fluorescence measurement in discrete regions of moving cells

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fluorescence measurement in discrete regions of moving cells

Thorsten Vetter
hi all,

 

I need to measure fluorescence intensities t-stackwise in different discrete
regions of cells (eg. cytosol, membrane). I would like to use the
"measurestack" plugin but my cells are moving significantly over time. Is
there any way to define ROIs in ImageJ which will be automatically
relocalized in every frame of a t-stack  by eg. a constant distance from the
edge of fluorescence which signifies the cellborder or any other means? As I
look at reasonable number of stacks any automated form of intensity
measurement would be very welcome.

 

thanks for any hints and help!

 

regards

 

--- thorsten

 

pharmacology,

university of würzburg, germany

 

 
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Re: fluorescence measurement in discrete regions of moving cells

Pedro J CamelloDr Pedro J Camello
Thorsten Vetter wrote:

>hi all,
>
>
>
>I need to measure fluorescence intensities t-stackwise in different discrete
>regions of cells (eg. cytosol, membrane). I would like to use the
>"measurestack" plugin but my cells are moving significantly over time. Is
>there any way to define ROIs in ImageJ which will be automatically
>relocalized in every frame of a t-stack  by eg. a constant distance from the
>edge of fluorescence which signifies the cellborder or any other means? As I
>look at reasonable number of stacks any automated form of intensity
>measurement would be very welcome.
>
>
>
>thanks for any hints and help!
>
>
>
>regards
>
>
>
>--- thorsten
>
>
>
>pharmacology,
>
>university of würzburg, germany
>
>
>
>
>
>  
>
I have the same problem: smooth muscle cells that, of course, contract
when stimulated. The movement is some times enough to spoil some
experiments (I make fura-2 measurement). What I was trying is this:
- I define a threshold for the cells
- I make a rectangular or circular ROI around each cell comprising the
cell and a piece of coverlip, making sure that the cell don´t "scape"
along my stack
- I make stack measurment in that ROI limited to the thresholded pixels

problems: 1) I really finish sooner if I draw rois for a part of the
cell with small of negligible movement, using the dedictaed software of
the lab. Only an automated plugin would finish this problem, but I have
no time to learn to do that (reading, making experiments, teaching,
analyzing results, thinking, .... :'( )
                2) I don´t know if the threshold must be set
independently for each frame of the stack (how to do that?) or if a
general threshold must be adjusted for all the slices after careful
"replay" of the stack while "playing" with the threshold menu (I felt it
very unconfortable and confusing)

If you find a solution for this, please let me know

Good luck (I´ll contact you if I find some solution)

Pedro
KP
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Re: fluorescence measurement in discrete regions of moving cells

KP
In reply to this post by Thorsten Vetter
Dear Thorsten,

I am having the same problem that you had regarding cells moving over time. Did you manage to find a way to relocalize each frame so that the cells end up stcking correctly over time?

Any info. would be greatly appreciated.

Thank you!

Kind regards,
Karen