Selective Plane Illumination Microscopy Using Non-spreading Airy Beams

Sammanfattning: Recently a new field of microscopy has emerged, known as selective plane illumination microscopy (SPIM). Using a SPIM setup the sample is illuminated with a thin light sheet from the side. The selective plane illumination overcomes many problems that conventional microscopes have. Conventional microscopes illuminate the sample axially and collects the light only from the focal plane of the microscope objective. Therefore parts of the sample which are out of focus are illuminated unnecessarily, leading to rapid fluorophore photobleaching and decrease of signal to noise ratio due to out of focus light. The $SPIM$ method overcomes these problems, because only the fluorophores in the focal plane of the objective are excited. Another problem with conventional microscopes is that they are capable of only two-dimensional imaging. The exception are confocal microscopes, but these microscopes are slow and the field of view is restricted to few hundred micrometers. The SPIM method significantly improves the three-dimensional imaging speed and can reach up to few hundred frames per second. This imaging speed enhancement is achieved, because the whole plane is captured with a single snapshot, instead of point by point scanning as in confocal microscopes. To take all the advantages of the SPIM method it is crucial to form a thin light sheet that extends for the whole field of view. Commercially available lasers have Gaussian beam. It is well known that tightly focused Gaussian beams spread out quickly, therefore there will be good axial resolution only in the central part of the image. Because of that the field of view will be restricted to the Rayleigh range of the Gaussian beam. In this work we present how to create non-spreading Airy beams using only two additional lens elements and how they can be implemented in the SPIM setup to significantly extend the field of view keeping high axial resolution. This is an important advancement since Airy beams would allow larger sized objects, e.g. optically cleared tissue specimens, to be imaged with high resolution. This thesis shows a novel design of a light sheet microscope that opens up new possibilities for biological and medical research.