Nile Red


#1

Hello –
I’m new to Cellprofiler and I’m trying to use it measure the fluorescence intensity of cells stained with Nile Red. I’m using the Nile Red assay to quantify the lipid accumulation in the cells. The lipids fluoresce in yellow and the rest of the cell in red. Is there a way to quantify the intensity in the whole cell and then differentiate between yellow and red fluorescence? If I’m only interested in the lipids, do I have to use a module to identify the lipid bodies first and then quantify the fluorescence in these objects? I was trying to avoid this because the lipids bodies don’t have a defined size or shape. Thanks for the help!


#2

Can you post your images? If they are color, we’ve had success (with H&E staining in tissue for example) splitting colors up to measure different fluorescence.


#3

Here is an image of some cells stained with bodipy. The lipid bodies fluoresce in green and the rest of the cell in red. I wanted to post a couple more images with Nile Red so that you can see that some times the lipid bodies are no so well defined but I get a message that the board attachment quota has been reached. The images files are not very big but still it doesn’t let me post them. Thanks for the help!
Bodipy 40X objective-1.zip (12.2 KB)


#4

Hi,

Some of our other users have had success posting in Picasa if they have an account already, and then posting the link to it here.

In any case, I have a question: Do the lipid bodies need to be individually identified within the cell for your purposes, or do you just need a total intensity measure of all the lipids within a cell?

Regards,
-Mark


#5

Hi, thanks for the tip. I posted the images in picasa:
picasaweb.google.com/ferdyluly/Images#
Regarding your question, I need the total intensity of all the lipids within the cell, I’m not interested in identifying/quantifying individual lipid bodies.
Thanks!


#6

Hi,

I’m attaching a tentative pipeline. Basically, it splits the color image into the 3 RGB channels. The red channel image is used for identifying the cells, and then a measurement module measures the intensity parameters in the green channel. One caveat: the parameters in the IdentifyPrimAuotmatic module may need adjusting depending on what magnification you choose. Is this what you are looking for?

Regards,
-Mark
2009_06_09_PIPE.mat (1006 Bytes)


#7

Thanks!! This is exactly what I needed. One last question. You can do the analysis this way because the lipids are green and the cells are red, right? If the lipids were yellow (like they are when you use Nile Red) could you still separate the yellow from the red? Or would you need to use a module to identify the lipids in each cell and then quantify their intensity?
Thanks again!!


#8

Hi,

Based on my exploration of your images with yellow pigment, the green channel may be able to stand in as a surrogate for the yellow color. To make sure this is correct, have a look at the output from the ColorToGray module for one of the yellow stained images and see if the green channel adequately reproduces the yellow lipid.

Regards,
-Mark