Development of a holoscopic imaging system and applied high-resolution fluorescence microscopy
Biomedical imaging helps extending our comprehension of ourselves and our environment. Advances in camera, laser and computation technologies have enabled an ever-increasing number of imaging technologies. Imaging with visible and infrared light has the advantage that it is less harmful than other radiation and its wavelength is in the order of magnitude of cells and subcellular components. Fluorescence microscopy provides good chemical contrast and multi-colour imaging can help elucidate cellular architecture. Incoherent superresolution methods permit us to bypass Abbe's diffraction limit of lateral resolution and visualize previously unnoticed details. Coherent imaging methods such as optical coherence tomography or holoscopy do not require any previous labelling and have the advantage that they record both the amplitude and phase of the light emitted from a scattering sample by interferometric superposition with a reference wave. Both incoherent an coherent imaging methods are used in this thesis. The results of two interdisciplinary research collaborations using different fluorescence microscopy methods, including superresolution methods, are presented. Podosomes in macrophages were studied with stimulated emission depletion microscopy, structured illumination microscopy and localisation microscopy and a distinctly polygonal shape in their vinculin rings was found. Image processing routines allowed for a quantitative analysis of the acquired images . In the second study, chlorophyll, the most prominent natural pigments, and digested chlorophyll metabolites were detected in gut section of the herbivorous Spodoptera littoralis larva. Widefield and high-resolution autofluorescence microscopy revealed that the brush border membranes of their gut are covered with the chlorophyllide binding protein tightly bound to the gut membrane. A function in defense against gut microbes is discussed . Optical coherence tomography (OCT) offers a slightly lower spatial resolution than light microscopy but generally better penetration depths. In order to use a higher numerical aperture for detection in OCT, the dilemma of the resulting reduced depth of field has to be overcome. Different extended focus possibilities are explored in this thesis. Bessel illumination is an established method to achieve an extended depth of field without compromising the lateral resolution. When broadband or multicolour imaging is required, wavelength-dependent changes in the radial profile of the Bessel illumination can however complicate further image processing and analysis. A solution for engineering a multicolour Bessel beam was implemented with a phase-only spatial light modulator in the image plane and an iterative Fourier Transformation algorithm . For higher acquisition speed, full-field recording is favourable to scanning the scattering sample with a Bessel beam. OCT can be combined with reconstruction methods from digital holography to achieve an extended focus numerically. A suitable experimental imaging setup and a custom-written reconstruction algorithm are presented.  M. Walde, J. Monypenny, R. Heintzmann, G. E. Jones, and S. Cox, “Vinculin binding angle in podosomes revealed by high resolution microscopy”, PLoS ONE, vol. 9, no. 2, 2014.  A. Badgaa, R. Büchler, N. Wielsch, M. Walde, R. Heintzmann, Y. Pauchet, A. Svatos, K. Ploss, and W. Boland, “The Green Gut: Chlorophyll Degradation in the Gut of Spodoptera littoralis”, Journal of Chemical Ecology, vol. 41, no. 11, pp. 965-974, 2015.  M. Walde, A. Jost, K. Wicker, and R. Heintzmann, “Engineering an achromatic Bessel beam using a phase-only spatial light modulator and an iterative Fourier transformation algorithm”, Optics Communications, vol. 383, pp. 64-68, 2017.