Overcoming the Challenges in Analysing Vesicle Trafficking

Challenge Background

Extracellular or intracellular vesicles perform vital functions in inter/intracellular communication. Not surprisingly, the de-regulation of the endocytic pathway affects numerous diseases, such as cancer, diabetes, cardiovascular diseases, among others. Moreover, the endocytic pathway can also be used as a valuable tool for therapeutic molecule delivery. Consequently, the deep understanding of vesicle trafficking, and the endocytic pathway is of extreme importance to promote a deeper understanding of the disease, and the development of new therapies.

Nevertheless, imaging vesicle trafficking effectively is a hard task, and researchers need to overcome several challenges to be able to do so. Key challenges to vesicle imaging are avoiding phototoxicity and photobleaching while fulfilling the requirement for high acquisition speed and high image resolution. These challenges do not end with image acquisition; post-acquisition vesicle tracking is also a demanding task.

Technology Solution

As discussed in earlier solution notes a camera-based confocal system is a better solution for live imaging of vesicles because it does not compromise cell viability due to phototoxicity, read more here. The equipment will need to acquire at high speeds and with high imaging resolution. Read our full solution note here. A post-acquisition software capable of analysing multiple vesicles simultaneously, calculating speeds, accelerations and trajectories is an essential requirement to extract the essential information from the acquired data. Automatic detection and quantification would be of key importance due to the large number of particles to be analysed. The software should also allow the generation of movies with different rendering (visualisation) options, including representation of detected objects and tracks.

Crucial requirements for analysing vesicle trafficking include:

• Automatic detection and quantification - Vesicles fuse with different target organelles and at different speeds. Hundreds or thousands of vesicles circulate in the cytoplasm with overlapping pathways. Simultaneous visualization of raw data and detected objects which can be color coded based on motion parameters such as speed, acceleration or localisation is a vital feature for analysis.
• Tracking vesicle movements – Cells are 3D structures, and vesicles move in the 3D intracellular space. To correctly analyse and track vesicles inside the cell, researchers need a software that can analyse particles in 3D space and time. The program should be able to automatically detect and track the movements of all particles. Researchers might need to correct for some overlapping tracks, so it should also have the flexibility to manually correct (or highlight) specific tracks.
• Generating animations – During analysis, the software should be able to generate key frame animations that can combine different visualisation and rendering modes. Overlaying volume rendering with detected objects and tracks and exporting it in an MP4 format is a crucial feature to create an insightful supplement to a presentation or a paper.

Andor’s Solution for Vesicle Analysis

As discussed in earlier solution notes, Dragonfly and the Sona sCMOS camera provide solutions to image intercellular trafficking. Andor´s combined solution for acquisition and post-acquisition image processing allows researchers to combine prime imaging conditions with the outstanding analysis and rendering tools available in Imaris. Moreover, the dData acquisition and analysis workflow is complete using Dragonfly Fusion & Imaris 3D/4D Image Analysis Software. The Fusion acquisition software saves the images in the highly efficient IMS file format and Imaris for Cell Biologists is the ideal package to visualize the data, track vesicle pathways and compute motion parameters. Imaris provides tools to automatically analyse moving object and generate quantitative information from the image data.