Re: fluorescence ratio

Posted by John Oreopoulos on Sep 03, 2009; 2:44pm
URL: http://imagej.273.s1.nabble.com/fluorescence-ratio-tp3691315p3691318.html

When drawing ROIs, it's not necessary to threshold since the  
measurement will only be applied to region enclosed in the ROI.

Thresholding is an alternative way to measure the pixel intensity  
value of your two different cell compartments that does not require  
ROIs. You can do the same thing as before by selecting "Limit to  
Threshold" in the Analyze->Set Measurements menu. This might actually  
be an easier way to do it if the intensity values between the two  
regions you're trying to investigate are well separated in intensity.

John Oreopoulos


On 2-Sep-09, at 8:11 PM, Giulia Falivelli wrote:

> Hi John,
> Thanks for helping.
> Sorry, should I set a threshold before calculate the mean  
> fluorescence?
>
> Giulia
>
> On Wed, Sep 2, 2009 at 4:53 PM, John Oreopoulos  
> <[hidden email]
>> wrote:
>
>> Giulia,
>>
>> First, go to Analyze->Set Measurements and make sure "Mean Gray  
>> Value" is
>> checked.
>> Next, draw a region of interest on your image with the Rectangular
>> Selection tool (or circular or freehand tools if your imaging  
>> situation
>> demands it. To get the mean pixel intensity value within your  
>> region of
>> interest, click Analyze->Measure, and the result will appear as a  
>> number in
>> a results window.
>>
>> Move the region of interest to your other cell compartment where  
>> you want
>> to measure the intensity value and do the same thing. Now you can  
>> take these
>> two numbers and calculate a ratio by simple division.
>>
>> Beyond that, if you require automation/repetition or more complex/
>> multiple
>> regions of interest to be examined, you may want to try writing a  
>> macro that
>> does this for you. Try looking through the long list of plugins  
>> available on
>> the main ImageJ website and see if there is any plugin out there that
>> already does this. None come to mind immediately, but perhaps  
>> there is a
>> calcium imaging plugin that might do the trick for you. Hopefully  
>> someone
>> else here on the server can suggest one.
>>
>> John Oreopoulos
>>
>>
>>
>> On 2-Sep-09, at 7:42 PM, Giulia Falivelli wrote:
>>
>>  Hi All,
>>>
>>> I am new in Image J.
>>> Does anybody know how to calculate the fluorescence ration  
>>> between two
>>> different cells compartement?
>>> Or how to calculate the mean grey value of a specific ROI?
>>> Thank you very much.
>>>
>>> Giulia
>>>
>>