How to Overcome the Challenges in Analysing Vesicle Trafficking
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.
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.
||Vesicle tracking & analysis solution: Dragonfly, Andor´s high QE cameras & Imaris
|Automatic detection and quantification of vesicles
||Imaris 3D image analysis software allows for reliable detection of vesicle structures and automated quantification of their parameters, such as number, position or distance to other organelles. Result 1 - Detect your vesicles in 3D using reliable algorithms in Imaris Spots model. Result 2 - Calculate total number of vesicles within the image or number of vesicles per cell. Result 3 - Get the position of each vesicle and calculate the distance between vesicles and other structures.
|Tracking vesicle movements in 3D
||Imaris provides fast and reliable tracking methods ideal to track vesicles pathways and analyse motion parameters. Result 1 - Automatically track moving objects and generate quantitative information from the image data. Result 2 - Handle thousands of objects and time points; represent their trajectory or displacement using multiple visualisation options. Result 3 - Calculate average or instantaneous speed and acceleration. Result 4 - Manually edit selected tracks.
|Customisation of analysis tools
||Imaris XT module is a gateway to customization. Developed for researchers with sophisticated and unique image analysis needs. Result 1 - Access to the pre-existing library of XTensions – custom image analysis scripts which can be modified for specific applications. Result 2 - Ability to integrate your own algorithms (via Matlab, Python or Fiji).
|Rendering and animation export
||Imaris provides a complete set of features for visualization of multi-channel microscopy datasets from static 2D images to 3D time series regardless of their size and format. Result 1 - Use premier 3D/4D volume rendering modes: (MIP, Blend, Shadow Projection, Normal Shading). Result 2 - Combine volume rendering, object representation, clipping planes and 2D slicers. Result 3 - Make high quality snapshots ready for presentations and publications. Result 4 - Easily make key-frame animations which include volume rendering, detected objects, annotations and object motion.