Re: Tiff format export from LSM broswer

Hi Martin. Thanks for the reply.


> Also, be aware that TIFF files can use different types of compression.
> (According to Wikipedia, LSM uses no compression.) You should avoid
> using lossy compression, such as JPEG, inside your TIFFs.
>

How do I check if the LSM browser is using such method? (Actually, I
guess I could call Zeiss and ask... ) Unless Tiff header contains such
informatin alreay...


> > Another related problem is background subtraction.
> >
> > Let's say you have an image that contains intensities in both green and
> > blue channels. Do you have to Bbackground subtract in both channels
> > separately? (By Splitting the image into two channels prior to
> > subtraction)
> >
> > Or Can I do a background subtraction on the entire image with two
> > colors with single ROI and then split the image into two channels?
> >
> > I hope I'm making myself clear...
>
> You are not making yourself very clear. I suspect you want to know
> whether image subtraction of two RGB images will subtract all three
> corresponding channels. The answer depends on the software package
> you are using, but would typically be yes.

This is big propblem for me.
http://news.gmane.org/find-root.php?message_id=%3cloom.20050913T013312%2d254%40post.gmane.org%3e

shows a post i made to IMAGEJ forum. IMAGEJ as you might know is a free
biomed imaging software written in java.

The point of the thread was that LSM file reader plugin implemented in
IMAGEJ scales the intensities in two channels(blue and green for me)
and to use them subsequently in background subtraction and ratiometric
imaging has been problematic. Different location on the same sample
will show up as having vastly different ratios of two channel
intensities.


What I was doing is choosing the same coordinates for definition of
ROI(region of interest)for both channels. But the background is uneven
in two channels. This results after background subtraction, in one of
the channel intentisies being underrepresented.

*And* to begin with the intensities are amplified.(even though they are
saying that this is only for the display)

So I thought I would take the two channels images that IMAGEJ generates
upon importing the LSM file, and then merge them to form an singleRGB
image. then ROI background subtract the merged image and split it into
two channels.(blue and green) This gets rid of one channel being
subracted more than the others. Both channels are subtracted by the
same amount. But I'm not sure if this is a valid way of background
subtraction in two channels with differing background. Two channel
intensities are obtained using two different sets of filters. (dichroic
mirrors)

one more question.

IMGAEJ has an implementation of rolling ball background subraction. Do
you know much about this? I've been told this is a better way to
background subtract if the background is uneven. (instead of using ROI
background subtraction.) But i have no idea what it is. it seems like
people use it for blending.. whatever that is...

.

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