3D projections
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I have a confocal data stack where the voxels are 0.06 x 0.06 x 0.5. When Z projected at a 90° angle, you get a series of interdigitated white and black lines unless you interpolate. I am curious as to why the data is represented this way. If the sections are 0.5µm thick, and stacked upon each other, where is the black space between coming from? I would have expected that looking from the side, you would see a white smear without a lot of surface depth cueing. Thanks- Dave
Dr. David Knecht Professor, Department of Molecular and Cell Biology University of Connecticut 91 N. Eagleville Rd. U-3125 Storrs, CT 06269-3125 860-486-2200 -- ImageJ mailing list: http://imagej.nih.gov/ij/list.html |
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This is just a guess, but I assume that ImageJ considers each slice to be
essentially infinitely thin, spaced at the step distance. Joel B. Sheffield, Ph.D Department of Biology Temple University Philadelphia, PA 19122 Voice: 215 204 8839 e-mail: [hidden email] Office: Biolife 311 URL: *https://bio.cst.temple.edu/~jbs/ <https://bio.cst.temple.edu/~jbs/> <http://tinyurl.com/khbouft>* On Mon, Mar 30, 2020 at 3:53 PM Knecht, David <[hidden email]> wrote: > I have a confocal data stack where the voxels are 0.06 x 0.06 x 0.5. When > Z projected at a 90° angle, you get a series of interdigitated white and > black lines unless you interpolate. I am curious as to why the data is > represented this way. If the sections are 0.5µm thick, and stacked upon > each other, where is the black space between coming from? I would have > expected that looking from the side, you would see a white smear without a > lot of surface depth cueing. Thanks- Dave > > Dr. David Knecht > Professor, Department of Molecular and Cell Biology > University of Connecticut > 91 N. Eagleville Rd. > U-3125 > Storrs, CT 06269-3125 > 860-486-2200 > > > -- > ImageJ mailing list: http://imagej.nih.gov/ij/list.html > -- ImageJ mailing list: http://imagej.nih.gov/ij/list.html |
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This has always confused me. After all, ImageJ does not treat PIXELS as infinitely small in x and y - so why should it consider VOXELS to be infinitely thin in z (but with finite width and height)?
-- Kenneth Sloan [hidden email] Vision is the art of seeing what is invisible to others. -- ImageJ mailing list: http://imagej.nih.gov/ij/list.html |
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I have seen systems, such as the Leica SP2 software. in which the contents
of each slice is extended through its depth, but that makes the assumption that the section is uniform through its depth. I wonder if that is valid. That is, does the image that is recorded include a full Z average of the psf? In the Leica, it makes the Z slice image very blocky. Joel B. Sheffield, Ph.D Department of Biology Temple University Philadelphia, PA 19122 Voice: 215 204 8839 e-mail: [hidden email] Office: Biolife 311 URL: *https://bio.cst.temple.edu/~jbs/ <https://bio.cst.temple.edu/~jbs/> <http://tinyurl.com/khbouft>* On Mon, Mar 30, 2020 at 5:49 PM Kenneth Sloan <[hidden email]> wrote: > This has always confused me. After all, ImageJ does not treat PIXELS as > infinitely small in x and y - so why should it consider VOXELS to be > infinitely thin in z (but with finite width and height)? > > -- > Kenneth Sloan > [hidden email] > Vision is the art of seeing what is invisible to others. > > -- > ImageJ mailing list: http://imagej.nih.gov/ij/list.html > -- ImageJ mailing list: http://imagej.nih.gov/ij/list.html |
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In reply to this post by Kenneth Sloan-2
"ImageJ does not treat PIXELS as infinitely small in x and y [...]"
If you would, which were correct, you wouldn't see anything. Therefore and in general, the cheapest interpolation is applied: Little squares or rectangles of constant value(s). Actually, such block images are incorrect as well, because the correct interpolation is a totally different one that is much more costly and cannot be realized by common display technology. Regards Herbie ::::::::::::::::::::::::::::::::::::::::::: Am 30.03.20 um 23:49 schrieb Kenneth Sloan: > This has always confused me. After all, ImageJ does not treat PIXELS as infinitely small in x and y - so why should it consider VOXELS to be infinitely thin in z (but with finite width and height)? > > -- > Kenneth Sloan > [hidden email] > Vision is the art of seeing what is invisible to others. > > -- > ImageJ mailing list: http://imagej.nih.gov/ij/list.html > -- ImageJ mailing list: http://imagej.nih.gov/ij/list.html |
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Understood. Now...why doesn't the same logic apply to image "planes"?
-- Kenneth Sloan [hidden email] Vision is the art of seeing what is invisible to others. Begin forwarded message: > From: Herbie <[hidden email]> > Subject: Re: 3D projections > Date: March 31, 2020 at 05:37:15 CDT > To: [hidden email] > Reply-To: [hidden email] > > "ImageJ does not treat PIXELS as infinitely small in x and y [...]" > > If you would, which were correct, you wouldn't see anything. > Therefore and in general, the cheapest interpolation is applied: > Little squares or rectangles of constant value(s). > Actually, such block images are incorrect as well, because the correct interpolation is a totally different one that is much more costly and cannot be realized by common display technology. > > Regards > > Herbie > > ::::::::::::::::::::::::::::::::::::::::::: > Am 30.03.20 um 23:49 schrieb Kenneth Sloan: >> This has always confused me. After all, ImageJ does not treat PIXELS as infinitely small in x and y - so why should it consider VOXELS to be infinitely thin in z (but with finite width and height)? >> -- >> Kenneth Sloan >> [hidden email] >> Vision is the art of seeing what is invisible to others. >> -- >> ImageJ mailing list: http://imagej.nih.gov/ij/list.html > > -- > ImageJ mailing list: http://imagej.nih.gov/ij/list.html -- ImageJ mailing list: http://imagej.nih.gov/ij/list.html |
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Good day Kenneth,
thanks for your reply! "Now...why doesn't the same logic apply to image "planes"?" With a stack of voxel-size 1x1x1 and when I use "Reslice..." (e.g. Rotate 90deg), I'm quite happy with the result. Not sure what's the original problem... Regards Herbie ::::::::::::::::::::::::::::::::::::::::::: Am 31.03.20 um 18:38 schrieb Kenneth Sloan: > Understood. Now...why doesn't the same logic apply to image "planes"? > -- > Kenneth Sloan > [hidden email] <mailto:[hidden email]> > Vision is the art of seeing what is invisible to others. > > Begin forwarded message: > >> From: Herbie <[hidden email] <mailto:[hidden email]>> >> >> Subject: Re: 3D projections >> >> Date: March 31, 2020 at 05:37:15 CDT >> >> To: [hidden email] <mailto:[hidden email]> >> >> Reply-To: [hidden email] <mailto:[hidden email]> >> >> >> "ImageJ does not treat PIXELS as infinitely small in x and y [...]" >> >> If you would, which were correct, you wouldn't see anything. >> Therefore and in general, the cheapest interpolation is applied: >> Little squares or rectangles of constant value(s). >> Actually, such block images are incorrect as well, because the correct >> interpolation is a totally different one that is much more costly and >> cannot be realized by common display technology. >> >> Regards >> >> Herbie >> >> ::::::::::::::::::::::::::::::::::::::::::: >> Am 30.03.20 um 23:49 schrieb Kenneth Sloan: >>> This has always confused me. After all, ImageJ does not treat PIXELS >>> as infinitely small in x and y - so why should it consider VOXELS to >>> be infinitely thin in z (but with finite width and height)? >>> -- >>> Kenneth Sloan >>> [hidden email] <mailto:[hidden email]> >>> Vision is the art of seeing what is invisible to others. >>> -- >>> ImageJ mailing list: http://imagej.nih.gov/ij/list.html >> >> -- >> ImageJ mailing list: http://imagej.nih.gov/ij/list.html -- ImageJ mailing list: http://imagej.nih.gov/ij/list.html |
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The problem is that confocal data is rarely cubic voxels. They are nearly always much larger in z and x-y. In the case of the data I was analyzing they were 0.06 x 0.06 x 0.5 µm and that is not unusual. So you would expect to see the side on projection as a smear given you are looking through elongated voxels. Dave
Dr. David Knecht Professor, Department of Molecular and Cell Biology University of Connecticut 91 N. Eagleville Rd. U-3125 Storrs, CT 06269-3125 860-486-2200 On Mar 31, 2020, at 1:37 PM, Herbie <[hidden email]<mailto:[hidden email]>> wrote: *Message sent from a system outside of UConn.* Good day Kenneth, thanks for your reply! "Now...why doesn't the same logic apply to image "planes"?" With a stack of voxel-size 1x1x1 and when I use "Reslice..." (e.g. Rotate 90deg), I'm quite happy with the result. Not sure what's the original problem... Regards Herbie ::::::::::::::::::::::::::::::::::::::::::: Am 31.03.20 um 18:38 schrieb Kenneth Sloan: Understood. Now...why doesn't the same logic apply to image "planes"? -- Kenneth Sloan [hidden email]<mailto:[hidden email]> <mailto:[hidden email]> Vision is the art of seeing what is invisible to others. Begin forwarded message: From: Herbie <[hidden email]<mailto:[hidden email]> <mailto:[hidden email]>> Subject: Re: 3D projections Date: March 31, 2020 at 05:37:15 CDT To: [hidden email]<mailto:[hidden email]> <mailto:[hidden email]> Reply-To: [hidden email]<mailto:[hidden email]> <mailto:[hidden email]> "ImageJ does not treat PIXELS as infinitely small in x and y [...]" If you would, which were correct, you wouldn't see anything. Therefore and in general, the cheapest interpolation is applied: Little squares or rectangles of constant value(s). Actually, such block images are incorrect as well, because the correct interpolation is a totally different one that is much more costly and cannot be realized by common display technology. Regards Herbie ::::::::::::::::::::::::::::::::::::::::::: Am 30.03.20 um 23:49 schrieb Kenneth Sloan: This has always confused me. After all, ImageJ does not treat PIXELS as infinitely small in x and y - so why should it consider VOXELS to be infinitely thin in z (but with finite width and height)? -- Kenneth Sloan [hidden email]<mailto:[hidden email]> <mailto:[hidden email]> Vision is the art of seeing what is invisible to others. -- ImageJ mailing list: https://nam10.safelinks.protection.outlook.com/?url=http%3A%2F%2Fimagej.nih.gov%2Fij%2Flist.html&data=02%7C01%7Cdavid.knecht%40uconn.edu%7Cd82c64e363bb4c479b7508d7d59a6a96%7C17f1a87e2a254eaab9df9d439034b080%7C0%7C0%7C637212731502269387&sdata=nzDYjWDdPdYvAbRrrJzPhKZx3ceDKN46W3%2FBWlUdtTw%3D&reserved=0 -- ImageJ mailing list: https://nam10.safelinks.protection.outlook.com/?url=http%3A%2F%2Fimagej.nih.gov%2Fij%2Flist.html&data=02%7C01%7Cdavid.knecht%40uconn.edu%7Cd82c64e363bb4c479b7508d7d59a6a96%7C17f1a87e2a254eaab9df9d439034b080%7C0%7C0%7C637212731502279382&sdata=SJU186JVMxsZqSKO9jcUk7CB%2FItPcStT7fFwKGoIShk%3D&reserved=0 -- ImageJ mailing list: https://nam10.safelinks.protection.outlook.com/?url=http%3A%2F%2Fimagej.nih.gov%2Fij%2Flist.html&data=02%7C01%7Cdavid.knecht%40uconn.edu%7Cd82c64e363bb4c479b7508d7d59a6a96%7C17f1a87e2a254eaab9df9d439034b080%7C0%7C0%7C637212731502279382&sdata=SJU186JVMxsZqSKO9jcUk7CB%2FItPcStT7fFwKGoIShk%3D&reserved=0 -- ImageJ mailing list: http://imagej.nih.gov/ij/list.html |
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Hi David,
which is the command that causes the problem? I tried with the "Confocal Series" sample and remove the second color channel, to get a 3D dataset (Image>Stacks>Tools>Make substack). Then, pixel size in x&y is ~0.05 um, in z it is 0.6 um. Image>Stacks>Orthogonal Views does it right. Image>Stacks>Reslice interpolates unless you check 'avoid interpolation' (then it shows it as if the voxels were cubes). Image>Stacks>zProject does not rotate. Among what I tried, the only one that shows thin slices is Image>Stacks>3D project - seems this is the one you are talking about? I had a look at it; the code (ij.plugin.projector) is not very easy to understand. (The relevant function seems to be 'doOneProjectionY') I fear that the author of the original Pascal version (Michael Castle, probably > 20 years ago in the times of NIH Image, at the University of Michigan Mental Health Research Institute) is not active in the ImageJ community. So I fear that the only easy solution is interpolation of the original data (probably easiest with "reslice"), which will, of course, create a much larger stack than what would be needed (9x the memory), then do the "Image>Stacks>3D project". Michael ___________________________________________________________________ On 31/03/2020 7:51 pm, Knecht, David wrote: > The problem is that confocal data is rarely cubic voxels. They are nearly always much larger in z and x-y. In the case of the data I was analyzing they were 0.06 x 0.06 x 0.5 µm and that is not unusual. So you would expect to see the side on projection as a smear given you are looking through elongated voxels. Dave > > Dr. David Knecht > Professor, Department of Molecular and Cell Biology > University of Connecticut > 91 N. Eagleville Rd. > U-3125 > Storrs, CT 06269-3125 > 860-486-2200 > > On Mar 31, 2020, at 1:37 PM, Herbie <[hidden email]<mailto:[hidden email]>> wrote: > > *Message sent from a system outside of UConn.* > > > Good day Kenneth, > > thanks for your reply! > > "Now...why doesn't the same logic apply to image "planes"?" > > With a stack of voxel-size 1x1x1 and when I use "Reslice..." (e.g. > Rotate 90deg), I'm quite happy with the result. > > Not sure what's the original problem... > > Regards > > Herbie > > ::::::::::::::::::::::::::::::::::::::::::: > Am 31.03.20 um 18:38 schrieb Kenneth Sloan: > Understood. Now...why doesn't the same logic apply to image "planes"? > -- > Kenneth Sloan > [hidden email]<mailto:[hidden email]> <mailto:[hidden email]> > Vision is the art of seeing what is invisible to others. > > Begin forwarded message: > > From: Herbie <[hidden email]<mailto:[hidden email]> <mailto:[hidden email]>> > > Subject: Re: 3D projections > > Date: March 31, 2020 at 05:37:15 CDT > > To: [hidden email]<mailto:[hidden email]> <mailto:[hidden email]> > > Reply-To: [hidden email]<mailto:[hidden email]> <mailto:[hidden email]> > > > "ImageJ does not treat PIXELS as infinitely small in x and y [...]" > > If you would, which were correct, you wouldn't see anything. > Therefore and in general, the cheapest interpolation is applied: > Little squares or rectangles of constant value(s). > Actually, such block images are incorrect as well, because the correct > interpolation is a totally different one that is much more costly and > cannot be realized by common display technology. > > Regards > > Herbie > > ::::::::::::::::::::::::::::::::::::::::::: > Am 30.03.20 um 23:49 schrieb Kenneth Sloan: > This has always confused me. After all, ImageJ does not treat PIXELS > as infinitely small in x and y - so why should it consider VOXELS to > be infinitely thin in z (but with finite width and height)? > -- > Kenneth Sloan > [hidden email]<mailto:[hidden email]> <mailto:[hidden email]> > Vision is the art of seeing what is invisible to others. > -- > ImageJ mailing list: https://nam10.safelinks.protection.outlook.com/?url=http%3A%2F%2Fimagej.nih.gov%2Fij%2Flist.html&data=02%7C01%7Cdavid.knecht%40uconn.edu%7Cd82c64e363bb4c479b7508d7d59a6a96%7C17f1a87e2a254eaab9df9d439034b080%7C0%7C0%7C637212731502269387&sdata=nzDYjWDdPdYvAbRrrJzPhKZx3ceDKN46W3%2FBWlUdtTw%3D&reserved=0 > > -- > ImageJ mailing list: https://nam10.safelinks.protection.outlook.com/?url=http%3A%2F%2Fimagej.nih.gov%2Fij%2Flist.html&data=02%7C01%7Cdavid.knecht%40uconn.edu%7Cd82c64e363bb4c479b7508d7d59a6a96%7C17f1a87e2a254eaab9df9d439034b080%7C0%7C0%7C637212731502279382&sdata=SJU186JVMxsZqSKO9jcUk7CB%2FItPcStT7fFwKGoIShk%3D&reserved=0 > > -- > ImageJ mailing list: https://nam10.safelinks.protection.outlook.com/?url=http%3A%2F%2Fimagej.nih.gov%2Fij%2Flist.html&data=02%7C01%7Cdavid.knecht%40uconn.edu%7Cd82c64e363bb4c479b7508d7d59a6a96%7C17f1a87e2a254eaab9df9d439034b080%7C0%7C0%7C637212731502279382&sdata=SJU186JVMxsZqSKO9jcUk7CB%2FItPcStT7fFwKGoIShk%3D&reserved=0 > > > -- > ImageJ mailing list: http://imagej.nih.gov/ij/list.html > -- ImageJ mailing list: http://imagej.nih.gov/ij/list.html |
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Hi Michael- You are correct. It is in creating rotation series with 3D project where one sees the problem. It is not a huge problem, as the orthogonal view rarely has anything useful in it. It is more that I am teaching this right now (online of course) and realized that I did not know how to explain that result to my students. In the “real world” I would almost never show that view, and if I needed to, I would interpolate or use Orthogonal views. The extra size is also not a big problem because the final result is not larger, just the memory it takes to get there and I have never run out of memory generating a rotation series. So I am definitely not arguing someone should spend lots of time fixing the code. Dave
Dr. David Knecht Professor, Department of Molecular and Cell Biology University of Connecticut 91 N. Eagleville Rd. U-3125 Storrs, CT 06269-3125 860-486-2200 On Mar 31, 2020, at 2:30 PM, Michael Schmid <[hidden email]<mailto:[hidden email]>> wrote: *Message sent from a system outside of UConn.* Hi David, which is the command that causes the problem? I tried with the "Confocal Series" sample and remove the second color channel, to get a 3D dataset (Image>Stacks>Tools>Make substack). Then, pixel size in x&y is ~0.05 um, in z it is 0.6 um. Image>Stacks>Orthogonal Views does it right. Image>Stacks>Reslice interpolates unless you check 'avoid interpolation' (then it shows it as if the voxels were cubes). Image>Stacks>zProject does not rotate. Among what I tried, the only one that shows thin slices is Image>Stacks>3D project - seems this is the one you are talking about? I had a look at it; the code (ij.plugin.projector) is not very easy to understand. (The relevant function seems to be 'doOneProjectionY') I fear that the author of the original Pascal version (Michael Castle, probably > 20 years ago in the times of NIH Image, at the University of Michigan Mental Health Research Institute) is not active in the ImageJ community. So I fear that the only easy solution is interpolation of the original data (probably easiest with "reslice"), which will, of course, create a much larger stack than what would be needed (9x the memory), then do the "Image>Stacks>3D project". Michael ___________________________________________________________________ On 31/03/2020 7:51 pm, Knecht, David wrote: The problem is that confocal data is rarely cubic voxels. They are nearly always much larger in z and x-y. In the case of the data I was analyzing they were 0.06 x 0.06 x 0.5 µm and that is not unusual. So you would expect to see the side on projection as a smear given you are looking through elongated voxels. Dave Dr. David Knecht Professor, Department of Molecular and Cell Biology University of Connecticut 91 N. Eagleville Rd. U-3125 Storrs, CT 06269-3125 860-486-2200 On Mar 31, 2020, at 1:37 PM, Herbie <[hidden email]<mailto:[hidden email]><mailto:[hidden email]>> wrote: *Message sent from a system outside of UConn.* Good day Kenneth, thanks for your reply! "Now...why doesn't the same logic apply to image "planes"?" With a stack of voxel-size 1x1x1 and when I use "Reslice..." (e.g. Rotate 90deg), I'm quite happy with the result. Not sure what's the original problem... Regards Herbie ::::::::::::::::::::::::::::::::::::::::::: Am 31.03.20 um 18:38 schrieb Kenneth Sloan: Understood. Now...why doesn't the same logic apply to image "planes"? -- Kenneth Sloan [hidden email]<mailto:[hidden email]><mailto:[hidden email]> <mailto:[hidden email]> Vision is the art of seeing what is invisible to others. Begin forwarded message: From: Herbie <[hidden email]<mailto:[hidden email]><mailto:[hidden email]> <mailto:[hidden email]>> Subject: Re: 3D projections Date: March 31, 2020 at 05:37:15 CDT To: [hidden email]<mailto:[hidden email]><mailto:[hidden email]> <mailto:[hidden email]> Reply-To: [hidden email]<mailto:[hidden email]><mailto:[hidden email]> <mailto:[hidden email]> "ImageJ does not treat PIXELS as infinitely small in x and y [...]" If you would, which were correct, you wouldn't see anything. Therefore and in general, the cheapest interpolation is applied: Little squares or rectangles of constant value(s). Actually, such block images are incorrect as well, because the correct interpolation is a totally different one that is much more costly and cannot be realized by common display technology. Regards Herbie ::::::::::::::::::::::::::::::::::::::::::: Am 30.03.20 um 23:49 schrieb Kenneth Sloan: This has always confused me. After all, ImageJ does not treat PIXELS as infinitely small in x and y - so why should it consider VOXELS to be infinitely thin in z (but with finite width and height)? -- Kenneth Sloan [hidden email]<mailto:[hidden email]><mailto:[hidden email]> <mailto:[hidden email]> Vision is the art of seeing what is invisible to others. -- ImageJ mailing list: https://nam10.safelinks.protection.outlook.com/?url=http%3A%2F%2Fimagej.nih.gov%2Fij%2Flist.html&data=02%7C01%7Cdavid.knecht%40uconn.edu%7C4e174ec6ef5640fea6fe08d7d5a1b9ec%7C17f1a87e2a254eaab9df9d439034b080%7C0%7C0%7C637212762910092666&sdata=0SPAlhIt9Ptz%2FEOxCuiTgwsNgGJFiffTcOTLDAPZw3A%3D&reserved=0 -- ImageJ mailing list: https://nam10.safelinks.protection.outlook.com/?url=http%3A%2F%2Fimagej.nih.gov%2Fij%2Flist.html&data=02%7C01%7Cdavid.knecht%40uconn.edu%7C4e174ec6ef5640fea6fe08d7d5a1b9ec%7C17f1a87e2a254eaab9df9d439034b080%7C0%7C0%7C637212762910102660&sdata=gWwGw8cyDrMnl5M6CuSHWhGTVmhHCLUdSfDvCLkUx%2Bo%3D&reserved=0 -- ImageJ mailing list: https://nam10.safelinks.protection.outlook.com/?url=http%3A%2F%2Fimagej.nih.gov%2Fij%2Flist.html&data=02%7C01%7Cdavid.knecht%40uconn.edu%7C4e174ec6ef5640fea6fe08d7d5a1b9ec%7C17f1a87e2a254eaab9df9d439034b080%7C0%7C0%7C637212762910102660&sdata=gWwGw8cyDrMnl5M6CuSHWhGTVmhHCLUdSfDvCLkUx%2Bo%3D&reserved=0 -- ImageJ mailing list: https://nam10.safelinks.protection.outlook.com/?url=http%3A%2F%2Fimagej.nih.gov%2Fij%2Flist.html&data=02%7C01%7Cdavid.knecht%40uconn.edu%7C4e174ec6ef5640fea6fe08d7d5a1b9ec%7C17f1a87e2a254eaab9df9d439034b080%7C0%7C0%7C637212762910102660&sdata=gWwGw8cyDrMnl5M6CuSHWhGTVmhHCLUdSfDvCLkUx%2Bo%3D&reserved=0 -- ImageJ mailing list: https://nam10.safelinks.protection.outlook.com/?url=http%3A%2F%2Fimagej.nih.gov%2Fij%2Flist.html&data=02%7C01%7Cdavid.knecht%40uconn.edu%7C4e174ec6ef5640fea6fe08d7d5a1b9ec%7C17f1a87e2a254eaab9df9d439034b080%7C0%7C0%7C637212762910102660&sdata=gWwGw8cyDrMnl5M6CuSHWhGTVmhHCLUdSfDvCLkUx%2Bo%3D&reserved=0 -- ImageJ mailing list: http://imagej.nih.gov/ij/list.html |
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