Invadopodia structure in 3d environment resolved by near‐infrared branding protocol combining correlative confocal and fib‐sem microscopy

Abstract

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.