Volume imaging of cancer with specific labelling

The main aim of the project is to obtain 3D reconstructions of tumor pieces resected during cancer operations with cellular resolution to improve pathological diagnostics. To make the images quickly useful for clinical diagnostics the cytoplasm and the nuclei should appear like in standard Haematoxilin/Eosin (HE) stainings which pathologists are used too. A problem in standard tumor diagnostics is that generally only one 4 m thick histological section is prepared per half cm slab of tumor tissue leading to an undersampling of 1:1000. Especially in situations where tumor free resection margins have to be confirmed this can lead to false negative results. Our optical sectioning provides a thousand times finer sampling rate than the standard histological procedure. We are thus convinced that our approach will lead to more secure cancer diagnostics. In addition the volume data generated by our 3D imaging should provide new insights into tumor biology. The 3D aspect of tumor development at the single cell level in tumor tissue has been nearly unaccessible by current techniques. Now it can be investigated in great detail. Besides the visualisation of cell nuclei and cytoplasm by HE like staining we would also like to visualize the blood vessel system inside the tumor. This could be of high diagnostic value as the development of new blood vessels is an important aspect of tumor development. This would be especially interesting for therapy directed against the development of new blood vessels . Although we can make cm large tumor pieces completely transparent by our clearing up to now we are mainly recording volumes with 1 mm lateral extent. To record larger parts of the tumor we want to stitch recordings together, use also objectives with a larger field of view a nd automatize the recording process. Besides staining tumor constituents with small molecule dyes we intend to establish also immunohistochemical staining (staning with antibodies). Immunohistochemistry is used in histological diagnostics in many situations where HE based diagnostics has to be confirmed. As antibodies are much larger than dye molecules, special protocols have to be worked out to allow immunostaining in a useful time frame. As for both HE and immunostaining time is an important factor in clinical diagnostics, additional techniques like the application of microwave energy will be investigated. With the guidance of our collaboration partners in pathology we will apply our technology to tumors for which 3D reconstructions are clinically especially relevant.