Image improvement by structured illumination in ultramicroscopy

Abstract

Ultramicroscopy is a technique, which allows three-dimensional imaging of macroscopic samples with micrometer resolution. In this kind of microscopy, the specimen is illuminated with a light sheet in the focal plane of the detection optics. Fluorescence is excited only within a thin 'optical' section of the specimen, which allows 3D reconstruction. However, imaging of large specimens is limited by light scattering and fluorescent background. Residual scattering in cleared specimen causes continuous blurring of the light sheet when propagating through the object. Additionally, emitted light is diffracted by proteins and small particles, which is detected as fluorescent background. Suppression of these sources of background could significantly improve the imaging performance. This diploma thesis describes a method, that improves the image quality by using structured illumination.<br />Structured illumination microscopy was developed for the widefield microscope and allows optical sectioning and contrast improvement. In this technique, the illumination path is modified to project a one-dimensional grid into the object plane. The setup, developed for this diploma thesis, is a combination of an ultramicroscope and a structured illumination microscope. Instead of exciting the sample directly, the light sheet is spatially filtered with a Nipkow disc and then projected into the sample. The modulation of the illumination intensity, followed by image processing, allows contrast enhancement.<br />For comparison of conventional ultramicroscopy and structured illumination ultramicroscopy, images from biological specimens were analyzed. In the present work, a mouse hippocampus, a mouse liver and an entire mouse brain were examined. All measurements showed significantly enhanced contrast, even hidden features were revealed from the background.