what is the reason for the difference seen here

[sean_incali]

hey everyone

[URL=http://imageshack.us][IMG]
http://img369.imageshack.us/img369/3179/compare6cb.jpg[/IMG][/URL]

Show two images of same lsm file opened in ImageJ and LSM broswer.

On the left is the lsm file opened using LSM import plug in, then the two
channels are merged using color>RGB merge.

On the right is the same file opened using lsm browser.

You can clearly see the intensity is amplified in the imageJ versin.

What is the reason for this?

[Jeff Brandenburg]

ImageJ does automatic contrast/brightness adjustment for display under
some circumstances.  If you examine the histogram for the image, you'll
find that it's unaltered.

On Sep 12, 2005, at 7:38 PM, sean_incali wrote:

> hey everyone
>
> [URL=http://imageshack.us][IMG]
> http://img369.imageshack.us/img369/3179/compare6cb.jpg[/IMG][/URL]
>
> Show two images of same lsm file opened in ImageJ and LSM broswer.
>
> On the left is the lsm file opened using LSM import plug in, then the
> two
> channels are merged using color>RGB merge.
>
> On the right is the same file opened using lsm browser.
>
> You can clearly see the intensity is amplified in the imageJ versin.
>
> What is the reason for this?
>

--
        -jeffB (Jeff Brandenburg, Duke Center for In-Vivo Microscopy)

[sean_incali]

> ImageJ does automatic contrast/brightness adjustment for display under
> some circumstances.  If you examine the histogram for the image, you'll
> find that it's unaltered.
>
http://img369.imageshack.us/img369/3179/compare6cb.jpg 


How do you use the histogram to check? Are you comparing the intensity
distributions? (I guess that's the only thing I can think of using Histogram to
check...)

Also I have a question about opening a lsm file using LSM plugin vs using an
image in Tiff format exported from LSM broswer.

If I open the "Tiff file" in ImageJ, is that same as the lsm file opened using
the plugin? (same, I mean in terms of intensity in two channels.)

If they are different, how different are they? in what respect?

[Jeff Brandenburg]

On Sep 14, 2005, at 6:58 PM, sean_incali wrote:

>> ImageJ does automatic contrast/brightness adjustment for display under
>> some circumstances.  If you examine the histogram for the image,
>> you'll
>> find that it's unaltered.
>>
> http://img369.imageshack.us/img369/3179/compare6cb.jpg
>
>
> How do you use the histogram to check? Are you comparing the intensity
> distributions? (I guess that's the only thing I can think of using
> Histogram to
> check...)

Here's what I did to verify what I thought I'd seen:

I opened a RAW image file in ImageJ, and noted its range of values (in
this case, 0-32767 or thereabouts).

I used Process: Math: Divide to divide all pixel values by 16.  The
resulting image was very dark, as I'd expect.  I saved this image in a
new file, again in RAW format.

I re-opened the original file and the divided-by-16 file.  Both images
*appeared* essentially identical as displayed by ImageJ.  However,
while the original image had a range of 0-32767, the new image had a
range of 0-2047.

I suspect that the image you're examining has no fully-saturated
pixels, and that LSM browser is displaying it without adjustment, while
ImageJ is automatically adjusting contrast and brightness to maximize
visual contrast.

If LSM browser lets you examine the histogram of your image, I'd
suggest examining the histogram there and in ImageJ.  I'm guessing that
you'll see the same values and the same distribution in both cases.

Note that I'm not an LSM user, so I can't vouch for this from personal
experience.

> Also I have a question about opening a lsm file using LSM plugin vs
> using an
> image in Tiff format exported from LSM broswer.
>
> If I open the "Tiff file" in ImageJ, is that same as the lsm file
> opened using
> the plugin? (same, I mean in terms of intensity in two channels.)
>
> If they are different, how different are they? in what respect?

Again, I don't know what LSM does.  However, I do believe that ImageJ
does the same autoscaling behavior with TIFFs that it does with RAW
data.
--
        -jeffB (Jeff Brandenburg, Duke Center for In-Vivo Microscopy)

[Gabriel Landini]

On Thursday 15 September 2005 15:44, Jeff Brandenburg wrote:
> Again, I don't know what LSM does.  However, I do believe that ImageJ
> does the same autoscaling behavior with TIFFs that it does with RAW
> data.

Is this the "Scale When Converting" option? I always thought that it is a bit
"dangerous" option :-), but very useful nevertheless.

Cheers,
G.

[Joel Sheffield]

Jeff, and others,

As your post suggests, I think that it is helpful to distinguish
between the actual values of the pixels and the display values.  
Remember that the monitor screen is actually able to display only a
limited range of values (256 comes to mind).  As a result, ImageJ
will set an output LUT (i.e. the range of display pixels) to display
the range of the data in 1/256 intensity steps.  Thus, even if you
use a 16-bit input file, your display will still be 8-bits.  This
will not affect your data, just how it appears.  You can manually set
the display to a given output scale with the b/c routine.  

However, once you click on "apply" the values are reassigned
(although this requires an 8-bit image) and your data is changed.  

> On Sep 14, 2005, at 6:58 PM, sean_incali wrote:
>
> >> ImageJ does automatic contrast/brightness adjustment for display
> >> under some circumstances.  If you examine the histogram for the
> >> image, you'll find that it's unaltered.
> >>
> > http://img369.imageshack.us/img369/3179/compare6cb.jpg
> >
> >
> > How do you use the histogram to check? Are you comparing the
> > intensity distributions? (I guess that's the only thing I can think
> > of using Histogram to check...)
>
> Here's what I did to verify what I thought I'd seen:
>
> I opened a RAW image file in ImageJ, and noted its range of values (in
> this case, 0-32767 or thereabouts).
>
> I used Process: Math: Divide to divide all pixel values by 16.  The
> resulting image was very dark, as I'd expect.  I saved this image in a
> new file, again in RAW format.
>
> I re-opened the original file and the divided-by-16 file.  Both images
> *appeared* essentially identical as displayed by ImageJ.  However,
> while the original image had a range of 0-32767, the new image had a
> range of 0-2047.
>
> I suspect that the image you're examining has no fully-saturated
> pixels, and that LSM browser is displaying it without adjustment,
> while ImageJ is automatically adjusting contrast and brightness to
> maximize visual contrast.
>
> If LSM browser lets you examine the histogram of your image, I'd
> suggest examining the histogram there and in ImageJ.  I'm guessing
> that you'll see the same values and the same distribution in both
> cases.
>
> Note that I'm not an LSM user, so I can't vouch for this from personal
> experience.
>
> > Also I have a question about opening a lsm file using LSM plugin vs
> > using an image in Tiff format exported from LSM broswer.
> >
> > If I open the "Tiff file" in ImageJ, is that same as the lsm file
> > opened using the plugin? (same, I mean in terms of intensity in two
> > channels.)
> >
> > If they are different, how different are they? in what respect?
>
> Again, I don't know what LSM does.  However, I do believe that ImageJ
> does the same autoscaling behavior with TIFFs that it does with RAW
> data. --
>  -jeffB (Jeff Brandenburg, Duke Center for In-Vivo Microscopy)

Joel B. Sheffield, Ph.D.
Biology Department, Temple University
1900 North 12th Street
Philadelphia, PA 19122
[hidden email]  
[phone], fax [phone]
http://astro.temple.edu/~jbs