Quantitating spots on an autorad
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Quantitating spots on an autorad
 
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Please HELP! I have spent hours and days trying to quantitate round spots of varying darkness on autorads, with no success. When I draw a circle of a certain size, and use it to get the density of the lighter background of my autorad, I measure an integrated density that is, say, 1,000,000. Then, when I move that circle to enclose a light gray spot, I get a SMALLER integrated density, say 550,000, and then when I move that circle to a dark black spot, I get an even smaller integrated density, say 130,000. This makes no sense to me. I have tried all sorts of combinations of image modifications and analyze modifications to no avail. It seems like I must reverse some parameter but I can't determine how to do that. I can't get over the fact that this should be easy to do. Autorad, light spot, dark spot. Light spot - lower density, dark spot- higher density.
I have tried the sample gel that can be downloaded. There you use the rectangular tool to identify lanes and you get peaks and assign a baseline and so forth. I see how that works and it does in fact give higher values for larger peaks (darker bands) but I am convinced there is another way that uses the circular tool that would be most appropriate for analyzing circlular spots on an autorad. Anyone have any suggestions? Frank J. Castora, Ph.D. Professor of Biochemistry Department of Physiological Sciences Eastern Virginia Medical School 700 W. Olney Road Norfolk, VA 23507 [hidden email]<mailto:[hidden email]> Frank J. Castora, Ph.D. Professor of Biochemistry Department of Physiological Sciences Eastern Virginia Medical School 700 W. Olney Road Norfolk, VA 23507 -- ImageJ mailing list: http://imagej.nih.gov/ij/list.html |
Re: Quantitating spots on an autorad
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Hi Frank,
The first problem is that ImageJ uses the convention that black pixels have the value of 0, and white pixels have the value of 255 (in an 8-bit system) Thus, as your samples become darker, the value that you get becomes smaller. You can correct this by inverting the image (Edit>invert) so that black is the new white (as it were). Second, have you taken a look at the "Analyze particles" routine? THis will allow you to set a threshold that wlll include all of the spots, and automatically calculate densities, areas, integrated densities for all. Joel On Thu, Jul 18, 2013 at 12:18 PM, Castora, Frank J. <[hidden email]>wrote: > Please HELP! I have spent hours and days trying to quantitate round spots > of varying darkness on autorads, with no success. When I draw a circle of > a certain size, and use it to get the density of the lighter background of > my autorad, I measure an integrated density that is, say, 1,000,000. Then, > when I move that circle to enclose a light gray spot, I get a SMALLER > integrated density, say 550,000, and then when I move that circle to a dark > black spot, I get an even smaller integrated density, say 130,000. This > makes no sense to me. I have tried all sorts of combinations of image > modifications and analyze modifications to no avail. It seems like I must > reverse some parameter but I can't determine how to do that. I can't get > over the fact that this should be easy to do. Autorad, light spot, dark > spot. Light spot - lower density, dark spot- higher density. > > I have tried the sample gel that can be downloaded. There you use the > rectangular tool to identify lanes and you get peaks and assign a baseline > and so forth. I see how that works and it does in fact give higher values > for larger peaks (darker bands) but I am convinced there is another way > that uses the circular tool that would be most appropriate for analyzing > circlular spots on an autorad. > > Anyone have any suggestions? > > Frank J. Castora, Ph.D. > Professor of Biochemistry > Department of Physiological Sciences > Eastern Virginia Medical School > 700 W. Olney Road > Norfolk, VA 23507 > [hidden email]<mailto:[hidden email]> > > > Frank J. Castora, Ph.D. > Professor of Biochemistry > Department of Physiological Sciences > Eastern Virginia Medical School > 700 W. Olney Road > Norfolk, VA 23507 > > > -- > ImageJ mailing list: http://imagej.nih.gov/ij/list.html > -- Joel B. Sheffield, Ph.D Department of Biology Temple University Philadelphia, PA 19122 Voice: 215 204 8839 e-mail: [hidden email] URL: http://astro.temple.edu/~jbs -- ImageJ mailing list: http://imagej.nih.gov/ij/list.html |
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In reply to this post by Castora, Frank J.
Hi,
bright background means low or even zero density, as I understand you. Than you have to measure in the inverted image. You could even try to apply the extinction transformation, see the thread on "Quantification of red in bright field image". To detect your spots a "top hat transformation" could be helpful. 1. Closing with appropriate size (larger than your spots) 2. Difference of closed and original, that you get a black (low value) background. 3. Thresholding 4. Particle analyzer redirected to the inverted or the extinction image. Regards Karsten Am 18.07.2013 um 18:18 schrieb "Castora, Frank J." <[hidden email]>: > Please HELP! I have spent hours and days trying to quantitate round spots of varying darkness on autorads, with no success. When I draw a circle of a certain size, and use it to get the density of the lighter background of my autorad, I measure an integrated density that is, say, 1,000,000. Then, when I move that circle to enclose a light gray spot, I get a SMALLER integrated density, say 550,000, and then when I move that circle to a dark black spot, I get an even smaller integrated density, say 130,000. This makes no sense to me. I have tried all sorts of combinations of image modifications and analyze modifications to no avail. It seems like I must reverse some parameter but I can't determine how to do that. I can't get over the fact that this should be easy to do. Autorad, light spot, dark spot. Light spot - lower density, dark spot- higher density. > > I have tried the sample gel that can be downloaded. There you use the rectangular tool to identify lanes and you get peaks and assign a baseline and so forth. I see how that works and it does in fact give higher values for larger peaks (darker bands) but I am convinced there is another way that uses the circular tool that would be most appropriate for analyzing circlular spots on an autorad. > > Anyone have any suggestions? > > Frank J. Castora, Ph.D. > Professor of Biochemistry > Department of Physiological Sciences > Eastern Virginia Medical School > 700 W. Olney Road > Norfolk, VA 23507 > [hidden email]<mailto:[hidden email]> > > > Frank J. Castora, Ph.D. > Professor of Biochemistry > Department of Physiological Sciences > Eastern Virginia Medical School > 700 W. Olney Road > Norfolk, VA 23507 > > > -- > ImageJ mailing list: http://imagej.nih.gov/ij/list.html Karsten [hidden email] -- ImageJ mailing list: http://imagej.nih.gov/ij/list.html |
Re: Quantitating spots on an autorad
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Thank you all for your feedback. With your suggestions I have been able to get past my brain block and analyze my autorads. I appreciate everyone who took time to respond.
Frank J. Castora, Ph.D. Professor of Biochemistry Department of Physiological Sciences Eastern Virginia Medical School 700 W. Olney Road Norfolk, VA 23507 -----Original Message----- From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of Karsten Sent: Thursday, July 18, 2013 1:23 PM To: [hidden email] Subject: Re: Quantitating spots on an autorad Hi, bright background means low or even zero density, as I understand you. Than you have to measure in the inverted image. You could even try to apply the extinction transformation, see the thread on "Quantification of red in bright field image". To detect your spots a "top hat transformation" could be helpful. 1. Closing with appropriate size (larger than your spots) 2. Difference of closed and original, that you get a black (low value) background. 3. Thresholding 4. Particle analyzer redirected to the inverted or the extinction image. Regards Karsten Am 18.07.2013 um 18:18 schrieb "Castora, Frank J." <[hidden email]>: > Please HELP! I have spent hours and days trying to quantitate round spots of varying darkness on autorads, with no success. When I draw a circle of a certain size, and use it to get the density of the lighter background of my autorad, I measure an integrated density that is, say, 1,000,000. Then, when I move that circle to enclose a light gray spot, I get a SMALLER integrated density, say 550,000, and then when I move that circle to a dark black spot, I get an even smaller integrated density, say 130,000. This makes no sense to me. I have tried all sorts of combinations of image modifications and analyze modifications to no avail. It seems like I must reverse some parameter but I can't determine how to do that. I can't get over the fact that this should be easy to do. Autorad, light spot, dark spot. Light spot - lower density, dark spot- higher density. > > I have tried the sample gel that can be downloaded. There you use the rectangular tool to identify lanes and you get peaks and assign a baseline and so forth. I see how that works and it does in fact give higher values for larger peaks (darker bands) but I am convinced there is another way that uses the circular tool that would be most appropriate for analyzing circlular spots on an autorad. > > Anyone have any suggestions? > > Frank J. Castora, Ph.D. > Professor of Biochemistry > Department of Physiological Sciences > Eastern Virginia Medical School > 700 W. Olney Road > Norfolk, VA 23507 > [hidden email]<mailto:[hidden email]> > > > Frank J. Castora, Ph.D. > Professor of Biochemistry > Department of Physiological Sciences > Eastern Virginia Medical School > 700 W. Olney Road > Norfolk, VA 23507 > > > -- > ImageJ mailing list: http://imagej.nih.gov/ij/list.html Karsten [hidden email] -- ImageJ mailing list: http://imagej.nih.gov/ij/list.html -- ImageJ mailing list: http://imagej.nih.gov/ij/list.html |
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