11 replies to this topic

[Aris]

Hi all,

I am trying to analyse my signal from some IFs that I did. is there a way to do densitometry of my fluorescence signal with the ImageJ? Any ideas? Is there another program except ImageJ that would eventually be better than that?

thank you all

[cellcounter]

Aris, on Jul 18 2009, 08:34 PM, said:

Hi all,

I am trying to analyse my signal from some IFs that I did. is there a way to do densitometry of my fluorescence signal with the ImageJ? Any ideas? Is there another program except ImageJ that would eventually be better than that?

thank you all

Check out the first search result. You will find many other methods with different queries, Including NIH Image.

[Aris]

cellcounter, on Jul 19 2009, 02:12 PM, said:

Aris, on Jul 18 2009, 08:34 PM, said:

Hi all,

I am trying to analyse my signal from some IFs that I did. is there a way to do densitometry of my fluorescence signal with the ImageJ? Any ideas? Is there another program except ImageJ that would eventually be better than that?

thank you all

Check out the first search result. You will find many other methods with different queries, Including NIH Image.

Thx for your post cellcounter

amazing link. I have gone through many adds but unfortunately I have not found any post specific for my problem. Could you please help me a little bit more in this??

thank you so much

[mdfenko]

if you have an image then imagej can be used to analyze it.

if you are looking at clear (or white) spots on a black background then you can invert the image to show positive values.

[Aris]

mdfenko, on Jul 20 2009, 12:20 PM, said:

if you have an image then imagej can be used to analyze it.

if you are looking at clear (or white) spots on a black background then you can invert the image to show positive values.

I have black background and green spots. How can i evaluate them?

[mdfenko]

Aris, on Jul 20 2009, 05:40 PM, said:

I have black background and green spots. How can i evaluate them?

i would invert the image then convert to grayscale before evaluating.

[Aris]

mdfenko, on Jul 21 2009, 09:20 AM, said:

Aris, on Jul 20 2009, 05:40 PM, said:

I have black background and green spots. How can i evaluate them?

i would invert the image then convert to grayscale before evaluating.

I did that and tried to evaluate but the values I get dont corespond to my observations. My upregulated images turn out downregulated in copmarison to normal...

[mdfenko]

did you invert before converting to grayscale?

if not then you will have high density for the background as well as the spot.

[polyfractal]

A bit of an aside, but if possible, I would attempt to get a program that specializes in western blot analysis. For instance, while ImageJ can perform background detection and subtraction (via rolling ball normalization), it does it to the whole blot. Specialized western blot analysis programs perform background subtraction on individual lanes and often have extra parameters for normalizing and quantifying bands.

[Aris]

polyfractal, on Jul 24 2009, 03:59 PM, said:

A bit of an aside, but if possible, I would attempt to get a program that specializes in western blot analysis. For instance, while ImageJ can perform background detection and subtraction (via rolling ball normalization), it does it to the whole blot. Specialized western blot analysis programs perform background subtraction on individual lanes and often have extra parameters for normalizing and quantifying bands.

Thx for the reply
which program would you suggest?
I have tried to invert and set to gray scale but still my readings are strange. Besides that what is the exact callibration i need to do b4 i start doing the measurments? Usually from what i have been shown for Western blot analysis, i have to set the ImageJ to the uncalibrated mode and set the scale to "pixel" and not "cm" which is the default setting. Now each time i try to do a measurement the scale setting goes back to cm. Is this the way it is supposed to be? And for IF images do i still have to do the Uncalibrated mode?
See the frustrating thing is that my upregulated images are for the naked eye really upregulated (20-20%). And when i try to measure with ImageJ all of a sudden they are downregulated. Strange strange strange....

[polyfractal]

Our lab uses "ImageQuant" to quantify western blots. Unfortunately, it is not a free program like ImageJ. It also has a slightly funky user interface. I would guess there are other good western blot softwares out there, I'm just not aware of them.

[Aris]

polyfractal, on Jul 25 2009, 12:22 PM, said:

Our lab uses "ImageQuant" to quantify western blots. Unfortunately, it is not a free program like ImageJ. It also has a slightly funky user interface. I would guess there are other good western blot softwares out there, I'm just not aware of them.

I downloaded ImageQuant. I have IFs and they arein color so i can not load them with this prog....