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Dear confocalists, I’d like to ask you for your opinion on a protocol for
analysing membrane dynamics. I work with human lymphoma cell lines, and
they change their shape and behaviour depending on an inducible RNAi
machinery: when I turn it on, cells loose their transformed aspect
(becoming round-shaped), they give up forming membrane protrusions and
stop their spontaneous and anarchic movement. I made more than 50 imaging
experiments, in different conditions, with different stimuli and different
cell lines: the data are strong, biochemically confirmed, and we’re really
confident. I have now to measure and quantify the change, during the
time-lapse experiments. I did the imaging with my TCS SP2 confocal, using
the “transmission” modality, further modified in order to minimize the
laser energy ( I can give you the details off-line).
I tried to track the membrane movements with Metamorph, but I failed using
the automatic tracking. This is due to the fact that the cells display an
adhesion point, around  which the whole membrane continuously rearrange
its shape, every few seconds. Sometimes they also migrate for short
distances. My aim is to measure the generation and movement of membrane
protrusions, quantifying the displacement of the leading edge.
I tracked manually, frame by frame, the leading edge of any single cell,
with the “track objects” option of Metamorph, moving the research frame
every time, updating the position and going on. The graphics are
convincing, but –during a recent oral presentation- I’ve been fairly
criticized: they told that, regardless to the fact that data are
absolutely reliable, my tracking procedure seems a bit odd.
Would you mind sending me suggestions and critics, before I send the paper?
I may send you a few photos of the cell movements, so you can take a look
at it, if you think it’s useful.
Thank you in advance for your help (I checked the archive, without any
real benefit).
Andrew


Andrea Manazza, MD PhD
Dept. of Biomedical sciences and human oncology
Molecular Oncology-CeRMS
Via Santena 7, 10126 Torino
Italia
tel: +39-11-6336859
fax: +39-11-6336887