Determining Mean Fluorescence Intensity

[ev34]

Hello,

I am new to ImageJ and trying to 1) how to figure out what the best
threshold value should be 2) how to determine the range of the particle size
that I need to set 3) how to calculate mean fluorescence intensity (how is
it related to mean pixel intensity, integrated density or mean particle
size? is it just the integrated particle size divided by the area?) I am
imaging intestinal fluorescence in C. elegans and need the program to pick
out the fluorescent granules. So far, it is picking up that and a lot of
other "junk" so that I have to manually scroll down the list to see which
integrated denisties have the highest values. My main concern is that I am
setting the threshold on "auto" run now without knowing if that's really the
best way to do it. Do all of my pictures have to have the same exposure time
if I want to compare them and keep the same threshold and particle size
settings? My other concern is that I am not sure what the lower limit of my
particle size should be.

Any help will be greatly appreciated. Thanks much!

Lena

[Liqun Zhang]

I have a similar question. How is the "auto" threshold determined? i.e.,
What is the algorithm behind it?
I'm no programmer, and do not understand the code.
Could some one enlighten me and/or point me to the right documents? Many
thanks.
Liqun

Lena wrote:

>Hello,
>
>I am new to ImageJ and trying to 1) how to figure out what the best
>threshold value should be 2) how to determine the range of the particle size
>that I need to set 3) how to calculate mean fluorescence intensity (how is
>it related to mean pixel intensity, integrated density or mean particle
>size? is it just the integrated particle size divided by the area?) I am
>imaging intestinal fluorescence in C. elegans and need the program to pick
>out the fluorescent granules. So far, it is picking up that and a lot of
>other "junk" so that I have to manually scroll down the list to see which
>integrated denisties have the highest values. My main concern is that I am
>setting the threshold on "auto" run now without knowing if that's really the
>best way to do it. Do all of my pictures have to have the same exposure time
>if I want to compare them and keep the same threshold and particle size
>settings? My other concern is that I am not sure what the lower limit of my
>particle size should be.
>
>Any help will be greatly appreciated. Thanks much!
>
>Lena
>  
>

[Volker Baecker]

Look here for information about the ImageJ auto-threshold:
http://imagejdocu.tudor.lu/imagej-documenation-wiki/faq/what-is-the-algorithm-used-to-threshold-images
Cheers,
Volker

Liqun Zhang a écrit :

> I have a similar question. How is the "auto" threshold determined?
> i.e., What is the algorithm behind it?
> I'm no programmer, and do not understand the code.
> Could some one enlighten me and/or point me to the right documents?
> Many thanks.
> Liqun
>
> Lena wrote:
>
>> Hello,
>>
>> I am new to ImageJ and trying to 1) how to figure out what the best
>> threshold value should be 2) how to determine the range of the
>> particle size
>> that I need to set 3) how to calculate mean fluorescence intensity
>> (how is
>> it related to mean pixel intensity, integrated density or mean particle
>> size? is it just the integrated particle size divided by the area?) I am
>> imaging intestinal fluorescence in C. elegans and need the program to
>> pick
>> out the fluorescent granules. So far, it is picking up that and a lot of
>> other "junk" so that I have to manually scroll down the list to see
>> which
>> integrated denisties have the highest values. My main concern is that
>> I am
>> setting the threshold on "auto" run now without knowing if that's
>> really the
>> best way to do it. Do all of my pictures have to have the same
>> exposure time
>> if I want to compare them and keep the same threshold and particle size
>> settings? My other concern is that I am not sure what the lower limit
>> of my
>> particle size should be.
>>
>> Any help will be greatly appreciated. Thanks much!
>>
>> Lena  
>>
>

--
passerelle antivirus du campus CNRS de Montpellier
--

[Liqun Zhang]

Hi,
I wonder if there are threshold plugins based background-symmetry
algorithm and triangle algorithm as mentioned on this page:
http://www.ph.tn.tudelft.nl/Courses/FIP/noframes/fip-Segmenta.html#Heading118

Thanks,
Liqun

[aeyu]

Does anyone have concise instructions for determining the mean fluorescent intensity for RGB images.  I already got my ROI measurments for each color.  I am doing a ratio of green to red with 4 different experimental groups.

Mean/Area?

Do I normalize to anything?

Best!
dr^2mom