Problem with "analyze particles" total area

[veraelisabeth]

I'm relatively new to imagej. Basically, I have to measure the total area of immunofluorescence microscopy images of cell cultures. My problem came out of the sudden: It's not possible to reproduce my measurements of the same image. I haven't changed any settings and have used the same threshold. Also, I did not change bits nor did I save any changes to the images. So does anyone know the problem? Could you help me? Thanks Vera

E.g.
count: yesterday:  705 --> today: 705
total area yesterday: 866788,0 --> today: 363361,2
average size: yesterday: 1230 --> 515,4
%area yesterday: 59,9 --> 59,9
mean yesterday: 389,7 --> 389,7

[Cameron Nowell-2]

Howdy,

Given that count, percent area and intensity are not changing but area is
I would guess that your images are calibrated differently (or one I not
calibrated at all). The other option is in the analyse particle box on one
of your runs you ticked the pixel units box.

To check image calibration look at the top on the image. It should tell
you if the image is calibrated or just in pixels.

Cheers
Cam


Cameron J. Nowell
Research Facilities Manager

Imaging, FACS and Analysis Core
Monash Institute of Pharmaceutical Sciences
Monash University
399 Royal Parade
(Mail address: 381 Royal Parade)
Parkville, VIC, 3052
Australia

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-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of
veraelisabeth
Sent: Tuesday, 31 March 2015 7:31 AM
To: [hidden email]
Subject: Problem with "analyze particles" total area

I'm relatively new to imagej. Basically, I have to measure the total area
of immunofluorescence microscopy images of cell cultures. My problem came
out of the sudden: It's not possible to reproduce my measurements of the
same image. I haven't changed any settings and have used the same
threshold.
Also, I did not change bits nor did I save any changes to the images. So
does anyone know the problem? Could you help me? Thanks Vera

E.g.
count: yesterday:  705 --> today: 705
total area yesterday: 866788,0 --> today: 363361,2 average size:
yesterday: 1230 --> 515,4 %area yesterday: 59,9 --> 59,9 mean yesterday:
389,7 --> 389,7



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[veraelisabeth]

Thank you for your quick response! I believe it's really been a problem with the conversion of pixels to microns. When opening the images the title gives microns and a number in brackets, which I believe is the size in pixels.  As it doesn't make any sense for me to measure microns, it would be easier to just measure the area in pixels. My question now would be if it is correct to open Image --> Properties --> and just change micron to pixel an set the pixel width and length to 1.00000 ??? Then the title of the image gives 1392x1040 pixels as size, which is exactly the number which is given in brackets when the image was set to microns.

By the way: I'm not sure if I use imageJ correctly: I've got 3 microscopy photos of each well of cell cultures. I set the threshold subjectively for each image and analyze particles, afterwards I would like to calculate the mean of the 3 measurements to have one single number for each well. But this seems VERY VERY subjective to me, because manual thresholding is linked very much to my "mental state on the day". I've tried some auto thresholding but it seems very random to me.

Kind regards and many thanks
Vera

[Michael Schmid]

On 2015-Mar-31, at 19:39, veraelisabeth wrote:

> As it doesn't make any sense for me to measure microns, it would be easier to
> just measure the area in pixels. My question now would be if it is correct
> to open Image --> Properties --> and just change micron to pixel an set the
> pixel width and length to 1.00000 ???

Hi Vera,

the easiest way to remove the spatial calibration: Analyze>Set Scale and press the "Click to remove scale" button.

> ... I set the threshold subjectively for
> each image and analyze particles, afterwards I would like to calculate the
> mean of the 3 measurements to have one single number for each well. But this
> seems VERY VERY subjective to me, because manual thresholding is linked very
> much to my "mental state on the day". I've tried some auto thresholding but
> it seems very random to me.

Did you try the 16 different threshold methods? They are in the choice of the 'Threshold' panel wher yo typically see 'Default', left of the 'Red' choice. For most image types, I find at least one them that works rather well.
[BTW, it would be better to start a new thread for a different question.]

Michael

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