bessel-like, non-diffracting beams which demonstrated the ability to penetrate deep inside tissue-like media and thereby yielded an imaging depth of nearly 650μm as compared to about 200μm for a state-of-the-art confocal microscope. The latter gave rise to light-sheet and it's extended version that is ideal for planar imaging at large penetration depths. Another development is the generation of multiple light-sheet illumination pattern that can simultaneously illuminate several planes of the specimen. The proposed multiple light-sheet illumination microscopy (MLSIM) technique may enable volume imaging in Fluorescence microscopy. The first two chapters of this thesis are introductory in nature and provides a general overview of the principles of Fluorescence microscopy and three state-of-the-art Fluorescence imaging techniques; namely confocal, multi-photon and light-sheet based microscopy. Confocal microscopes are widely considered as a standard tool for biologists and this discussion shows that even though they have made signi ficant contributions in the fields of biophysics, biophotonics and nanoscale imaging, their inability to achieve better penetration depth has prevented their use in thick, scattering samples such as biological tissue. The system PSF of a confocal microscope broadens as it goes deeper in-side a scattering sample resulting in poor-resolution thereby destroying the very concept of high resolution, noise-free imaging. Additionally, confocal microscopy suffers from in-creased photo-bleaching due to o -layer (above and below the focal plane) excitation and low temporal resolution since it requires point-by-point scanning mechanism. On the other hand, multi-photon microscopy offers several advantages over confocal microscopy such as reduced photo-bleaching and inherent optical sectioning ability, however, it still lacks in providing high temporal resolution. Light-sheet based microscopy have gained popularity in recent years and promises to deliver high spatio-temporal resolution with minimized photo-bleaching. Recently, a considerable amount of research has been dedicated to further develop this promising technique for a variety of applications. The ability to look deeper inside a biological specimen has profound implications. How-ever, at depths of hundreds of microns, several effects (such as scattering, PSF distortion and noise) deteriorates the image quality and prohibits detailed study of key biological phenomenon. Chapter 3 of this thesis describes the original research work which experimentally addresses to this issue. Here, Bessel-like beam is employed in conjugation with an orthogonal detection scheme to achieve imaging at large penetration depth. Bessel beams are penetrative, non-di ffracting and have self-reconstruction properties making them a natural choice for imaging scattering prone specimens which are otherwise inaccessible by other microscopy imaging techniques such as, Widefield, CLSM, 4PI, Structural illumination microscopy and others. In this case such a Bessel-like beam is generated by masking the back-aperture of the excitation objective with a ring-like spatial filter. The proposed excitation scheme allow continuous scanning by simply translating the detection optics. Additionally, only a pencil-like region of the specimen can be illuminated at a given instance thereby reducing premature photobleaching of neighboring regions. This illumination scheme coupled with orthogonal detection shows the ability of selective imaging from a desired plane deep inside the specimen. In such a configuration, the lateral resolution of the illumination arm determines the axial resolution of the overall imaging system. Such an imaging system is a boon for obtaining depth information from any desired specimen layer that includes nano-particle tracking in thick tissue. Experiments performed by imaging the Fluorescent polymer tagged-CaCO3 particles and yeast cell in a tissue-like gel-matrix demonstrates penetration depth that extends up to 650 m. This will advance the field of fluorescence imaging microscopy and imaging. Similar to the ability to observe deep inside a sample, simultaneous 3D monitoring of whole specimens play a vital role in understanding many developmental process in Biology. At present, light-sheet based microscopy is the prime candidate amongst the various microscopy techniques, that is capable of providing high signal-to-background-ratio as far as planar imaging is concerned. Since spatial filtering technique was found to successfully give rise to novel features (such as large penetration depth) in a fluorescence microscope setup, a logical extension would be to implement a similar approach with a light-sheet based microscope setup. These implementations are discussed in Chapter 4 of this thesis where spatial filtering is employed with cylindrical lenses. For facilitating computational and experimental studies, a vectorial formalism was derived to give an explicit computable integral solution of the electric field generated at the focal region of a cylindrical lens. This representation is based on vectorial diffraction theory and further enables the computation of the point spread function of a cylindrical lens. Commonly used assumptions are made in the derivation such as no back-scattering and negligible contribution from evanescent fields. Stationary phase approximation along with the Fresnel transmission coefficients are employed for evaluating the polarization dependent electric field components. Computational studies were carried out to determine the polarization effects and calculate the system resolution. Experimental comparison of light-sheet intensity pro les show good agreement with the theoretical calculations and hence validate the model. This formalism was derived as a first step since it gives the essential understanding of tightly focused E-fields of a high N.A. cylindrical lens systems and thereby helps in further understanding the effect of spatial filtering. As the next step, generation of extended light-sheet for fluorescence microscopy is pro-posed by introducing a specially designed double-window spatial filter at the back-aperture of a cylindrical lens. The filter allows the light to pass through the periphery and center of a cylindrical lens. When illuminated with a plane wave, the proposed filter results in an extended depth-of-focus along with side-lobes which are due to other interferences in the transverse focal plane. Computational studies show a maximum extension of light-sheet by 3:38 times for single photon excitation, and 3:68 times for multi-photon excitation as compared to state-of-art single plane illumination microscopy (SPIM) system and essentially implies a larger field of view. Finally, generation of multiple light-sheet pattern is proposed and demonstrated using a different spatial filter placed at the back aperture of a cylindrical lens. A complete imaging setup consisting of multiple light-sheets for illumination and an orthogonal detection arm, is implemented for volume imaging in fluorescence microscopy. This proposed scheme is a single shot technique that enables whole volume imaging by simultaneously exciting multiple specimen layers. Experimental results confirm the generation of multiple light-sheets of thickness 6:6 m with an inter-sheet spacing of 13:4 m. Imaging of 3 5 m sized fluorescently coated Yeast cells (encaged in Agarose gel-matrix) is per-formed and conclusively demonstrates the usefulness and potential of multiple light-sheet illumination microscopy (MLSIM) for volume imaging. As part of the future scope of the research work presented in this thesis, the Bessel-beam based improved depth microscopy technique may attract applications in particle tracking deep inside tissues and optical injection apart from fluorescence imaging applications. The vectorial formalism derived for cylindrical lens can be used to predict other, complex optical setups involving cylindrical lenses. Extended light-sheet generation proposed in this work by using appropriate spatial filtering with a cylindrical lens, complements the existing and popular selective plane illumination microscopy technique and may facilitate the study of large biological specimens (such as, full-grown Zebra sh and tissue) with high spatial resolution and reduced photobleaching. Finally, the MLSIM technique presented in this thesis may accelerate the field of developmental biology, cell biology, fluorescence imaging and 3D optical data storage.

Correct layering (lamination) of neurons in the central nervous system (CNS) is critical for the tissue functionality. Neuronal lamination is established during development, when the majority of neurons have to move from their birthplace to the appropriate layer, where they function. Therefore, to grasp the logic of CNS development, it is essential to understand the kinetics and modes of the variety of neuronal translocation events. Most of our knowledge about neuronal translocation has been gained using fixed tissue or ex vivo imaging, which is not ideal for such a dynamic process heavily dependent on the surrounding environment. To avoid these limitations, I combined translucent zebrafish embryos with light sheet fluorescence microscopy, which together enabled gentle in toto imaging of neuronal translocation. I studied the translocation of retinal ganglion cells (RGCs) across the developing zebrafish retina. RGCs are the first neurons that differentiate in the vertebrate retina and are born in a proliferative zone at the retinal apical side. From here, they move basally, spanning the complete apico-basal length of the tissue. They are destined to occupy the most basal layer, where their axons form the optic nerve. Although it was described that RGCs move their soma while being attached to both apical and basal sides of the retina, the kinetics and cell biological mechanisms of somal translocation remained unknown. Extracting single cell behavior of RGCs from high-resolution movies of their translocation allowed for quantitative analysis of RGC movement. I revealed that RGCs cross the retina in less than two hours in a directionally persistent manner. The movement of RGC soma is a cell autonomously generated process, which requires intact microtubules and actin-dependent basal attachment of cells for speed and efficiency. Unexpectedly, interference with somal translocation leads to a shift towards a multipolar migratory mode, previously not observed for RGCs, in which they temporarily lose both apical and basal attachment and apico-basal polarity. The multipolar mode is overall slower and less directionally persistent, but still allows RGCs to reach the basal retina. However, when RGC translocation is inhibited completely, they differentiate ectopically in the center of the retina, which in turn triggers the formation of ectopic layers of later born neurons. These results highlight the importance of establishing the basal layer of ganglion cells for ensuing retinal lamination. Overall, I generated important advances in the understanding of neuronal translocation and lamination, which might be relevant for other parts of the CNS.

Chronic kidney disease is a progressive kidney disease that affects approximately one tenth of the world population. In almost all cases of progressive, end-stage kidney disease, fibrosis is seen. Renal fibrosis is a condition of the kidneys in which collagens and other proteins accumulate in the extracellular matrix of the kidneys. The aim of the project was to explore the use of Fast Green to visualize collagen in renal tissue using light-sheet microscopy. A healthy sample and a diseased sample of renal tissue was imaged in aqueous and anhydrous conditions and a comparison of the difference in staining pattern between the conditions was performed. There was a clear difference in staining pattern between the two conditions, where the samples prepared in anhydrous conditions showed a higher staining specificity to collagen as described previously for other types of tissue. An evaluation of the differences in collagen expression between the healthy and the diseased sample was performed as well. There was a visible difference between them where a higher expression of collagen was observed in numerous tubuli, indicative of pathological scarring. / Kronisk njursjukdom är en progressiv njursjukdom som påverkar approximativt en tiondel av världens befolkning. I alla progressiva njursjukdomar ses så kallad njurfibros. Njurfibros är ett tillstånd då kollagen och andra proteiner ansamlas i njurarnas extracellulära matris. Målet med projektet var att utforska användningen av Fast Green för att visualisera kollagen i njurvävnad med ”Light sheet”-mikroskopi. Vävnadsproverfrån frisk vävnad och sjuk vävnad avbildades i vattenhaltiga och vattenfria förhållanden och en jämförelse av inmärkningen mellan förhållandena utfördes. Det var en tydlig skillnad i inmärkningen mellan de två förhållandena, där det i de vattenfria proven observerades att Fast Green märkte in kollagen med högre specificitet. En jämförelse av skillnaderna i kollagenuttryck mellan det friska och det sjuka provet utfördes. Det var tydliga skillnader mellan proven, där ett stort antalkollagen-påverkade tubuli kunde observeras i det sjuka provet.

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