Fluorescence quantification

[Sofia Domingues]

Hi there,
I am working with GFP astrocytes and I would like to quantify the loss of
fluorescence in the process of apoptosis, as when they die they detach from
the wells and become very small round green cells.
Does anyone know wether there is a plug-in on Image J to quantify the "green
signal" per area?
Thanks
Sofia

[John Oreopoulos]

Hi Sofia,

If you draw a rectangular region of interest with the rectangular  
selection tool in ImageJ and then click Analyze->Measure, you'll get  
a results table that lists the average pixel intensity ("green  
signal") over the area you have drawn. On a more technical/
experimental note, I would caution you against trying to make a  
correlation between how close to death a cell is (via apoptosis) and  
the amount of greet signal. There are many other factors in a live  
cell fluorescence imaging experiment that can lead to changes in the  
amount of signal. For instance, See Jim Pawley's "39 steps" article  
in Biotechniques.

John Oreopoulos


On 19-Jun-08, at 12:00 PM, Sofia Domingues wrote:

> Hi there,
> I am working with GFP astrocytes and I would like to quantify the  
> loss of
> fluorescence in the process of apoptosis, as when they die they  
> detach from
> the wells and become very small round green cells.
> Does anyone know wether there is a plug-in on Image J to quantify  
> the "green
> signal" per area?
> Thanks
> Sofia

[George Sharonov]

If cells become round the cytoplasmic volume that is projected to one CCD
pixel increase. Thus changes in fluorescence intensity may be due to cell
shape changes and not due to florescence quantum yield changes. To avoid
this for some extent you should use confocal microscopy with carefully
adjusted focal plane position and pinhole settings. Even with confocal
microscopy, quantification of intracellular fluorescence has a lot of
pitfalls. I can advice you an article on this topic:

"Confocal microscopy: quantitative analytical capabilities." Dobrucki
JW. Methods
Cell Biol. 2004;75:41-72. (it's from the book: "Cytometry: New Developments,
4th Ed." )


On Thu, Jun 19, 2008 at 8:33 PM, John Oreopoulos <
[hidden email]> wrote:

> Hi Sofia,
>
> If you draw a rectangular region of interest with the rectangular selection
> tool in ImageJ and then click Analyze->Measure, you'll get a results table
> that lists the average pixel intensity ("green signal") over the area you
> have drawn. On a more technical/experimental note, I would caution you
> against trying to make a correlation between how close to death a cell is
> (via apoptosis) and the amount of greet signal. There are many other factors
> in a live cell fluorescence imaging experiment that can lead to changes in
> the amount of signal. For instance, See Jim Pawley's "39 steps" article in
> Biotechniques.
>
> John Oreopoulos
>
>
>
> On 19-Jun-08, at 12:00 PM, Sofia Domingues wrote:
>
>  Hi there,
>> I am working with GFP astrocytes and I would like to quantify the loss of
>> fluorescence in the process of apoptosis, as when they die they detach
>> from
>> the wells and become very small round green cells.
>> Does anyone know wether there is a plug-in on Image J to quantify the
>> "green
>> signal" per area?
>> Thanks
>> Sofia
>>
>

... [show rest of quote]

--

George Sharonov,
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry
Moscow, Russia