• confocal line scanner
• fluorescence microscopy
• imaging
• sofi
• super-resolution microscopy
• super-resolution optical fluctuation imaging

This thesis was born as a collaboration between the center of research “E. Piaggio” from University of Pisa and the Lab of Nanobiology from Katholieke Universiteit Leuven. We studied the last phase of a construction of a new fluorescence microscope. This represents a line scanner microscope that can reach a resolution equals to a normal confocal one but can acquire images faster. The objectives of this thesis are:
- Acquisition of the first SOFI images through this new microscope after a reconstruction phase. This phase was necessary to validate the construction process. Last step of construction is the alignment phase which is characterized by a refined positioning of the components. This phase can be done by analyzing the images acquired during the positioning through a new Graphic User Interface (GUI) that can extract the system Point Spread Function (PSF) starting from beads samples. The GUI shows a surface reconstruction of the PSF allowing users to understand how to vary the position of the components to reach a perfect image. To capture images suitable to the super resolution process, the DNA painting technique was used. The dye highlights the microtubules inside the cells.
- Analysis of the images acquired with the microscope to describe useful parameters that can be used in future. In addition to the PSF analysis a script has been developed to evaluate values like resolution and signal to noise ratio. The program can also approximate the distribution of noise and signal in order to facilitate future analysis with the new microscope.