Hello I try to improve my images before doing an Auto Threshold for the rest of the quantifications. I have a significant background noise related to the Fibrin gel in which the cells develop. In the attached images, the arrows point to areas I would like to "erase" and the cells are circled in green. [cid:image001.jpg@01D6FBA4.EB60B440] [cid:image005.jpg@01D6FBA4.EB60B440] Any help will be welcome. Patricia _______________________________________ Email : [hidden email]<mailto:[hidden email]> -- ImageJ mailing list: http://imagej.nih.gov/ij/list.html image002.jpg (199K) Download Attachment image004.jpg (180K) Download Attachment image001.jpg (18K) Download Attachment image005.jpg (16K) Download Attachment original_IJ.jpg (1M) Download Attachment |
If this is autofluorescence of some sort, it is often broad wavelength, so one simple way to remove it is to take an image with a different fluorescence channel than your true signal and subtract the background image (noise) from the signal image. Dave
Dr. David Knecht Professor, Department of Molecular and Cell Biology University of Connecticut 91 N. Eagleville Rd. U-3125 Storrs, CT 06269-3125 On Feb 5, 2021, at 3:54 AM, OBEID Patricia 154904 <[hidden email]<mailto:[hidden email]>> wrote: *Message sent from a system outside of UConn.* Hello I try to improve my images before doing an Auto Threshold for the rest of the quantifications. I have a significant background noise related to the Fibrin gel in which the cells develop. In the attached images, the arrows point to areas I would like to "erase" and the cells are circled in green. [cid:image001.jpg@01D6FBA4.EB60B440] [cid:image005.jpg@01D6FBA4.EB60B440] Any help will be welcome. Patricia _______________________________________ Email : [hidden email]<mailto:[hidden email]><mailto:[hidden email]> -- ImageJ mailing list: https://nam10.safelinks.protection.outlook.com/?url=http%3A%2F%2Fimagej.nih.gov%2Fij%2Flist.html&data=04%7C01%7Cdavid.knecht%40uconn.edu%7C9ea5fa1ebedd40e111a108d8c9b3e4b6%7C17f1a87e2a254eaab9df9d439034b080%7C0%7C0%7C637481121791222348%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000&sdata=f4NwslLQ5kRCinSwd95hgRXgG1bl7NhIDi0PTND2fgY%3D&reserved=0 <image002.jpg><image004.jpg><image001.jpg><image005.jpg><original_IJ.jpg> -- ImageJ mailing list: http://imagej.nih.gov/ij/list.html |
In reply to this post by OBEID Patricia 154904
It looks like the blurs are out of focus cells deep in the gel. A
pinhole confocal might yield the best data (or maybe SPIM). Cleaning up what you have might be a partial work around. A rolling ball subtraction (paraboloid 6) or a bandpassFFT (8,0,autoscale) can lessen the scattered background. The uneven label will pose the next problem for threshold-- skeletonizing the membrane vs picking up the entire cell. Using a gamma 0.6 to make the scatter more uniform, ditto the label, before the rolling ball, seems to pick up most cells with Li threshold. Better data = better results. regards On 2/5/2021 2:54 AM, OBEID Patricia 154904 wrote: > Hello > I try to improve my images before doing an Auto Threshold for the rest of the quantifications. > I have a significant background noise related to the Fibrin gel in which the cells develop. > In the attached images, the arrows point to areas I would like to "erase" and the cells are circled in green. > > [cid:image001.jpg@01D6FBA4.EB60B440] > > > > > > > > > > > > > > > > [cid:image005.jpg@01D6FBA4.EB60B440] > > > > > > > > > > > > > > > > > Any help will be welcome. > Patricia > _______________________________________ > > Email : [hidden email]<mailto:[hidden email]> > > > > > > > > > -- > ImageJ mailing list: https://urldefense.com/v3/__http://imagej.nih.gov/ij/list.html__;!!OaZRxjSJGsACmqMyc8s!YlLHfww6a7aO8kT-pE2XQK787tWdfI-Ec3qbB1Z66RphMGm8oJeLSAsOpcBpHm3mre6D3No$ > > ---------------------------------------------------------------------- > “This message was received from outside of the organization. Please pay special attention and practice care when clicking on any links, or providing any information to the sender. Cyber attacks commonly attempt to trick you in to thinking the sender is a reputable individual who you can trust.” > __ Vytas Bindokas, M.S., Ph.D. Research Assoc. Prof., Director, BSD Light Microscopy Core Facility phone: 773-702-4875 [alt's: 773-834-9040 or 773-834-2639] [address for letters ONLY (see shipping addr below):] Dept Pharmacol Physiol Sci MC0926 947 E 58th Street The University of Chicago Chicago IL 60637 Room Abbott 129 shipping address (main KCBD site): V. Bindokas 900 E 57th Street KCBD room 1250, Microscopy Core The University of Chicago Chicago IL 60637 email [hidden email] web site for LMCF: http://digital.uchicago.edu -- ImageJ mailing list: http://imagej.nih.gov/ij/list.html |
I got some decent results with Labkit (https://imagej.net/Labkit), I guess they would even become better with some pre-filteren etc.
Best wishes Kees -----Original Message----- From: vbindokas Sent: 05 February 2021 14:05 Subject: Re: [EXTERNAL] Help to remove background It looks like the blurs are out of focus cells deep in the gel. A pinhole confocal might yield the best data (or maybe SPIM). Cleaning up what you have might be a partial work around. A rolling ball subtraction (paraboloid 6) or a bandpassFFT (8,0,autoscale) can lessen the scattered background. The uneven label will pose the next problem for threshold-- skeletonizing the membrane vs picking up the entire cell. Using a gamma 0.6 to make the scatter more uniform, ditto the label, before the rolling ball, seems to pick up most cells with Li threshold. Better data = better results. regards On 2/5/2021 2:54 AM, OBEID Patricia 154904 wrote: > Hello > I try to improve my images before doing an Auto Threshold for the rest of the quantifications. > I have a significant background noise related to the Fibrin gel in which the cells develop. > In the attached images, the arrows point to areas I would like to "erase" and the cells are circled in green. > > [cid:image001.jpg@01D6FBA4.EB60B440] > > > > > > > > > > > > > > > > [cid:image005.jpg@01D6FBA4.EB60B440] > > > > > > > > > > > > > > > > > Any help will be welcome. > Patricia > _______________________________________ > > Email : [hidden email]<mailto:[hidden email]> > > > > > > > > > -- > ImageJ mailing list: > https://eur03.safelinks.protection.outlook.com/?url=https%3A%2F%2Furld > efense.com%2Fv3%2F__http%3A%2F%2Fimagej.nih.gov%2Fij%2Flist.html__%3B! > !OaZRxjSJGsACmqMyc8s!YlLHfww6a7aO8kT-pE2XQK787tWdfI-Ec3qbB1Z66RphMGm8o > JeLSAsOpcBpHm3mre6D3No%24&data=04%7C01%7Ckrs5%40leicester.ac.uk%7C > 546b6a052df74e159f1c08d8c9df2f64%7Caebecd6a31d44b0195ce8274afe853d9%7C > 0%7C0%7C637481307725778552%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDA > iLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=slaO5 > vO9%2BLZPfeTRtSHgndBwCD3BhYUHSDvVoAeP3mI%3D&reserved=0 > > ---------------------------------------------------------------------- > "This message was received from outside of the organization. Please pay special attention and practice care when clicking on any links, or providing any information to the sender. Cyber attacks commonly attempt to trick you in to thinking the sender is a reputable individual who you can trust." > __ Vytas Bindokas, M.S., Ph.D. Research Assoc. Prof., Director, BSD Light Microscopy Core Facility phone: 773-702-4875 [alt's: 773-834-9040 or 773-834-2639] [address for letters ONLY (see shipping addr below):] Dept Pharmacol Physiol Sci MC0926 947 E 58th Street The University of Chicago Chicago IL 60637 Room Abbott 129 shipping address (main KCBD site): V. Bindokas 900 E 57th Street KCBD room 1250, Microscopy Core The University of Chicago Chicago IL 60637 email [hidden email] web site for LMCF: https://eur03.safelinks.protection.outlook.com/?url=http%3A%2F%2Fdigital.uchicago.edu%2F&data=04%7C01%7Ckrs5%40leicester.ac.uk%7C546b6a052df74e159f1c08d8c9df2f64%7Caebecd6a31d44b0195ce8274afe853d9%7C0%7C0%7C637481307725778552%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=COl3zmz0jTBxq2jcIqshTqZy93W4sSBxj%2Bxg1p1JK1g%3D&reserved=0 -- ImageJ mailing list: https://eur03.safelinks.protection.outlook.com/?url=http%3A%2F%2Fimagej.nih.gov%2Fij%2Flist.html&data=04%7C01%7Ckrs5%40leicester.ac.uk%7C546b6a052df74e159f1c08d8c9df2f64%7Caebecd6a31d44b0195ce8274afe853d9%7C0%7C0%7C637481307725778552%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=Iryg4YB%2Fjbd7s6iC51ejKaVnK%2FVttxqAguo8dqttawY%3D&reserved=0 -- ImageJ mailing list: http://imagej.nih.gov/ij/list.html |
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