Methods and Protocols
Imaging and quantitative analysis of sprouting angiogenesis
Sprouting angiogenesis is the formation of new blood vessels from existing ones and depends on endothelial tip cells. In this paper we present methods for time-lapse imaging of sprouting angiogenesis and tip cell formation. We also include a fast, robust and reproducible protocol for imaging of angiogenesis by confocal microscopy (ideal for screening purposes), and a script for high-throughput quantitative analysis (developed by bas Ponsioen).
Image: Processing steps for the analysis of sprouting on confocal image stacks.
Protocols for live imaging of cell-matrix adhesions
Cell-matrix adhesions are dynamic structures that are continuously assembling and disassembling, especially in migrating cells. Here we describe methods and protocols for live-cell imaging of cell-matrix adhesions by widefield, confocal, total internal reflection fluorescence microscopy (TIRF), and fluorescence recovery after photobleaching (FRAP).
Movie: Live-cell TIRF imaging of focal adhesion dynamics.
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Quantitative analysis of focal adhesions
Focal adhesions can differ widely in size, number, and appearance. Here we present a script for the quantitative analysis of focal adhesions imaged by confocal or total internal reflection fluorescence (TIRF) microscopy (developed by Andrew Clark).
Image: Segmentation and identification of focal adhesions in cultured cells.
Multi-channel xyzt imaging in 2D/3D with minimal phototoxicity
Deep multichannel fluorescent imaging of rapid and dynamic cell behaviour over time requires recordings of image stacks at short intervals, which can cause photobleaching and phototoxicity. In this work, we present methods for multicolor live-cell labeling, followed by multi-position time-lapse imaging in 2D and in 3D protein matrices. We also present a protocol for long-term imaging at low laser intensity, followed by post-imaging signal enhancement using artificial intelligence (AI).
Image: Live-cell labeling of nuclei and actin filaments and images from a time-lapse sequence (projections of confocal image stacks).
Quantitative analysis of endothelial cell dynamics and extracellular matrix remodeling by label-free live-cell imaging
Label-free imaging prevents photobleaching and phototoxicity during long-term time-lapse experiments but comes at the cost of lower resolution. We are currently developing a pipeline for processing and quantitative analysis of prolonged (days) label-free live-cell image series. In addition, we develop tools to analyze the deformation of extracellular matrix during cell migration.
Movie: Time lapse phase-contrast microscopy of endothelial cells migrating from a bead into matrix. The individual branches and junction points are detected and tracked. The ECM deformation is detected, and the direction is visualized with color hue as shown in the top right.
Cell migration in three dimensions
Cell migration plays an essential role in many (patho-) physiological processes and is therefore widely studied in different fields from basic cell biology to regenerative medicine.
In this book, we compile current state-of-the-art methods and protocols to investigate cell migration in model systems ranging from cells, organoids, tissue explants and microfluidic systems to Drosophila, zebrafish, and mice. Furthermore, methods are included for advanced image analysis and quantitative assessment of diverse parameters related to cell migration.
The 24 chapters, written by experts in the field, are organized into 4 major themes; 1) the cell biology of cell migration, 2) developmental model systems to assess cell migration during morphogenesis, 3) cell migration in cancers and the tumor micro-environment, and 4) blood vessel formation and interactions.