May 30, 2016 at 7:33 am #3332
- New User
I’m starting to set up a nanoparticle internalization protocol using a lysosome marker. Which parameters must I have into account in order to see my nanoparticles inside my lysosomes? Is there anything in the literature related with this colocalization procedure? Which is the best way to analyze this datacubes in order to find out the lysosome spectral library and the nanoparticle (inside the lysosome) spectral library?
Thanks in advance!
Paula ZamoraJune 1, 2016 at 8:44 am #3338
If you do not have the fluorescence module (DMF) to correlate nanoparticle spectral mapping to fluorescently labeled subcellular location, then you can use the Peak Location Classifier (PLC) tool in ENVI. This feature is under ‘CytoViva Analysis’ in the ENVI menu bar and will allow you to “map” pixels based on their peak wavelength. You will indicate your peak wavelength in nm and tolerance based on the fluorescent emission of your lysosome tracker. The noise floor may need to be raised if you notice that you are picking up noisy pixels.
You will save this file which is a Classification file (similar to a SAM file) under Output Result to File. Hit the ‘double arrows’ (No Output Rule Image) and hit “OK”. Overlay this PLC Classification file the same way you would a SAM Classification. As long as the fluorescent tracker is detected with HSI, this is a quick way to locate your lysosomes. Then, you can build a spectral library of lysosomes based on the PLC results. Also create a nanoparticle spectral library and filter both against the negative control and against each other (careful with naming conventions here). Finally, you can map both libraries onto the datacube (see related thread on mapping 2+ spectral libraries).
For related published papers, you can type “Cytoviva Lysotracker” or similar key words into Google Scholar.
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