I want to study protein localization in single cell level by Time-Lapse microscope. Please kindly suggest me the proper methodology or protocol to study the live cell. Thanks in advance for your valuable opinion.
The paper, which you mentioned is not free to access from my institute. Please, if it is possible send me the paper. Thank you.
Hi Aniruddha,
Its very simple and straight forward. Fuse your protein to a fluorescent marker ( this can vary based on your specific experiment), express your protein using transfection, transduction methods. visualize your protein expression in simple fluorescent microscope to confirm, plate cells in a Wilco or similar microscopy grade dishes to semi confluency, the next day go under a epi-fluorescent microscope that has a 100x water immersion objective ( has the advantage over oil immersion by overcoming abberation problems). Then based on the type of microscope and filter sets and magnification glasses choose the best combinations to have high zoom. Depending on your protein dynamics set the time frame in which you might want to capture images ( the more exposure of light will result in bleaching of the fluorophore, so be cautious) additionally you might want to acquire time-lapse images over 3D ( namely X,Y and Z) set your Z steps between 0.5 and 1um distances over 40-20 steps respectively ( or from the top to bottom of the cell) these specifications can be modified based on your sample. Take time-lapse images over your desired time frame and then you will have to deconvolve your images ( huygens essential is a good software to achieve this, but there many free wares and other softwares). Make sure you enter your de-convolution parameters correctly as this is important for a proper correction of images ( test your signal to noise ratio (S/N) experimentally using beads ).
At this step u might have one or more cells in your time-lapse images. Crop your image to one cell, re-construct in 4D (X,Y,Z over t) and then you can monitor your protein localization at different time points or over time during treatment.
There are a lot of papers in pubmed describing these techniques. you can refer to them for specifics, but what I have written here is the basics of time lapse microscopy.
All the best
If you have access to them, you may want to use microscopic systems that provide optical sectioning like confocal or spinning disk systems. This will allow you to isolate optically a specific plane within your cell and these are best for 3D colocalization. Spinning disk would go quite faster than confocal by the way.
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