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Structured illumination in optical Tomography

Final Activity Report Summary - STRIPES (Structured illumination in optical tomography)

Development of novel types of microscopes to meet the requirements of biologists and physicians is essential, because there is a high demand of increasingly precise structural information about objects as small as single proteins up to in-vivo imaging of whole animals. The focus of the research performed in this project lies on the improvement of different types of microscopes working with visible light. To this end, a novel imaging technique called 'Optical projection tomography' (OPT) has been advanced and successfully applied to imaging of live and fixed animals, including model organisms of biology such as Caenorhabditis elegans (roundworm), which are used because it is expected that discoveries made will also provide insight into the functioning of other organisms. Furthermore, the microscope has been enhanced to also allow imaging of larger samples, such as organs, not of humans, but of another model organism, of mice.

Many of the project tasks aim at improving the optical resolution achievable with the respective microscopes in order to warrant smaller details to become visible to the scientist and clinician. For this, a particular illumination called structured illumination is used. When specific target regions of animals are analysed, these regions are usually labelled with fluorophores, which allow high contrast imaging by suppressing details from other, non-labelled parts of the animals. Using structured illumination, only a portion of the fluorophores of the target region is visible at a time, making sub-structure information of the target region visible, even if the microscope cannot detect and resolve these substructures otherwise.

Great effort has been put into the development of algorithms for the extraction of this additional information. The problem always lies in visualising and measuring the additional features (missing completely in images without structured illumination) which cannot be seen by simply looking at the recorded images. In particular, the project was focused on the development of 3D reconstruction algorithms for optical sections tomography and OPT when using structured illumination. For both types of microscopes, the corresponding data analysis software was written, yielding in both cases a significant resolution improvement. The concept of structured illumination microscopy has first been developed due to the need of highest resolution optical imaging on the cellular level.

Throughout the project, collaborations with the secondary host, the King's College in London, and with the University of Heidelberg, have continued and successful applications of the respective microscopes to study sub-cellular and sub-nuclear complexes have been performed and resulted in several publications. Thus not only a major training aspect formulated in the original proposal was completed, but also a strong and vital basis for the development of the 3D reconstruction software for optical sections tomography and for OPT was provided.

Another problem related to the achievable optical resolution involves the mechanical stability of the microscope setup, especially when trying to increase the amount of object details visible. Already small vibrations, drifts, and movements of the hardware components and of the specimens themselves, particularly when they are alive, can reduce the image quality drastically. These problems were addressed and resolved in different ways: By stabilising and redesigning the hardware components, and by developing algorithms which deal with and correct for such movements when it cannot be suppressed otherwise, the OPT device was developed into a ready-to-use microscope for a whole range of biological applications.