Pilger, Christian: Development of novel Optics and Analysis Tools for enhancing Biomedical Imaging by Coherent Raman Scattering. 2019
Inhalt
- List of Figures
- Introduction
- Theoretical background
- Introduction to Raman scattering
- Coherent Raman scattering
- Concept of structured illumination for resolution enhancement
- Materials and Methods
- Experimental setup
- Solid-state laser system
- Optical parametric oscillator
- Spatio-temporal overlap with laser diagnostics
- Acousto-optic modulator
- Setup for a customized CRS-microscope
- SRS modulation and detection scheme
- Structured illumination for resolution enhancement
- Results
- Investigation of osteogenic differentiation in human stem cells
- Exploring label-free CARS microscopy as suitable tool for visualizing the successful differentiation of ITSCs in clinical research
- Design of the experiments: CARS and SHG imaging of ITSCs and control group
- CARS and SHG data are compared to spontaneous Raman spectra and Alizarin Red staining
- CARS and SHG imaging leads to successful visualization of the ITSCs differentiation also applicable to clinical research
- In vivo analysis of intracellular lipid droplets in Monoraphidium neglectum
- Identification and characterization of lipid rich microalgae as non-food feedstock in bio-diesel production
- Established methods for analyzing the lipid content of microalgae are laborious and partly not feasible due to rigid cell walls
- Raman-based techniques speed up the analysis process of M. neglectum
- Design of experiments: modulation approach excludes two-photon excited fluorescence enabling CARS imaging of lipid droplets
- Comparing the results concerning the lipid content acquired by volumetric CARS imaging to established methods
- Studying the degree of unsaturation of fatty acids in lipid droplets of M. neglectum by Raman micro-spectroscopy
- Successful CARS imaging of lipid contents in M. neglectum under nitrogen starvation conditions indicates optimal harvesting time point enabling comparison to other types of microalgae
- Developing a hyperspectral CARS imaging modality
- Exploring hyperspectral CARS for enhancing the information content of samples by their spectral response
- Improving the stability of the microscopy setup enables high fidelity HS-CARS data sets
- Customized software controls entire HS-CARS scan process ensuring undistorted spectra
- Pulse length variation induces spectral distortions in hyperspectral CRS-microscopy
- Identifying the OPO parameters influencing the pulse length during wavelengths scanning
- Influence of pulse length induced distortions on HS-CARS spectra shown in methanol
- Investigating the effect of non-controlled OPO wavelengths tuning on HS-CARS spectra in U2OS cell sample
- Applying HS-CARS imaging to biological research questions
- Analyzing the influence of copper on the fitness parameters of Caenorhabditis elegans
- Volumetric CARS imaging reveals changes of the lipid storage in C. elegans
- Hyperspectral CARS scanning assessing false positive identification of lipid contents
- Visualizing the differentiation of primary human alveolar epithelial cells by label-free imaging techniques
- Volumetric CARS imaging enables z-sectioning and shows differentiation process over time
- Non-negative matrix factorization applied on HS-CARS scans reveals cellular compartments
- Resolution enhancement utilizing structured illumination in scanning-based non-linear microscopy
- Generating striped illumination pattern by addressing the galvanometric scanning mirrors
- Enhancing the resolution and contrast by striped illumination of fluorescent bead samples in two-photon excitation
- Exploiting autofluorescence signals of Convallaria majalis for combining z-scanning with striped illumination pattern via MAP-SIM
- Investigating the potential of a striped two-photon illumination in tissue utilizing a stained murine kidney section
- SI-CARS: investigating the potential of resolution enhancement by striped illumination patterns
- Structured illumination utilizing galvanometric scanning mirrors proves to be a versatile tool for two-photon fluorescence and CARS microscopy
- Developing a self-synchronized two-color fiber laser for fast, high contrast CRS-microscopy
- Utilizing highly doped ytterbium and erbium non-linear fibers for the low noise, high power two-color laser in CRS-microscopy
- Characterizing the temporal and spectral properties of the all fiber-based CRS-laser source
- All fiber-based two-color laser system enables simultaneous SRS and CARS imaging on living human cells
- Investigating the performance of the all fiber-based laser on tissue samples utilizing simultaneous SRS and CARS contrast
- Fs-laser pulses of Stokes cavity are utilized for SHG and two-photon excited fluorescence imaging broadening the range of application
- Extending the range of accessible wavenumbers with a third laser cavity enabling the visualization of alkyne tagged samples in the future
- Summary and outlook
- List of publications
- Statutory declaration
- Acknowledgments
- References