I plan to visualize trans-Golgi movement dynamics by confocal analysis of a TGN localized protein tagged with GFP. I am looking for a recent plugin (with reference or paper) that can do particle tracking of the individual GFP tagged protein across multiple time point confocal images. A preliminary search yielded some instances like virus movement but nothing with organelle motility, or maybe I am missing keyword in searches completely.
Any help/pointers would be much appreciated.
One of my colleagues at the University of Virginia School of Medicine, Dr. John Gildea is an expert in confocal microscopy. I would contact him and ask his opinion. [email protected]. Good luck!
Hi Rahul,
Could you please make your question more exact? You are interested in particle movement, but tags mention organelle shape and protein tracking. These three tasks require different imaging protocols.
1. Accuracy of tracking: what is target centroid displacement and is particle shape the thing you are interested in?
2. What is the cell to be imaged/analyzed? 2D or 3D? If not a plant cell imaged, WF scopes are a better choice
3. Semi-manual or fully automatic tracking? There are several plugins for user-assisted tracking, i.e. Manual Tracking plugin, but fully automatic plugins' performance is usually dubious. esp. if they are not adjusted for the exact experiment
When speaking about cell/organelle tracking, there is a trick: take a image with DIC/Phase contrast and than switch to fluorescence and take a short-exposure (sometimes with binning) FL image. The one to be analyzed is a overlayed image of BF and preprocessed fluor image. This helps to avoid bleaching during timelapse.
http://rsb.info.nih.gov/ij/plugins/track/track.html
I see some good suggestions added already. It may also help to characterize the living and dead cells with even still images. A sound approach in doing this is a good start and can extended to cell movements. The color characteristics (even in regular lighting) might be different for the living and dead cells. Images in stack simulates the movement. Good luck.
We used a technique called kymograph, where you mark the path of the organelles and then lay themeasured lines or areas of each timepoint on top of each other. In this way you get a nice visualization of the movment, directionality and even movment speed in a way that can also be printed in an understandable way. It also works in multiple colours. You can find an example in Bittins CM, et al. 2010, Cell Mol Neurobiol
Good luck! Cheers Tilo
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