Dr. Katarina Wolf, RIMLS, Dept. of Cell Biology, Radboudumc Nijmegen
Nonlinear optical microscopy by multiphoton excitation has developed into a powerful approach that combines different excitation and emission techniques for the three-dimensional (3D) reconstruction of biological specimens over time (4D microscopy). The speciﬁc approaches include detection of two- and three-photon excited ﬂuorescence, two-photon excited ﬂuorescence lifetime (FLIM), and the second and third harmonic generation for the visualization of native tissue structures. Cancer progression imaging is performed in different models such as collagen lattices in vitro, mouse imaging window models in vivo and human skin models ex vivo. Using dynamic imaging examples of cancer invasion for mapping of single-cell and collective invasion mode, and the underlying guidance principles by tissue structures together with therapeutic interventions, the power of multiphoton microscopy will be demonstrated.
Benjamin B. Bouchet
The ability of cells to migrate through a 3D matrix is associated with cancer invasion and metastasis. While it is recognized that the actin cytoskeleton plays a key role in interstitial migration, the role of microtubules in this process is still poorly understood. Most of the 3D cell culture setups consist in embedding cells in thick collagen I-based gels. In this type of cell culture system, the vast majority of optics available for confocal fluorescence imaging cannot be used for high-resolution observation and recording of cytoskeleton dynamics. By combining spinning disk-based confocal illumination with a new generation of high numerical aperture long working distance objective, we were able to perform high-resolution fluorescent imaging of microtubules in live cells that were fully embedded in a 3D matrix. The observations made with this new imaging setup revealed aspects of microtubule dynamics in physiologically relevant cell migration that are absent in conventional 2D cell culture systems.