Re: background subtraction
Posted by
Knecht, David on
Feb 09, 2011; 12:37pm
URL: http://imagej.273.s1.nabble.com/background-subtraction-tp3685750p3685752.html
Hi Aryeh- I don't understand your fundamental concern. We compare patterns of fluorescence in different channels all the time. How is this different from comparing two channels in a co-localization analysis? It is the relative amount of signal in different parts of the same cell that is in question. Dave
On Feb 8, 2011, at 1:38 AM, Aryeh Weiss wrote:
> I doubt that there is
>
> On 2/8/11 5:44 AM, David Knecht charter wrote:
>> We are trying to determine the distribution of a GFP-fusion protein
>> in a cell. All the images are 16 bit and confocal. If one probes with
>> GFP alone, there are areas of the cell that appear significantly
>> brighter than other areas because they are vesicle free (so called
>> hyaline cytoplasm) and so the concentration of free cytoplasmic
>> proteins is higher. Our fusion protein probe localizes to one of
>> these vesicle free regions. So if you double label a cell with
>> GFP-fusion protein and RFP, the same areas appear higher than
>> background, but the ratio of hyaline localized to normal cytoplasm is
>> higher for the fusion protein than the FP alone. Therefore, we want
>> to use the unfused protein as "background" and subtract that signal
>> from the fusion protein signal in order to correct for the
>> non-uniform background. My plan is to set the non-localized
>> cytoplasmic area of the cell to similar intensity values and then
>> subtract with the Image Calculator. So lets say the RFP image values
>> range from 50 to 1000 and the GFP values range from 200-2000. I am
>> unsure how to correct the images before subtraction. You cannot
>> "apply" a look up table to the 16 bit images to correct for the two
>> images not being collected at that same intensity level. What is the
>> most appropriate way to "adjust" the intensity levels of a 16 bit
>> image to equalize two images? THanks- Dave
>>
>
> I doubt that there is any validity to comparing fluorescence of two
> different FPs measured in two different channels. However, it might be
> appropriate to normalize the RFP to its value in the vesicle free
> area, and this may provide a multiplicative correction factor for your
> GFP fusion.
>
> --aryeh
> --
> Aryeh Weiss
> School of Engineering
> Bar Ilan University
> Ramat Gan 52900 Israel
>
> Ph: 972-3-5317638
> FAX: 972-3-7384051
Dr. David Knecht
Department of Molecular and Cell Biology
Co-head Flow Cytometry and Confocal Microscopy Facility
U-3125
91 N. Eagleville Rd.
University of Connecticut
Storrs, CT 06269
860-486-2200
860-486-4331 (fax)