Imaging and quantification of the tumor microenvironment of triple negative breast cancer using TPEF and scanning laser optical tomography.

Triple-negative breast cancer is an aggressive subtype of breast cancer that has a poor five-year survival rate. The tumor's extracellular matrix is a major compartment of its microenvironment and influences the proliferation, migration and the formation of metastases. The study of such dependencies requires methods to analyze the tumor matrix in its native form. In this work, the limits of SHG-microscopy, namely limited penetration depth, sample size and specificity, are addressed by correlative three-dimensional imaging. We present the combination of scanning laser optical tomography (SLOT) and multiphoton microscopy, to depict the matrix collagen on different scales. Both methods can be used complementarily to generate full-volume views and allow for in-depth analysis. Additionally, we explore the use of SHG as a contrast mechanism for complex samples in SLOT. It was possible to depict the overall collagen structure and specific fibers using marker free imaging on different scales. An appropriate sample preparation enables the fixation of the structures while simultaneously conserving the fluorescence of antibody staining. We find that SHG is a suitable contrast mechanism to depict matrix collagen even in complex samples and using SLOT. The insights presented here shall further facilitate the study of the tumor extracellular matrix by correlative 3d imaging.

Scanning laser optical tomography resolves developmental neurotoxic effects on pioneer neurons.

Developmental neurotoxic compounds impair the developing human nervous system at lower doses than those affecting adults. Standardized test methods for assessing developmental neurotoxicity (DNT) require the use of high numbers of laboratory animals. Here, we use a novel assay that is based on the development of an intact insect embryo in serum-free culture. Neural pathways in the leg of embryonic locusts are established by a pair of afferent pioneer neurons, extending axons along a well-defined pathway to the central nervous system. After exposure to test chemicals, we analyze pioneer neuron shape with conventional fluorescence microscopy and compare it to 3D images, obtained by scanning laser optical tomography (SLOT) and processed by a segmentation algorithm. The segmented SLOT images resolve the 3D structure of the pioneers, recognize pathfinding defects and are thus advantageous for detecting DNT-positive compounds. The defects in axon elongation and pathfinding of pioneer axons caused by two DNT-positive reference compounds (methylmercury chloride; sodium(meta)arsenite) are compared to the biochemically measured general viability of the embryo. Using conventional fluorescence microscopy to establish concentration-response curves of axon elongation, we show that this assay identifies methylmercury chloride and the pro-apoptotic compound staurosporine as developmental neurotoxicants.