A eukaryotic cell resembles a big city with several districts specialized in particular roles. In order to work properly, the cell is regulated by a complex infrastructure of “highways” which carry out the communication among the different cell districts. Materials are continuously transported via membrane‐bound, tiny packets called vesicles throughout all of our cells. These vesicles are attached to special molecular motors that haul them along components of the cytoskeleton, such as microtubules and actin filaments.
This process is called intracellular trafficking and it is crucial for the normal functioning of cells. Any defect in regulation may cause a vast variety of diseases including neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases.
In this blog post, we would like to show you how Real-Time 4D microscopy has enormous potential to increase our understanding of this biological process. Nanolive’s 3D Cell Explorer permits accurate and quantitative 4D spatio-temporal monitoring of nano-sized vesicular structures moving along the cytoskeleton network in living cells. It allows discrimination of vesicles (violet) and other cellular structures (cellular membrane in green) based on their specific refractive index, allowing for marker-free studies of this fascinating phenomenon.
In this case, the distribution of intracellular vesicles at the cell edge was monitored over time to evaluate their accumulation in this region of interest (ROI).
Mouse skin melanoma cancer cells (B16, p35) were grown to 40% confluency in complete DMEM medium (Dulbecco’s Modified Eagle Medium) in 35mm glass bottom culture dishes (FluoroDishes™ WPI, #FD35-100). The time-lapse imaging experiment was conducted with a standard top-stage incubator set to 37°C and 5% CO2 for 8 hours, capturing images every minute.
Cell’s ROI. Intracellular vesicle quantification over time. The number of vesicles was counted at different time points: t0; t1h; t2h; t3h.