[Ramin Vismeh]

:laugh: will double check with them…

[Ramin Vismeh]

I have to talk to people from Prosolia. But I remember I had this talk with them before and they think that this is the BioMap issue. This is part of the email I received from them:

” Unfortunately, the issue has to do with BioMAP. BioMAP has an intrinsic data limitation of 32,767 points in any dimension (i.e. intensity, x, y, m/z). What you need to do is limit the m/z domain to a smaller mass window around the ion of interest within firefly prior to conversion. Unfortunately, due to the same limitation the intensities will truncate at 32, 767. In a new release coming soon (FireFly 2.0) we remove the limitation to allow customers to import data into Matlab. ”

So, number of data points can be reduced but the intensity issue is still there. Thats what they think!

Ramin

[Ramin Vismeh]

All right Markus..
Thanks. I will let you know my progress!

[Ramin Vismeh]

All right Markus…
Thanks. I will also check with Prosolia since I am using their Firefly software for coverting the raw data to image files. I will let you guys know the results. But you dont think that the intensities are being cut if they are above 32767 right?

[Ramin Vismeh]

I am using X-Calibur (version 2.1.0 1139) from thermo. The instrument is Orbitrap XL and I am doing DESI imaging! My BioMap is also 3.8.0.3.

[Ramin Vismeh]

Hi Guys,

I did exactly the same thing as Markus said. That right! The number that we get from Analysis od ROI, is actualy the intensity of ions/pixels. So, it is nomalized to the number of pixels. I got the exact same numbers…
BUT….
What I am woried about is that in that 4 pixels which I chose, my ion of interest had an ion intensity of 32767! I am sure thats not the real intensity of my ion. for example if these 4 pixels that Markus talked about have all intensities above 32767, they all will be shown az 32767! I dont see any intensity higher than 32767 for any pixel when I do pixel scan. That means 32767 is also the limit for intensity!

Markus:
I am curios to see what are the intensities of those pixels you chose? Arent they all 32767 or below?

Ramin

[Ramin Vismeh]

Dodge and Markus,

Thanks. So you guys think that the intensities from ROI regions CAN be used as a base for quantification as they are not cut or scaled?

Dodge,
WhenI say the intensities do not match, I am talking about the average. When I average my raw data (say for the whole image) for a specific m/z, teh average is most of the times more than 32767! But if I select the whole image as one ROI, teh intensity for that specific m/z would not be the same in the ROI intensity panel! Is’nt the intensity divided by the number of pixels for the ROIs? another words: the ion intensity that I get from Analysys—>Plot—>ROI is the ion intensity/pixel?

Thanks

[Ramin Vismeh]

Thanks so much Markus for the Reply.:)
The other thing is the intensity of ions. I understand that the 32767 is the limit for parameters like data points. That’s why when creating an image; I use a narrow range of masses so that I end up with less # of data points. But I am not sure what BioMap does to the intensities. Is it going to cut the intensities that are above 32767 or is it dividing them all by a number so that the highest one becomes 32767 and scales the rest of them with the same ratio? I am talking about the numbers in Analysis—>plot—->ROI panel. I know when I look at my raw data, the intensities are higher than 32767 but when I generate ROIs, I get different intensities in the ROIs plot! My goal is to see if I really can trust the intensities in the ROI plot for ion intensities and use them as a basis for Quantification!

Thanks,
Ramin

[Ramin Vismeh]

Basically its the number of Data points

[Ramin Vismeh]

Based on what I observed, N depends on the bin size and your mass range. For example when you convert your raw data files to an image, if your mass range is from 200-400 Da and your bin size is 0.1, then N=1 means m/z is 200, N=2 means m/z is 200.1, N=3 means m/z is 200.2 and …

Ramin

[Ramin Vismeh]

Hi Dave,

Same here. N in the Stats window is one more than the N in the main window. But I am not sure if N is m/z value! I am not sure what N is. I see that it depends on the number of scans and number of data points in the image.

Ramin

[Ramin Vismeh]

Dave,

I am using DESI. So, my spot size is not known actually. I can have an estimation but it wont be accurate. Thats why I am trying to assess the area using the image. How do you usually generate the standard curves? I assume you use something like ion intensity vs. ng/area for some spots of standard that you put on tissues. Is that right? When you image the tissue, well most of the times the compound you are looking for is not probably in one nice circle spot. It is usually scattered all over the tissue. So, I assume for quantification the right unit to use is ng/area. Am I right?

Thanks

[Ramin Vismeh]

[Ramin Vismeh]

Hi Dave,

Thats right. that is actually the number of pixels of ROI. But the thing is apparently it assumes each pixel is 1 mm^2. I guess the real size PROBABLY depends on the pixel size that you set up in your imaging experiment. Please correct me if I am wrong.

Ramin

[Ramin Vismeh]

Thanks Dave for replying…
Yes, that makes sense…Do you know how we can get the area for the ROI?

Thanks,
Ramin

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