Dear all,
I am currently a graduate student working on my thesis project. Using
Fiji64, I have been calculating ROIs to quantify changes in the ratio of
fluorophore intensity for cells' plasma membrane to cytosol staining.
Specifically, I am trying to look at changes in intensity over time as
various agents are added to the media. The image sequence data (movie)
shows clear differences, with changes in intensity between the membrane and
cytostol. Yet, when I calculate ROIs, the graphs of delta F/F vs time show
no change. I was hoping to get advice on how to standardize my method, but
still show an effect. Using the same ROI to calculate the membrane and
cytosol does not work because the cytosolic changes are much greater than
can be seen in an ROI that encloses the membrane. Although the peak
membrane and cytosol intensity seem to equate, one possibility is to make a
larger ROI for the cytoplasm to better capture the intensity changes. Would
it be valid then to compare different sized and shaped ROIs for the
cytoplasm and plasma membrane (keeping the ROIs consistent from cell to
cell, experiment to experiment)?
Your help is much appreciated.
Sincerely,
Ryan Anderson
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