Histogram scale change when importing .ZVI files

Posted by javila0624 on Jul 15, 2014; 6:33pm
URL: http://imagej.273.s1.nabble.com/Histogram-scale-change-when-importing-ZVI-files-tp5008753.html

Hello all,
I'm having a difficult time understanding why histogram scales change when I import .zvi using the bio-formats importer. I'd like to convert all of my files to .tif format and use ImageJ for analysis but I am concerned any subsequent analysis will not be valid as a result of this rescaling.



Here's what I have done:
I save a 16-bit .zvi file and load onto Mac OSX. (I know it is 16-bit because my Axiovision camera captures at this depth and the software confirms in the file information.)
Using the bio-formats import option, I open the 3-channel .zvi file with the Standard-ImageJ stack option selected. The 3 channels are split; autoscaling is off, and color mode is in gray-scale. I have also tried different variations of this by using hyperstack options, and color-mode on default.

The result has been the same: Upon clicking on the histogram function, I see that instead of seeing a 0-65535 range on my intensity distribution scale, images from both different channels and different samples can vary in the intensity scaling. Example: sample 1, channel 1 shows a 0-65520 scale; sample 1 channel 3 shows a 0-65520 scale ;sample 2, channel 1 shows a 0-65520 scale; sample 2, channel 3 shows a 0-58890 scale.

In an attempt to confirm that the images should be on a 0-65535 scale, opening the files without bio-formats importer and then clicking on the histogram function shows me that this is in fact the case: the scale is 0-65535. (this is not an option for me however, as images areas are scrambled on the ImageJ window, i.e. the image quadrants/relative positions are 'scrambled' on the image.)

It seems like a straightforward scaling or binning problem, but how can the importer be set to bin equivalently?


Can anyone help me understand why this happens? How can I maintain the 16-bit scale (0-65535)?

I've played around with converting to 32-bit and re-converting back to 16-bit. I'd rather avoid this step if at all necessary and retain the original data scale.


Thank in advance for any help!!