saving images as they appear by eye

classic Classic list List threaded Threaded
4 messages Options
Reply | Threaded
Open this post in threaded view
|  
Report Content as Inappropriate

saving images as they appear by eye

Julia Edgar
Hi list members

When using micromanager for image capture I have to choose 'auto' on the histogram function to see anything on the screen (fluorescence images). Then I have to change the 'curve' on the histogram to get an image on the screen that looks anything like what I see by eye - if I don't adjust the diagonal line to an upward curve, faint cellular processes are invisible.

After doing this I see something on the screen that resembles what I see by eye. But when I then 'snap' and save as a tif file and then open the tif file in image j, almost all but the brightest pixels have disappeared. I can adjust the brightness and contrast using image j (which helps to some extent) but I cannot restore the original (natural looking) image.

Can you please help?

Thank you
Julia

------------------------------------------------------------------------------
Check out the vibrant tech community on one of the world's most
engaging tech sites, Slashdot.org! http://sdm.link/slashdot
_______________________________________________
micro-manager-general mailing list
[hidden email]
https://lists.sourceforge.net/lists/listinfo/micro-manager-general
Reply | Threaded
Open this post in threaded view
|  
Report Content as Inappropriate

Re: saving images as they appear by eye

m.losen

Dear Julia

 

To answer briefly: if your image is saved in a 16 bit format (rather than 8 bit, which is the intensity range of a monitor), you also need to adjust the display contrast in ImageJ after opening the image. This is done in the imageJ menu “Image”-“Adjust”-“Brightness/Contrast”. In the case of a 16-bit image, this does not change the pixel intensities, but just the way they are shown on the screen.

If you already use a 8 bit format, you probably need to enhance the exposure time of the camera, to increase signal intensities and reduce noise.

 

Hope this helps,

Mario


------------------------------------------------------------------------------
Check out the vibrant tech community on one of the world's most
engaging tech sites, Slashdot.org! http://sdm.link/slashdot
_______________________________________________
micro-manager-general mailing list
[hidden email]
https://lists.sourceforge.net/lists/listinfo/micro-manager-general
Reply | Threaded
Open this post in threaded view
|  
Report Content as Inappropriate

Re: saving images as they appear by eye

Kesavan Subburam
In reply to this post by Julia Edgar
Hi Julia,

If I understand correctly, I think what you're dealing with is called contrast stretching or histogram stretching. In most cases, because of the way the images are displayed, you won't be able to see the details of the image until you adjust the contrast, and in most cases, linearly.

When you're opening the raw tif file in ImageJ, the software does not automatically stretch the image like most commercial software packages do. Unless your image is 8bit, in which case the intensity values are in the range of 0 - 2^8, (0 being the darkest or black and 255 being the brightest or white), you will not be able to view the image 'naturally'. But all the data are still there -- the bright pixels are still brighter compared to the other pixels in the image, it just doesn't look bright to us. If you point your cursor over the image, you can see the absolute intensity values for that region displayed in ImageJ along with the X and Y coordinates. You might notice that the values remain the same even after adjusting the contrast automatically from Image > Adjust > Brightness/Contrast > Auto.

In case you want to adjust the contrast and use the image in a presentation as it is being displayed in the software, you'll have to "Apply" the values and save the image as a png file, which can be directly inserted in a presentation, and the bright pixels will look bright against the dark pixels. But it's always a good idea to keep the raw data stored in the .tif files as such.


I hope I have not misread your question,

Best,

Kesavan Subburam

On 2 June 2017 at 04:19, Julia Edgar <[hidden email]> wrote:
Hi list members

When using micromanager for image capture I have to choose 'auto' on the histogram function to see anything on the screen (fluorescence images). Then I have to change the 'curve' on the histogram to get an image on the screen that looks anything like what I see by eye - if I don't adjust the diagonal line to an upward curve, faint cellular processes are invisible.

After doing this I see something on the screen that resembles what I see by eye. But when I then 'snap' and save as a tif file and then open the tif file in image j, almost all but the brightest pixels have disappeared. I can adjust the brightness and contrast using image j (which helps to some extent) but I cannot restore the original (natural looking) image.

Can you please help?

Thank you
Julia

------------------------------------------------------------------------------
Check out the vibrant tech community on one of the world's most
engaging tech sites, Slashdot.org! http://sdm.link/slashdot
_______________________________________________
micro-manager-general mailing list
[hidden email]
https://lists.sourceforge.net/lists/listinfo/micro-manager-general




--
Kesavan

------------------------------------------------------------------------------
Check out the vibrant tech community on one of the world's most
engaging tech sites, Slashdot.org! http://sdm.link/slashdot
_______________________________________________
micro-manager-general mailing list
[hidden email]
https://lists.sourceforge.net/lists/listinfo/micro-manager-general
Reply | Threaded
Open this post in threaded view
|  
Report Content as Inappropriate

Re: saving images as they appear by eye

Sam Lord
In reply to this post by Julia Edgar
Julia Edgar wrote
Then I have to change the 'curve' on the histogram to get an image on the screen that looks anything like what I see by eye - if I don't adjust the diagonal line to an upward curve, faint cellular processes are invisible.
The other responses are partially correct. But specifically what you are referring to when you change the curve of the line is gamma:
https://micro-manager.org/wiki/Micro-Manager_User%27s_Guide#Gamma_Function

Changing gamma changes the display from a linear function (increasing intensity scales linearly with increasing white level on the screen) to a nonlinear function. Typically, it would be appropriate/necessary to state the gamma adjustment in a figure caption if you make the image display nonlinear.

Also, remember that eyes (+brain) are amazing detectors!

-Sam
Loading...