Cancer cell invasion through tissue barriers is the intrinsic feature of metastasis, the most life-threatening aspect of cancer. Detailed observation and analysis of cancer cell behaviour in a 3D environment is essential for a full understanding of the mechanisms of cancer cell invasion. The inherent limits of optical microscopy resolution do not allow to for in-depth observation of intracellular structures, such as invadopodia of invading cancer cells. The required resolution can be achieved using electron microscopy techniques such as FIB-SEM. However, visualising cells in a 3D matrix using FIB-SEM is challenging due to difficulties with localisation of a specific cell deep within the resin block. We have developed a new protocol based on the near-infrared branding (NIRB) procedure that extends the pattern from the surface grid deep inside the resin. This 3D burned pattern allows for precise trimming followed by targeted 3D FIB-SEM. Here we present detailed 3D CLEM results combining confocal and FIB-SEM imaging of cancer cell invadopodia that extend deep into the collagen meshwork.

Biotechnology and Biomedicine Centre of the Academy of Sciences and Charles University Průmyslová 595 25242 Vestec u Prahy Czech Republic

Department of Cell Biology Charles University Viničná 7 12800 Prague Czech Republic

Department of Physical Chemistry Charles University Hlavova 8 12800 Prague Czech Republic

Imaging Methods Core Facility at BIOCEV Faculty of Science Charles University Průmyslová 595 25242 Vestec u Prahy Czech Republic

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