seeking "reliable literature" on correct imaging
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Cammer, Michael |
seeking "reliable literature" on correct imaging
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Increasingly we have found that microscope users do not understand proper imaging. A few examples: Confocal images are saturated and have the offset set to negative numbers intentionally "to reduce background". Exposures are changed in fluorescent micrographs to make each one "look good". 16 bit raw data with no saturation or clipping are set to 8 bits with a lot of saturated and clipped pixels at the extremes and these are what are considered raw data. JPG compression is great because images can be emailed. People assume (and get angry when this doesn't work) that computers can automatically segment and count anything regardless of experimental design, image quality, etc. Even if they have saved everything as TIF with no metadata and forgot what magnifications they used. A student who recognizes these problems and wants to learn the right way emailed me yesterday asking for "any reliable literature you recommend if I'd like to read more about correct imaging." I did Google searches and, after skimming a bunch of web pages, realized that I didn't really have an answer. I could send to this page and to this page, but it's not a coherent narrative. And I don't have a library; most of what I know about this was picked up willy-nilly up in the 1980s and '90s. For instance knowledge of color separation from writing animations for the Apple II in machine code, working in a cable TV studio in the 1980s, and from an art history class in college where we discussed 19th C travel photos and poly/monochromatic film chemistries of the periods. Later I learned fluorescence quantification from a cell biologist intent on making sure we had true linear responses for readouts of f-actin mass and concentration, I was fortunate to attend both a Woods Hole microscopy course and at NCSU John Russ's course on image analysis, and BioRad MRC 600 training course at their offices in Cambridge, MA covered a lot of material on this too. So, do people have favorite texts that cover these topics in a manner digestible by new generations of students? Of biology students. Keep in mind many don't know what a byte is; when I teach I ask the class if anyone knows why an image is coded with white as 255 or 4095 or 16383 and typically one or two hands go up but the rest of the class really doesn't know. Looking forward to suggestions of easy to read definitive texts! Thank you. Cheers- ------------------------------------------------------------ This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email. ================================= |
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*****
To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi Michael, We recently ran a workshop where I gave a 3-hour talk on various kinds of quantification in microscopy. I can send you a PowerPoint file but don't have a text. Mike Model -----Original Message----- From: Confocal Microscopy List <[hidden email]> On Behalf Of Cammer, Michael Sent: Friday, November 16, 2018 11:50 AM To: [hidden email] Subject: seeking "reliable literature" on correct imaging ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Increasingly we have found that microscope users do not understand proper imaging. A few examples: Confocal images are saturated and have the offset set to negative numbers intentionally "to reduce background". Exposures are changed in fluorescent micrographs to make each one "look good". 16 bit raw data with no saturation or clipping are set to 8 bits with a lot of saturated and clipped pixels at the extremes and these are what are considered raw data. JPG compression is great because images can be emailed. People assume (and get angry when this doesn't work) that computers can automatically segment and count anything regardless of experimental design, image quality, etc. Even if they have saved everything as TIF with no metadata and forgot what magnifications they used. A student who recognizes these problems and wants to learn the right way emailed me yesterday asking for "any reliable literature you recommend if I'd like to read more about correct imaging." I did Google searches and, after skimming a bunch of web pages, realized that I didn't really have an answer. I could send to this page and to this page, but it's not a coherent narrative. And I don't have a library; most of what I know about this was picked up willy-nilly up in the 1980s and '90s. For instance knowledge of color separation from writing animations for the Apple II in machine code, working in a cable TV studio in the 1980s, and from an art history class in college where we discussed 19th C travel photos and poly/monochromatic film chemistries of the periods. Later I learned fluorescence quantification from a cell biologist intent on making sure we had true linear responses for readouts of f-actin mass and concentration, I was fortunate to attend both a Woods Hole microscopy course and at NCSU John Russ's course on image analysis, and BioRad MRC 600 training course at their offices in Cambridge, MA covered a lot of material on this too. So, do people have favorite texts that cover these topics in a manner digestible by new generations of students? Of biology students. Keep in mind many don't know what a byte is; when I teach I ask the class if anyone knows why an image is coded with white as 255 or 4095 or 16383 and typically one or two hands go up but the rest of the class really doesn't know. Looking forward to suggestions of easy to read definitive texts! Thank you. Cheers- ------------------------------------------------------------ This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email. ================================= |
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Gulpreet Kaur |
Re: seeking "reliable literature" on correct imaging
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hello Michael, I’m a recent graduate student from University of Wisconsin Madison and I will be using a lot of imaging techniques in he future. Can you send me a copy of the PowerPoint too? Thanks, Gul Gulpreet Kaur Research Assistant - Lakkaraju Lab Department of Ophthalmology and Visual Sciences Program in Cellular and Molecular Biology University of Wisconsin - Madison > On Nov 16, 2018, at 11:00 AM, MODEL, MICHAEL <[hidden email]> wrote: > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Hi Michael, > > We recently ran a workshop where I gave a 3-hour talk on various kinds of quantification in microscopy. I can send you a PowerPoint file but don't have a text. > > Mike Model > > -----Original Message----- > From: Confocal Microscopy List <[hidden email]> On Behalf Of Cammer, Michael > Sent: Friday, November 16, 2018 11:50 AM > To: [hidden email] > Subject: seeking "reliable literature" on correct imaging > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Increasingly we have found that microscope users do not understand proper imaging. > > A few examples: > Confocal images are saturated and have the offset set to negative numbers intentionally "to reduce background". > Exposures are changed in fluorescent micrographs to make each one "look good". > 16 bit raw data with no saturation or clipping are set to 8 bits with a lot of saturated and clipped pixels at the extremes and these are what are considered raw data. > JPG compression is great because images can be emailed. > People assume (and get angry when this doesn't work) that computers can automatically segment and count anything regardless of experimental design, image quality, etc. > Even if they have saved everything as TIF with no metadata and forgot what magnifications they used. > > A student who recognizes these problems and wants to learn the right way emailed me yesterday asking for "any reliable literature you recommend if I'd like to read more about correct imaging." > > I did Google searches and, after skimming a bunch of web pages, realized that I didn't really have an answer. I could send to this page and to this page, but it's not a coherent narrative. And I don't have a library; most of what I know about this was picked up willy-nilly up in the 1980s and '90s. For instance knowledge of color separation from writing animations for the Apple II in machine code, working in a cable TV studio in the 1980s, and from an art history class in college where we discussed 19th C travel photos and poly/monochromatic film chemistries of the periods. Later I learned fluorescence quantification from a cell biologist intent on making sure we had true linear responses for readouts of f-actin mass and concentration, I was fortunate to attend both a Woods Hole microscopy course and at NCSU John Russ's course on image analysis, and BioRad MRC 600 training course at their offices in Cambridge, MA covered a lot of material on this too. > > So, do people have favorite texts that cover these topics in a manner digestible by new generations of students? Of biology students. Keep in mind many don't know what a byte is; when I teach I ask the class if anyone knows why an image is coded with white as 255 or 4095 or 16383 and typically one or two hands go up but the rest of the class really doesn't know. > Looking forward to suggestions of easy to read definitive texts! > > Thank you. > > Cheers- > > > ------------------------------------------------------------ > This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email. > ================================= |
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VERMEREN Matthieu |
Re: seeking "reliable literature" on correct imaging
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In reply to this post by Cammer, Michael
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Dear Michael, I've been reading Pete Bankhead's excellent "Analyzing fluorescence microscopy images with ImageJ" (see https://petebankhead.gitbooks.io/imagej-intro/content/). I think the first part of the course covers what you describe. At least it's a good start. Sincerely, Matthieu -- The University of Edinburgh is a charitable body, registered in Scotland, with registration number SC005336. -----Original Message----- From: Confocal Microscopy List <[hidden email]> On Behalf Of Cammer, Michael Sent: Friday, 16 November, 2018 16:50 To: [hidden email] Subject: seeking "reliable literature" on correct imaging ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Increasingly we have found that microscope users do not understand proper imaging. A few examples: Confocal images are saturated and have the offset set to negative numbers intentionally "to reduce background". Exposures are changed in fluorescent micrographs to make each one "look good". 16 bit raw data with no saturation or clipping are set to 8 bits with a lot of saturated and clipped pixels at the extremes and these are what are considered raw data. JPG compression is great because images can be emailed. People assume (and get angry when this doesn't work) that computers can automatically segment and count anything regardless of experimental design, image quality, etc. Even if they have saved everything as TIF with no metadata and forgot what magnifications they used. A student who recognizes these problems and wants to learn the right way emailed me yesterday asking for "any reliable literature you recommend if I'd like to read more about correct imaging." I did Google searches and, after skimming a bunch of web pages, realized that I didn't really have an answer. I could send to this page and to this page, but it's not a coherent narrative. And I don't have a library; most of what I know about this was picked up willy-nilly up in the 1980s and '90s. For instance knowledge of color separation from writing animations for the Apple II in machine code, working in a cable TV studio in the 1980s, and from an art history class in college where we discussed 19th C travel photos and poly/monochromatic film chemistries of the periods. Later I learned fluorescence quantification from a cell biologist intent on making sure we had true linear responses for readouts of f-actin mass and concentration, I was fortunate to attend both a Woods Hole microscopy course and at NCSU John Russ's course on image analysis, and BioRad MRC 600 training course at their offices in Cambridge, MA covered a lot of material on this too. So, do people have favorite texts that cover these topics in a manner digestible by new generations of students? Of biology students. Keep in mind many don't know what a byte is; when I teach I ask the class if anyone knows why an image is coded with white as 255 or 4095 or 16383 and typically one or two hands go up but the rest of the class really doesn't know. Looking forward to suggestions of easy to read definitive texts! Thank you. Cheers- ------------------------------------------------------------ This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email. ================================= |
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kspencer007 |
Re: seeking "reliable literature" on correct imaging
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In reply to this post by mmodel
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi Mike; Could I get your PowerPoint as well? I have the same issues with my users. I'm "retraining" all of them with an open-software quiz, to see if they understand basic concepts. Many are complaining and reluctant, because they don't see the issues with their images. This after one 6-year post-doc's data was totally discarded because she saved everything as JPG....I certainly NEVER taught her to do THAT. Your PowerPoint should hold more credibility that I obviously have.... Best; Kathy -----Original Message----- From: Confocal Microscopy List <[hidden email]> On Behalf Of MODEL, MICHAEL Sent: Friday, November 16, 2018 9:00 AM To: [hidden email] Subject: Re: seeking "reliable literature" on correct imaging ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi Michael, We recently ran a workshop where I gave a 3-hour talk on various kinds of quantification in microscopy. I can send you a PowerPoint file but don't have a text. Mike Model -----Original Message----- From: Confocal Microscopy List <[hidden email]> On Behalf Of Cammer, Michael Sent: Friday, November 16, 2018 11:50 AM To: [hidden email] Subject: seeking "reliable literature" on correct imaging ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Increasingly we have found that microscope users do not understand proper imaging. A few examples: Confocal images are saturated and have the offset set to negative numbers intentionally "to reduce background". Exposures are changed in fluorescent micrographs to make each one "look good". 16 bit raw data with no saturation or clipping are set to 8 bits with a lot of saturated and clipped pixels at the extremes and these are what are considered raw data. JPG compression is great because images can be emailed. People assume (and get angry when this doesn't work) that computers can automatically segment and count anything regardless of experimental design, image quality, etc. Even if they have saved everything as TIF with no metadata and forgot what magnifications they used. A student who recognizes these problems and wants to learn the right way emailed me yesterday asking for "any reliable literature you recommend if I'd like to read more about correct imaging." I did Google searches and, after skimming a bunch of web pages, realized that I didn't really have an answer. I could send to this page and to this page, but it's not a coherent narrative. And I don't have a library; most of what I know about this was picked up willy-nilly up in the 1980s and '90s. For instance knowledge of color separation from writing animations for the Apple II in machine code, working in a cable TV studio in the 1980s, and from an art history class in college where we discussed 19th C travel photos and poly/monochromatic film chemistries of the periods. Later I learned fluorescence quantification from a cell biologist intent on making sure we had true linear responses for readouts of f-actin mass and concentration, I was fortunate to attend both a Woods Hole microscopy course and at NCSU John Russ's course on image analysis, and BioRad MRC 600 training course at their offices in Cambridge, MA covered a lot of material on this too. So, do people have favorite texts that cover these topics in a manner digestible by new generations of students? Of biology students. Keep in mind many don't know what a byte is; when I teach I ask the class if anyone knows why an image is coded with white as 255 or 4095 or 16383 and typically one or two hands go up but the rest of the class really doesn't know. Looking forward to suggestions of easy to read definitive texts! Thank you. Cheers- ------------------------------------------------------------ This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email. ================================= |
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Lemasters, John J. |
Re: seeking "reliable literature" on correct imaging
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi Mike, I would like a copy of the PowerPoint as well. This is a very important issue that Michael Cammer raises. Trainees (and even more senior folks) continue to make these mistakes even as you emphatically tell them not to. Thanks again, John -- John J. Lemasters, MD, PhD Professor and GlaxoSmithKline Distinguished Endowed Chair Director, Center for Cell Death, Injury & Regeneration Departments of Drug Discovery & Biomedical Sciences and Biochemistry & Molecular Biology Medical University of South Carolina DD504 Drug Discovery Building 70 President Street, MSC 139 Charleston, SC 29425 Office: 843-876-2360 Lab: 843-876-2354 Fax: 843-876-2353 Email: [hidden email] http://academicdepartments.musc.edu/ccdir -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Kathryn Spencer Sent: Friday, November 16, 2018 12:32 PM To: [hidden email] Subject: Re: seeking "reliable literature" on correct imaging CAUTION: External ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi Mike; Could I get your PowerPoint as well? I have the same issues with my users. I'm "retraining" all of them with an open-software quiz, to see if they understand basic concepts. Many are complaining and reluctant, because they don't see the issues with their images. This after one 6-year post-doc's data was totally discarded because she saved everything as JPG....I certainly NEVER taught her to do THAT. Your PowerPoint should hold more credibility that I obviously have.... Best; Kathy -----Original Message----- From: Confocal Microscopy List <[hidden email]> On Behalf Of MODEL, MICHAEL Sent: Friday, November 16, 2018 9:00 AM To: [hidden email] Subject: Re: seeking "reliable literature" on correct imaging ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi Michael, We recently ran a workshop where I gave a 3-hour talk on various kinds of quantification in microscopy. I can send you a PowerPoint file but don't have a text. Mike Model -----Original Message----- From: Confocal Microscopy List <[hidden email]> On Behalf Of Cammer, Michael Sent: Friday, November 16, 2018 11:50 AM To: [hidden email] Subject: seeking "reliable literature" on correct imaging ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Increasingly we have found that microscope users do not understand proper imaging. A few examples: Confocal images are saturated and have the offset set to negative numbers intentionally "to reduce background". Exposures are changed in fluorescent micrographs to make each one "look good". 16 bit raw data with no saturation or clipping are set to 8 bits with a lot of saturated and clipped pixels at the extremes and these are what are considered raw data. JPG compression is great because images can be emailed. People assume (and get angry when this doesn't work) that computers can automatically segment and count anything regardless of experimental design, image quality, etc. Even if they have saved everything as TIF with no metadata and forgot what magnifications they used. A student who recognizes these problems and wants to learn the right way emailed me yesterday asking for "any reliable literature you recommend if I'd like to read more about correct imaging." I did Google searches and, after skimming a bunch of web pages, realized that I didn't really have an answer. I could send to this page and to this page, but it's not a coherent narrative. And I don't have a library; most of what I know about this was picked up willy-nilly up in the 1980s and '90s. For instance knowledge of color separation from writing animations for the Apple II in machine code, working in a cable TV studio in the 1980s, and from an art history class in college where we discussed 19th C travel photos and poly/monochromatic film chemistries of the periods. Later I learned fluorescence quantification from a cell biologist intent on making sure we had true linear responses for readouts of f-actin mass and concentration, I was fortunate to attend both a Woods Hole microscopy course and at NCSU John Russ's course on image analysis, and BioRad MRC 600 training course at their offices in Cambridge, MA covered a lot of material on this too. So, do people have favorite texts that cover these topics in a manner digestible by new generations of students? Of biology students. Keep in mind many don't know what a byte is; when I teach I ask the class if anyone knows why an image is coded with white as 255 or 4095 or 16383 and typically one or two hands go up but the rest of the class really doesn't know. Looking forward to suggestions of easy to read definitive texts! Thank you. Cheers- ------------------------------------------------------------ This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email. ================================= ------------------------------------------------------------------------- This message was secured via TLS by MUSC. |
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Julio Vazquez-2 |
Re: seeking "reliable literature" on correct imaging
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In reply to this post by kspencer007
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** For the basics, I used the book by John Russ (The Image Procesing Handbook), but this is quite expensive for students to purchase, and overkill, although the introductory chapters would be useful. I also found the following articles useful: Douglas Cromey: Digital Imaging Ethics (Google Digital Imaging Ethics), and references in there. Rossner and Yamada: What’s in a picture? The temptation of image manipulation J Cell Biology 166: 11-15 These papers will give some basic info about digital images and also point to the dangerous boundary between poor image acquisition/processing practices and data falsification. -- Julio Vazquez, PhD Director of Cellular Imaging (206) 667-1215 (206) 667-4205 Fred Hutchinson Cancer Research Center 1100 Fairview Ave.N., Mailstop DE-512 Seattle, WA 98109 On Nov 16, 2018, at 9:31 AM, Kathryn Spencer <[hidden email]<mailto:[hidden email]>> wrote: ***** To join, leave or search the confocal microscopy listserv, go to: https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIF-g&c=eRAMFD45gAfqt84VtBcfhQ&r=ttNYkfRwLB_uJJmzFu8cIdRdnibIA0UXGXDLv4MFuvU&m=P-6UuvOKGqAi_cIaRLp3TgvtgmGUOG7tRli3BkF00dA&s=5B3zF6g2x4sYF2f3fSIusceTEzZCgVhT_jwlWOoqYw0&e= Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIF-g&c=eRAMFD45gAfqt84VtBcfhQ&r=ttNYkfRwLB_uJJmzFu8cIdRdnibIA0UXGXDLv4MFuvU&m=P-6UuvOKGqAi_cIaRLp3TgvtgmGUOG7tRli3BkF00dA&s=7OeCl1ldMz036QeWxyEIq7Fd9FzFkyes_PqYy5R-PDY&e= and include the link in your posting. ***** Hi Mike; Could I get your PowerPoint as well? I have the same issues with my users. I'm "retraining" all of them with an open-software quiz, to see if they understand basic concepts. Many are complaining and reluctant, because they don't see the issues with their images. This after one 6-year post-doc's data was totally discarded because she saved everything as JPG....I certainly NEVER taught her to do THAT. Your PowerPoint should hold more credibility that I obviously have.... Best; Kathy -----Original Message----- From: Confocal Microscopy List <[hidden email]> On Behalf Of MODEL, MICHAEL Sent: Friday, November 16, 2018 9:00 AM To: [hidden email] Subject: Re: seeking "reliable literature" on correct imaging ***** To join, leave or search the confocal microscopy listserv, go to: https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIF-g&c=eRAMFD45gAfqt84VtBcfhQ&r=ttNYkfRwLB_uJJmzFu8cIdRdnibIA0UXGXDLv4MFuvU&m=P-6UuvOKGqAi_cIaRLp3TgvtgmGUOG7tRli3BkF00dA&s=5B3zF6g2x4sYF2f3fSIusceTEzZCgVhT_jwlWOoqYw0&e= Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIF-g&c=eRAMFD45gAfqt84VtBcfhQ&r=ttNYkfRwLB_uJJmzFu8cIdRdnibIA0UXGXDLv4MFuvU&m=P-6UuvOKGqAi_cIaRLp3TgvtgmGUOG7tRli3BkF00dA&s=7OeCl1ldMz036QeWxyEIq7Fd9FzFkyes_PqYy5R-PDY&e= and include the link in your posting. ***** Hi Michael, We recently ran a workshop where I gave a 3-hour talk on various kinds of quantification in microscopy. I can send you a PowerPoint file but don't have a text. Mike Model -----Original Message----- From: Confocal Microscopy List <[hidden email]> On Behalf Of Cammer, Michael Sent: Friday, November 16, 2018 11:50 AM To: [hidden email] Subject: seeking "reliable literature" on correct imaging ***** To join, leave or search the confocal microscopy listserv, go to: https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIF-g&c=eRAMFD45gAfqt84VtBcfhQ&r=ttNYkfRwLB_uJJmzFu8cIdRdnibIA0UXGXDLv4MFuvU&m=P-6UuvOKGqAi_cIaRLp3TgvtgmGUOG7tRli3BkF00dA&s=5B3zF6g2x4sYF2f3fSIusceTEzZCgVhT_jwlWOoqYw0&e= Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIF-g&c=eRAMFD45gAfqt84VtBcfhQ&r=ttNYkfRwLB_uJJmzFu8cIdRdnibIA0UXGXDLv4MFuvU&m=P-6UuvOKGqAi_cIaRLp3TgvtgmGUOG7tRli3BkF00dA&s=7OeCl1ldMz036QeWxyEIq7Fd9FzFkyes_PqYy5R-PDY&e= and include the link in your posting. ***** Increasingly we have found that microscope users do not understand proper imaging. A few examples: Confocal images are saturated and have the offset set to negative numbers intentionally "to reduce background". Exposures are changed in fluorescent micrographs to make each one "look good". 16 bit raw data with no saturation or clipping are set to 8 bits with a lot of saturated and clipped pixels at the extremes and these are what are considered raw data. JPG compression is great because images can be emailed. People assume (and get angry when this doesn't work) that computers can automatically segment and count anything regardless of experimental design, image quality, etc. Even if they have saved everything as TIF with no metadata and forgot what magnifications they used. A student who recognizes these problems and wants to learn the right way emailed me yesterday asking for "any reliable literature you recommend if I'd like to read more about correct imaging." I did Google searches and, after skimming a bunch of web pages, realized that I didn't really have an answer. I could send to this page and to this page, but it's not a coherent narrative. And I don't have a library; most of what I know about this was picked up willy-nilly up in the 1980s and '90s. For instance knowledge of color separation from writing animations for the Apple II in machine code, working in a cable TV studio in the 1980s, and from an art history class in college where we discussed 19th C travel photos and poly/monochromatic film chemistries of the periods. Later I learned fluorescence quantification from a cell biologist intent on making sure we had true linear responses for readouts of f-actin mass and concentration, I was fortunate to attend both a Woods Hole microscopy course and at NCSU John Russ's course on image analysis, and BioRad MRC 600 training course at their offices in Cambridge, MA covered a lot of material on this too. So, do people have favorite texts that cover these topics in a manner digestible by new generations of students? Of biology students. Keep in mind many don't know what a byte is; when I teach I ask the class if anyone knows why an image is coded with white as 255 or 4095 or 16383 and typically one or two hands go up but the rest of the class really doesn't know. Looking forward to suggestions of easy to read definitive texts! Thank you. Cheers- ------------------------------------------------------------ This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email. ================================= |
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John Oreopoulos |
Re: seeking "reliable literature" on correct imaging
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In reply to this post by Cammer, Michael
*****
To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Where to begin... Well, here are the most modern books I can think of off the top of my head, but there are other good ones out there. I have an entire library of "basic microscopy" articles collected over the years which I can share with you offline as well. But in terms of a total/coherent narrative... Start with this paper by Pawley. This will put the fear of changing exposure levels arbitrarily into anyone: https://www.future-science.com/doi/10.2144/00285bt01 Here are some good modern books on microscopy imaging: https://www.elsevier.com/books/digital-microscopy/sluder/978-0-12-407761-4 https://www.sciencedirect.com/bookseries/methods-in-cell-biology/vol/123 https://onlinelibrary.wiley.com/doi/book/10.1002/9781118382905 https://www.springer.com/gp/book/9780306484681 https://www.springer.com/gp/book/9780387259215 And in terms of digital imaging ethics, EVERYONE should read Cromey's article on this topic: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4114110/ John Oreopoulos Staff Scientist Andor Technology PLEASE NOTE OUR NEW ADDRESS: 250 West Beaver Creek Rd., Unit 1 Richmond Hill, Ontario Canada L4B 1C7 Tel: (905) 326-5040 ext. 412 Email: [hidden email] WWW site: www.andor.com Quoting "Cammer, Michael" <[hidden email]>: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Increasingly we have found that microscope users do not understand > proper imaging. > > A few examples: > Confocal images are saturated and have the offset set to negative > numbers intentionally "to reduce background". > Exposures are changed in fluorescent micrographs to make each one > "look good". > 16 bit raw data with no saturation or clipping are set to 8 bits > with a lot of saturated and clipped pixels at the extremes and these > are what are considered raw data. > JPG compression is great because images can be emailed. > People assume (and get angry when this doesn't work) that computers > can automatically segment and count anything regardless of > experimental design, image quality, etc. > Even if they have saved everything as TIF with no metadata and > forgot what magnifications they used. > > A student who recognizes these problems and wants to learn the right > way emailed me yesterday asking for "any reliable literature you > recommend if I'd like to read more about correct imaging." > > I did Google searches and, after skimming a bunch of web pages, > realized that I didn't really have an answer. I could send to this > page and to this page, but it's not a coherent narrative. And I > don't have a library; most of what I know about this was picked up > willy-nilly up in the 1980s and '90s. For instance knowledge of > color separation from writing animations for the Apple II in machine > code, working in a cable TV studio in the 1980s, and from an art > history class in college where we discussed 19th C travel photos and > poly/monochromatic film chemistries of the periods. Later I > learned fluorescence quantification from a cell biologist intent on > making sure we had true linear responses for readouts of f-actin > mass and concentration, I was fortunate to attend both a Woods Hole > microscopy course and at NCSU John Russ's course on image analysis, > and BioRad MRC 600 training course at their offices in Cambridge, > MA covered a lot of material on this too. > > So, do people have favorite texts that cover these topics in a > manner digestible by new generations of students? Of biology > students. Keep in mind many don't know what a byte is; when I teach > I ask the class if anyone knows why an image is coded with white as > 255 or 4095 or 16383 and typically one or two hands go up but the > rest of the class really doesn't know. > Looking forward to suggestions of easy to read definitive texts! > > Thank you. > > Cheers- > > > ------------------------------------------------------------ > This email message, including any attachments, is for the sole use > of the intended recipient(s) and may contain information that is > proprietary, confidential, and exempt from disclosure under > applicable law. Any unauthorized review, use, disclosure, or > distribution is prohibited. If you have received this email in error > please notify the sender by return email and delete the original > message. Please note, the recipient should check this email and any > attachments for the presence of viruses. The organization accepts no > liability for any damage caused by any virus transmitted by this > email. > ================================= > |
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Nancy Costigliola |
Re: seeking "reliable literature" on correct imaging
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In reply to this post by kspencer007
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi Mike, I am in industry, and would also really like to have a powerpoint on this for new users. Thanks! Nancy -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Kathryn Spencer Sent: Friday, November 16, 2018 12:32 PM To: [hidden email] Subject: Re: seeking "reliable literature" on correct imaging ***** To join, leave or search the confocal microscopy listserv, go to: https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIF-g&c=TzEZu9LIcihmW37vx9Ah6w&r=6yV8Jm8gBKxW3FelQyszarVcemf9nkXOKBO_nLK4fvg&m=Naz6rLjdo8BFyk0-vCP0btGPpFRLROu8ISI-YsK0KyA&s=DUKV1yCfWn9cWLDKFG46ViE1YatWxZ6e89zok-3__qE&e= Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIF-g&c=TzEZu9LIcihmW37vx9Ah6w&r=6yV8Jm8gBKxW3FelQyszarVcemf9nkXOKBO_nLK4fvg&m=Naz6rLjdo8BFyk0-vCP0btGPpFRLROu8ISI-YsK0KyA&s=BKJbIASgny5fT8vcyLqiawQJQnFihx7oIu9LGbadiMI&e= and include the link in your posting. ***** Hi Mike; Could I get your PowerPoint as well? I have the same issues with my users. I'm "retraining" all of them with an open-software quiz, to see if they understand basic concepts. Many are complaining and reluctant, because they don't see the issues with their images. This after one 6-year post-doc's data was totally discarded because she saved everything as JPG....I certainly NEVER taught her to do THAT. Your PowerPoint should hold more credibility that I obviously have.... Best; Kathy -----Original Message----- From: Confocal Microscopy List <[hidden email]> On Behalf Of MODEL, MICHAEL Sent: Friday, November 16, 2018 9:00 AM To: [hidden email] Subject: Re: seeking "reliable literature" on correct imaging ***** To join, leave or search the confocal microscopy listserv, go to: https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIF-g&c=TzEZu9LIcihmW37vx9Ah6w&r=6yV8Jm8gBKxW3FelQyszarVcemf9nkXOKBO_nLK4fvg&m=Naz6rLjdo8BFyk0-vCP0btGPpFRLROu8ISI-YsK0KyA&s=DUKV1yCfWn9cWLDKFG46ViE1YatWxZ6e89zok-3__qE&e= Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIF-g&c=TzEZu9LIcihmW37vx9Ah6w&r=6yV8Jm8gBKxW3FelQyszarVcemf9nkXOKBO_nLK4fvg&m=Naz6rLjdo8BFyk0-vCP0btGPpFRLROu8ISI-YsK0KyA&s=BKJbIASgny5fT8vcyLqiawQJQnFihx7oIu9LGbadiMI&e= and include the link in your posting. ***** Hi Michael, We recently ran a workshop where I gave a 3-hour talk on various kinds of quantification in microscopy. I can send you a PowerPoint file but don't have a text. Mike Model -----Original Message----- From: Confocal Microscopy List <[hidden email]> On Behalf Of Cammer, Michael Sent: Friday, November 16, 2018 11:50 AM To: [hidden email] Subject: seeking "reliable literature" on correct imaging ***** To join, leave or search the confocal microscopy listserv, go to: https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIF-g&c=TzEZu9LIcihmW37vx9Ah6w&r=6yV8Jm8gBKxW3FelQyszarVcemf9nkXOKBO_nLK4fvg&m=Naz6rLjdo8BFyk0-vCP0btGPpFRLROu8ISI-YsK0KyA&s=DUKV1yCfWn9cWLDKFG46ViE1YatWxZ6e89zok-3__qE&e= Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIF-g&c=TzEZu9LIcihmW37vx9Ah6w&r=6yV8Jm8gBKxW3FelQyszarVcemf9nkXOKBO_nLK4fvg&m=Naz6rLjdo8BFyk0-vCP0btGPpFRLROu8ISI-YsK0KyA&s=BKJbIASgny5fT8vcyLqiawQJQnFihx7oIu9LGbadiMI&e= and include the link in your posting. ***** Increasingly we have found that microscope users do not understand proper imaging. A few examples: Confocal images are saturated and have the offset set to negative numbers intentionally "to reduce background". Exposures are changed in fluorescent micrographs to make each one "look good". 16 bit raw data with no saturation or clipping are set to 8 bits with a lot of saturated and clipped pixels at the extremes and these are what are considered raw data. JPG compression is great because images can be emailed. People assume (and get angry when this doesn't work) that computers can automatically segment and count anything regardless of experimental design, image quality, etc. Even if they have saved everything as TIF with no metadata and forgot what magnifications they used. A student who recognizes these problems and wants to learn the right way emailed me yesterday asking for "any reliable literature you recommend if I'd like to read more about correct imaging." I did Google searches and, after skimming a bunch of web pages, realized that I didn't really have an answer. I could send to this page and to this page, but it's not a coherent narrative. And I don't have a library; most of what I know about this was picked up willy-nilly up in the 1980s and '90s. For instance knowledge of color separation from writing animations for the Apple II in machine code, working in a cable TV studio in the 1980s, and from an art history class in college where we discussed 19th C travel photos and poly/monochromatic film chemistries of the periods. Later I learned fluorescence quantification from a cell biologist intent on making sure we had true linear responses for readouts of f-actin mass and concentration, I was fortunate to attend both a Woods Hole microscopy course and at NCSU John Russ's course on image analysis, and BioRad MRC 600 training course at their offices in Cambridge, MA covered a lot of material on this too. So, do people have favorite texts that cover these topics in a manner digestible by new generations of students? Of biology students. Keep in mind many don't know what a byte is; when I teach I ask the class if anyone knows why an image is coded with white as 255 or 4095 or 16383 and typically one or two hands go up but the rest of the class really doesn't know. Looking forward to suggestions of easy to read definitive texts! Thank you. Cheers- ------------------------------------------------------------ This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email. ================================= This email message and any attachments are confidential and intended for use by the addressee(s) only. If you are not the intended recipient, please notify me immediately by replying to this message, and destroy all copies of this message and any attachments. Thank you. |
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Oshel, Philip Eugene |
Re: seeking "reliable literature" on correct imaging
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In reply to this post by kspencer007
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** I would also like a copy of your powerpoint slides Michael. They would be useful in our classes. A couple of articles we've used: What’s in a picture? The temptation of image manipulation. Mike Rossner and Kenneth M. Yamada The Journal of Cell Biology, Volume 166, Number 1, July 5, 2004 11–15 and Avoiding Twisted Pixels: Ethical Guidelines for the Appropriate Use and Manipulation of Scientific Digital Images. Douglas W. Cromey Science and Engineering Ethics December 2010, Volume 16, Issue 4, pp 639–667 John Russ's Image Processing Handbook is also useful, but probably too expense to have as anything but one copy for a lab reference. There are other imaging books as well. The Microscopy Society of America's statement on digital imaging: "The MSA position on digital image processing has been approved as follows: "Ethical digital imaging requires that the original uncompressed image file be stored on archival media (e.g., CD-R) without any image manipulation or processing operation. All parameters of the production and acquisition of this file, as well as any subsequent processing steps, must be documented and reported to ensure reproducibility. Generally, acceptable (non-reportable) imaging operations include gamma correction, histogram stretching, and brightness and contrast adjustments. All other operations (such as Unsharp-Masking, Gaussian Blur, etc.) must be directly identified by the author as part of the experimental methodology. However, for diffraction data or any other image data that is used for subsequent quantification, all imaging operations must be reported." This policy was formulated by the Digital Image Processing & Ethics Group of the MSA Education Committee and was adopted as MSA policy at the Summer Council meeting August 2-3, 2003." https://www.microscopy.org/resources/digital_imaging.cfm This hasn't changed since implemented. As for what file format ... TIFF. Other lossless image format is also OK, and many microscope companys' proprietary formats are really TIFF images with extra stuff in the header. Like Nikon's ND2 format for Elements. Saving as TIFF (or other lossless format) you just have to pound into people's heads Phil ------------- Philip Oshel Imaging Facility Director Biology Department 1304 Biosciences 1455 Calumet Ct. Central Michigan University Mt. Pleasant, MI 48859 989 774-3576 office 989 774-7567 lab -----Original Message----- From: Confocal Microscopy List <[hidden email]> on behalf of Kathryn Spencer <[hidden email]> Reply-To: Confocal Microscopy List <[hidden email]> Date: Friday, 16November, 2018 at 12:32 To: "[hidden email]" <[hidden email]> Subject: Re: seeking "reliable literature" on correct imaging ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi Mike; Could I get your PowerPoint as well? I have the same issues with my users. I'm "retraining" all of them with an open-software quiz, to see if they understand basic concepts. Many are complaining and reluctant, because they don't see the issues with their images. This after one 6-year post-doc's data was totally discarded because she saved everything as JPG....I certainly NEVER taught her to do THAT. Your PowerPoint should hold more credibility that I obviously have.... Best; Kathy -----Original Message----- From: Confocal Microscopy List <[hidden email]> On Behalf Of MODEL, MICHAEL Sent: Friday, November 16, 2018 9:00 AM To: [hidden email] Subject: Re: seeking "reliable literature" on correct imaging ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi Michael, We recently ran a workshop where I gave a 3-hour talk on various kinds of quantification in microscopy. I can send you a PowerPoint file but don't have a text. Mike Model -----Original Message----- From: Confocal Microscopy List <[hidden email]> On Behalf Of Cammer, Michael Sent: Friday, November 16, 2018 11:50 AM To: [hidden email] Subject: seeking "reliable literature" on correct imaging ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Increasingly we have found that microscope users do not understand proper imaging. A few examples: Confocal images are saturated and have the offset set to negative numbers intentionally "to reduce background". Exposures are changed in fluorescent micrographs to make each one "look good". 16 bit raw data with no saturation or clipping are set to 8 bits with a lot of saturated and clipped pixels at the extremes and these are what are considered raw data. JPG compression is great because images can be emailed. People assume (and get angry when this doesn't work) that computers can automatically segment and count anything regardless of experimental design, image quality, etc. Even if they have saved everything as TIF with no metadata and forgot what magnifications they used. A student who recognizes these problems and wants to learn the right way emailed me yesterday asking for "any reliable literature you recommend if I'd like to read more about correct imaging." I did Google searches and, after skimming a bunch of web pages, realized that I didn't really have an answer. I could send to this page and to this page, but it's not a coherent narrative. And I don't have a library; most of what I know about this was picked up willy-nilly up in the 1980s and '90s. For instance knowledge of color separation from writing animations for the Apple II in machine code, working in a cable TV studio in the 1980s, and from an art history class in college where we discussed 19th C travel photos and poly/monochromatic film chemistries of the periods. Later I learned fluorescence quantification from a cell biologist intent on making sure we had true linear responses for readouts of f-actin mass and concentration, I was fortunate to attend both a Woods Hole microscopy course and at NCSU John Russ's course on image analysis, and BioRad MRC 600 training course at their offices in Cambridge, MA covered a lot of material on this too. So, do people have favorite texts that cover these topics in a manner digestible by new generations of students? Of biology students. Keep in mind many don't know what a byte is; when I teach I ask the class if anyone knows why an image is coded with white as 255 or 4095 or 16383 and typically one or two hands go up but the rest of the class really doesn't know. Looking forward to suggestions of easy to read definitive texts! Thank you. Cheers- ------------------------------------------------------------ This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email. ================================= |
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Adeela Syed |
Re: seeking "reliable literature" on correct imaging
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In reply to this post by mmodel
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hello Mike, I would like as copy of the PowerPoint as well, this would be very helpful for our confocal core users! Kind regards, Adeela -- Adeela Syed, Ph.D. Associate Project Scientist Optical Biology Facility Manager Dept. Developmental and Cell Biology University of California Irvine Irvine, CA 92697-2300 949-824-3856 email: [hidden email] obc.bio.uci.edu -----Original Message----- From: Confocal Microscopy List <[hidden email]> On Behalf Of MODEL, MICHAEL Sent: Friday, November 16, 2018 9:00 AM To: [hidden email] Subject: Re: seeking "reliable literature" on correct imaging ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi Michael, We recently ran a workshop where I gave a 3-hour talk on various kinds of quantification in microscopy. I can send you a PowerPoint file but don't have a text. Mike Model -----Original Message----- From: Confocal Microscopy List <[hidden email]> On Behalf Of Cammer, Michael Sent: Friday, November 16, 2018 11:50 AM To: [hidden email] Subject: seeking "reliable literature" on correct imaging ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Increasingly we have found that microscope users do not understand proper imaging. A few examples: Confocal images are saturated and have the offset set to negative numbers intentionally "to reduce background". Exposures are changed in fluorescent micrographs to make each one "look good". 16 bit raw data with no saturation or clipping are set to 8 bits with a lot of saturated and clipped pixels at the extremes and these are what are considered raw data. JPG compression is great because images can be emailed. People assume (and get angry when this doesn't work) that computers can automatically segment and count anything regardless of experimental design, image quality, etc. Even if they have saved everything as TIF with no metadata and forgot what magnifications they used. A student who recognizes these problems and wants to learn the right way emailed me yesterday asking for "any reliable literature you recommend if I'd like to read more about correct imaging." I did Google searches and, after skimming a bunch of web pages, realized that I didn't really have an answer. I could send to this page and to this page, but it's not a coherent narrative. And I don't have a library; most of what I know about this was picked up willy-nilly up in the 1980s and '90s. For instance knowledge of color separation from writing animations for the Apple II in machine code, working in a cable TV studio in the 1980s, and from an art history class in college where we discussed 19th C travel photos and poly/monochromatic film chemistries of the periods. Later I learned fluorescence quantification from a cell biologist intent on making sure we had true linear responses for readouts of f-actin mass and concentration, I was fortunate to attend both a Woods Hole microscopy course and at NCSU John Russ's course on image analysis, and BioRad MRC 600 training course at their offices in Cambridge, MA covered a lot of material on this too. So, do people have favorite texts that cover these topics in a manner digestible by new generations of students? Of biology students. Keep in mind many don't know what a byte is; when I teach I ask the class if anyone knows why an image is coded with white as 255 or 4095 or 16383 and typically one or two hands go up but the rest of the class really doesn't know. Looking forward to suggestions of easy to read definitive texts! Thank you. Cheers- ------------------------------------------------------------ This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email. ================================= |
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Carol Heckman |
Re: seeking "reliable literature" on correct imaging
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In reply to this post by Cammer, Michael
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** It's actually worse than this. A 16-bit image is stored, high bits and low bits, in different order in different formats. The best way to be sure you have the same thing when translated into a new software suite is to compare the histograms. This gets tedious if you have a lot of images to be processed.... Carol Heckman Center for Microscopy & Microanalysis Bowling Green State University ________________________________ From: Confocal Microscopy List <[hidden email]> on behalf of Cammer, Michael <[hidden email]> Sent: Friday, November 16, 2018 11:50 AM To: [hidden email] Subject: seeking "reliable literature" on correct imaging ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy LISTSERV 16.0 - CONFOCALMICROSCOPY List at LISTS.UMN.EDU - Listserv Lists at the University of Minnesota<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> lists.umn.edu [hidden email]: listserv archives. confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Increasingly we have found that microscope users do not understand proper imaging. A few examples: Confocal images are saturated and have the offset set to negative numbers intentionally "to reduce background". Exposures are changed in fluorescent micrographs to make each one "look good". 16 bit raw data with no saturation or clipping are set to 8 bits with a lot of saturated and clipped pixels at the extremes and these are what are considered raw data. JPG compression is great because images can be emailed. People assume (and get angry when this doesn't work) that computers can automatically segment and count anything regardless of experimental design, image quality, etc. Even if they have saved everything as TIF with no metadata and forgot what magnifications they used. A student who recognizes these problems and wants to learn the right way emailed me yesterday asking for "any reliable literature you recommend if I'd like to read more about correct imaging." I did Google searches and, after skimming a bunch of web pages, realized that I didn't really have an answer. I could send to this page and to this page, but it's not a coherent narrative. And I don't have a library; most of what I know about this was picked up willy-nilly up in the 1980s and '90s. For instance knowledge of color separation from writing animations for the Apple II in machine code, working in a cable TV studio in the 1980s, and from an art history class in college where we discussed 19th C travel photos and poly/monochromatic film chemistries of the periods. Later I learned fluorescence quantification from a cell biologist intent on making sure we had true linear responses for readouts of f-actin mass and concentration, I was fortunate to attend both a Woods Hole microscopy course and at NCSU John Russ's course on image analysis, and BioRad MRC 600 training course at their offices in Cambridge, MA covered a lot of material on this too. So, do people have favorite texts that cover these topics in a manner digestible by new generations of students? Of biology students. Keep in mind many don't know what a byte is; when I teach I ask the class if anyone knows why an image is coded with white as 255 or 4095 or 16383 and typically one or two hands go up but the rest of the class really doesn't know. Looking forward to suggestions of easy to read definitive texts! Thank you. Cheers- ------------------------------------------------------------ This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email. ================================= |
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Ben Abrams |
Re: seeking "reliable literature" on correct imaging
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In reply to this post by Cammer, Michael
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi Michael, I routinely cite "Fundamentals of Light Microscopy and Electronic Imaging" by Douglas Murphy and Michael Davidson when I lecture. It covers a broad range of topics and is accessible. -Ben On 11/16/18 8:50 AM, Cammer, Michael wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Increasingly we have found that microscope users do not understand proper imaging. > > A few examples: > Confocal images are saturated and have the offset set to negative numbers intentionally "to reduce background". > Exposures are changed in fluorescent micrographs to make each one "look good". > 16 bit raw data with no saturation or clipping are set to 8 bits with a lot of saturated and clipped pixels at the extremes and these are what are considered raw data. > JPG compression is great because images can be emailed. > People assume (and get angry when this doesn't work) that computers can automatically segment and count anything regardless of experimental design, image quality, etc. > Even if they have saved everything as TIF with no metadata and forgot what magnifications they used. > > A student who recognizes these problems and wants to learn the right way emailed me yesterday asking for "any reliable literature you recommend if I'd like to read more about correct imaging." > > I did Google searches and, after skimming a bunch of web pages, realized that I didn't really have an answer. I could send to this page and to this page, but it's not a coherent narrative. And I don't have a library; most of what I know about this was picked up willy-nilly up in the 1980s and '90s. For instance knowledge of color separation from writing animations for the Apple II in machine code, working in a cable TV studio in the 1980s, and from an art history class in college where we discussed 19th C travel photos and poly/monochromatic film chemistries of the periods. Later I learned fluorescence quantification from a cell biologist intent on making sure we had true linear responses for readouts of f-actin mass and concentration, I was fortunate to attend both a Woods Hole microscopy course and at NCSU John Russ's course on image analysis, and BioRad MRC 600 training course at their offices in Cambridge, MA covered a lot of material on this too. > > So, do people have favorite texts that cover these topics in a manner digestible by new generations of students? Of biology students. Keep in mind many don't know what a byte is; when I teach I ask the class if anyone knows why an image is coded with white as 255 or 4095 or 16383 and typically one or two hands go up but the rest of the class really doesn't know. > Looking forward to suggestions of easy to read definitive texts! > > Thank you. > > Cheers- > > > ------------------------------------------------------------ > This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email. > ================================= -- Benjamin Abrams, Ph.D. Facility Manager UCSC Life Sciences Microscopy Center University of California, Santa Cruz 1156 High Street 150 Sinsheimer labs Santa Cruz, CA 95064 Office/voicemail: (831) 459-3999 Mobile: (831) 332-0911 http://stemcell.soe.ucsc.edu/facilities/microscopy Training Request Form: http://goo.gl/forms/M9jd1ZHyohTnj0xk1 |
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Glen MacDonald |
Re: seeking "reliable literature" on correct imaging
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In reply to this post by John Oreopoulos
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** thanks for posting this excellent list John. the ethics articles are helpful, but I think most bad images are made by not understanding how an image is created as well as what constitutes a digital image. And all the points from sample preparation to quantitation/publication where the informational content may be compromised. FWIW, Alan Hibb’s book is dated but the information remains quite useful. Before his premature death, he had shipped some books to me in preparation of a visit to North America. I thought all were sold, but I just found one more copy while cleaning my study. Original price was US$125, I will sell it for half price plus shipping. Proceeds are remitted to his family. regards, Glen MacDonald > On Nov 16, 2018, at 9:54 AM, [hidden email] wrote: > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Where to begin... Well, here are the most modern books I can think of off the top of my head, but there are other good ones out there. I have an entire library of "basic microscopy" articles collected over the years which I can share with you offline as well. But in terms of a total/coherent narrative... > > Start with this paper by Pawley. This will put the fear of changing exposure levels arbitrarily into anyone: > > https://www.future-science.com/doi/10.2144/00285bt01 > > > Here are some good modern books on microscopy imaging: > > https://www.elsevier.com/books/digital-microscopy/sluder/978-0-12-407761-4 > > https://www.sciencedirect.com/bookseries/methods-in-cell-biology/vol/123 > > https://onlinelibrary.wiley.com/doi/book/10.1002/9781118382905 > > https://www.springer.com/gp/book/9780306484681 > > https://www.springer.com/gp/book/9780387259215 > > > And in terms of digital imaging ethics, EVERYONE should read Cromey's article on this topic: > > https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4114110/ > > > John Oreopoulos > Staff Scientist > Andor Technology > > PLEASE NOTE OUR NEW ADDRESS: > > 250 West Beaver Creek Rd., Unit 1 > Richmond Hill, Ontario Canada L4B 1C7 > Tel: (905) 326-5040 ext. 412 > Email: [hidden email] > WWW site: www.andor.com > > Quoting "Cammer, Michael" <[hidden email]>: > >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> Post images on http://www.imgur.com and include the link in your posting. >> ***** >> >> Increasingly we have found that microscope users do not understand proper imaging. >> >> A few examples: >> Confocal images are saturated and have the offset set to negative numbers intentionally "to reduce background". >> Exposures are changed in fluorescent micrographs to make each one "look good". >> 16 bit raw data with no saturation or clipping are set to 8 bits with a lot of saturated and clipped pixels at the extremes and these are what are considered raw data. >> JPG compression is great because images can be emailed. >> People assume (and get angry when this doesn't work) that computers can automatically segment and count anything regardless of experimental design, image quality, etc. >> Even if they have saved everything as TIF with no metadata and forgot what magnifications they used. >> >> A student who recognizes these problems and wants to learn the right way emailed me yesterday asking for "any reliable literature you recommend if I'd like to read more about correct imaging." >> >> I did Google searches and, after skimming a bunch of web pages, realized that I didn't really have an answer. I could send to this page and to this page, but it's not a coherent narrative. And I don't have a library; most of what I know about this was picked up willy-nilly up in the 1980s and '90s. For instance knowledge of color separation from writing animations for the Apple II in machine code, working in a cable TV studio in the 1980s, and from an art history class in college where we discussed 19th C travel photos and poly/monochromatic film chemistries of the periods. Later I learned fluorescence quantification from a cell biologist intent on making sure we had true linear responses for readouts of f-actin mass and concentration, I was fortunate to attend both a Woods Hole microscopy course and at NCSU John Russ's course on image analysis, and BioRad MRC 600 training course at their offices in Cambridge, MA covered a lot of material on this too. >> >> So, do people have favorite texts that cover these topics in a manner digestible by new generations of students? Of biology students. Keep in mind many don't know what a byte is; when I teach I ask the class if anyone knows why an image is coded with white as 255 or 4095 or 16383 and typically one or two hands go up but the rest of the class really doesn't know. >> Looking forward to suggestions of easy to read definitive texts! >> >> Thank you. >> >> Cheers- >> >> >> ------------------------------------------------------------ >> This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email. >> ================================= >> |
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Cromey, Douglas W - (dcromey) |
Re: seeking "reliable literature" on correct imaging
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** We see the same problems here. Once a year, right before the fall semester starts, we do a half-day workshop (http://microscopy.arizona.edu/dig_image_workshop/index) on scientific digital images. We start with the basics and work our way up through bit depth, color, file types, ethics and some hands-on demos in Photoshop & ImageJ. We advertise widely, and pull attendees from almost all the science disciplines and engineering (sometimes other colleges as well). Attendees range from undergrads to senior faculty. In the post-workshop evaluations we get a range of responses from complaints about wasting time covering the basics, people wishing there was more in-depth information on a particular topic, and the occasional (and rewarding) "I learned so much! Thank you". The workshop doesn't solve every problem, but I regularly meet people who remember a few key concepts and come to us questions about what they should do. I have often been inspired by David Piston's commentary in Nature from a few years ago, that I need to help people understand how things work (https://www.nature.com/articles/484440a). Sadly, in these days of "kit biology", the ability to thoughtfully solve problems and learn something new along the way seems to be disappearing. Doug FYI: http://microscopy.arizona.edu/learn/digital-image-ethics (The workshop wasn't all my idea, a tip of the hat to colleagues Carl Boswell, Chip Hedgecock, David Elliott, Brooke Massani, and Ben Cromey who have all added significantly to the success of the workshop over the 10+ years we have been doing it here at the University of Arizona. It all started when a couple of microscopy core managers got together for lunch...) ------------------------------------------------------------------------------------------ Douglas W. Cromey, M.S. - Associate Scientific Investigator Dept. of Cellular & Molecular Medicine, University of Arizona 1501 N. Campbell Ave, Tucson, AZ 85724-5044 USA office: LSN 463 email: [hidden email] voice: 520-626-2824 fax: 520-626-2097 http://microscopy.arizona.edu/learn/microscopy-imaging-resources-www Home of: "Microscopy and Imaging Resources on the WWW" UA Microscopy Alliance - http://microscopy.arizona.edu -----Original Message----- From: Confocal Microscopy List <[hidden email]> On Behalf Of Glen MacDonald Sent: Friday, November 16, 2018 12:56 PM To: [hidden email] Subject: Re: seeking "reliable literature" on correct imaging ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** thanks for posting this excellent list John. the ethics articles are helpful, but I think most bad images are made by not understanding how an image is created as well as what constitutes a digital image. And all the points from sample preparation to quantitation/publication where the informational content may be compromised. FWIW, Alan Hibb’s book is dated but the information remains quite useful. Before his premature death, he had shipped some books to me in preparation of a visit to North America. I thought all were sold, but I just found one more copy while cleaning my study. Original price was US$125, I will sell it for half price plus shipping. Proceeds are remitted to his family. regards, Glen MacDonald > On Nov 16, 2018, at 9:54 AM, [hidden email] wrote: > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Where to begin... Well, here are the most modern books I can think of off the top of my head, but there are other good ones out there. I have an entire library of "basic microscopy" articles collected over the years which I can share with you offline as well. But in terms of a total/coherent narrative... > > Start with this paper by Pawley. This will put the fear of changing exposure levels arbitrarily into anyone: > > https://www.future-science.com/doi/10.2144/00285bt01 > > > Here are some good modern books on microscopy imaging: > > https://www.elsevier.com/books/digital-microscopy/sluder/978-0-12-407761-4 > > https://www.sciencedirect.com/bookseries/methods-in-cell-biology/vol/123 > > https://onlinelibrary.wiley.com/doi/book/10.1002/9781118382905 > > https://www.springer.com/gp/book/9780306484681 > > https://www.springer.com/gp/book/9780387259215 > > > And in terms of digital imaging ethics, EVERYONE should read Cromey's article on this topic: > > https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4114110/ > > > John Oreopoulos > Staff Scientist > Andor Technology > > PLEASE NOTE OUR NEW ADDRESS: > > 250 West Beaver Creek Rd., Unit 1 > Richmond Hill, Ontario Canada L4B 1C7 > Tel: (905) 326-5040 ext. 412 > Email: [hidden email] > WWW site: www.andor.com > > Quoting "Cammer, Michael" <[hidden email]>: > >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> Post images on http://www.imgur.com and include the link in your posting. >> ***** >> >> Increasingly we have found that microscope users do not understand proper imaging. >> >> A few examples: >> Confocal images are saturated and have the offset set to negative numbers intentionally "to reduce background". >> Exposures are changed in fluorescent micrographs to make each one "look good". >> 16 bit raw data with no saturation or clipping are set to 8 bits with a lot of saturated and clipped pixels at the extremes and these are what are considered raw data. >> JPG compression is great because images can be emailed. >> People assume (and get angry when this doesn't work) that computers can automatically segment and count anything regardless of experimental design, image quality, etc. >> Even if they have saved everything as TIF with no metadata and forgot what magnifications they used. >> >> A student who recognizes these problems and wants to learn the right way emailed me yesterday asking for "any reliable literature you recommend if I'd like to read more about correct imaging." >> >> I did Google searches and, after skimming a bunch of web pages, realized that I didn't really have an answer. I could send to this page and to this page, but it's not a coherent narrative. And I don't have a library; most of what I know about this was picked up willy-nilly up in the 1980s and '90s. For instance knowledge of color separation from writing animations for the Apple II in machine code, working in a cable TV studio in the 1980s, and from an art history class in college where we discussed 19th C travel photos and poly/monochromatic film chemistries of the periods. Later I learned fluorescence quantification from a cell biologist intent on making sure we had true linear responses for readouts of f-actin mass and concentration, I was fortunate to attend both a Woods Hole microscopy course and at NCSU John Russ's course on image analysis, and BioRad MRC 600 training course at their offices in Cambridge, MA covered a lot of material on this too. >> >> So, do people have favorite texts that cover these topics in a manner digestible by new generations of students? Of biology students. Keep in mind many don't know what a byte is; when I teach I ask the class if anyone knows why an image is coded with white as 255 or 4095 or 16383 and typically one or two hands go up but the rest of the class really doesn't know. >> Looking forward to suggestions of easy to read definitive texts! >> >> Thank you. >> >> Cheers- >> >> >> ------------------------------------------------------------ >> This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email. >> ================================= >> |
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Dr. K N Ganesh |
Re: seeking "reliable literature" on correct imaging
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In reply to this post by Lemasters, John J.
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi I do visit various institutions to teach basics on microscopy and imaging always struggle convey the right message in the absence of a proper material.. Your presentation may be really a good tool for me to convey correct message and make the next generation scientist fool proof.. Kindly provide me the PPT .. Reg Ganesh Kadasoor Bangalore India Sent from my iPhone > On 16-Nov-2018, at 11:06 PM, Lemasters, John J. <[hidden email]> wrote: > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Hi Mike, > > I would like a copy of the PowerPoint as well. This is a very important issue that Michael Cammer raises. Trainees (and even more senior folks) continue to make these mistakes even as you emphatically tell them not to. > > Thanks again, > > John > > -- > John J. Lemasters, MD, PhD > Professor and GlaxoSmithKline Distinguished Endowed Chair > Director, Center for Cell Death, Injury & Regeneration > Departments of Drug Discovery & Biomedical Sciences and Biochemistry & Molecular Biology > Medical University of South Carolina > DD504 Drug Discovery Building > 70 President Street, MSC 139 > Charleston, SC 29425 > > Office: 843-876-2360 > Lab: 843-876-2354 > Fax: 843-876-2353 > Email: [hidden email] > http://academicdepartments.musc.edu/ccdir > > -----Original Message----- > From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Kathryn Spencer > Sent: Friday, November 16, 2018 12:32 PM > To: [hidden email] > Subject: Re: seeking "reliable literature" on correct imaging > > CAUTION: External > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Hi Mike; > Could I get your PowerPoint as well? I have the same issues with my users. I'm "retraining" all of them with an open-software quiz, to see if they understand basic concepts. Many are complaining and reluctant, because they don't see the issues with their images. This after one 6-year post-doc's data was totally discarded because she saved everything as JPG....I certainly NEVER taught her to do THAT. > Your PowerPoint should hold more credibility that I obviously have.... > Best; > Kathy > > > > -----Original Message----- > From: Confocal Microscopy List <[hidden email]> On Behalf Of MODEL, MICHAEL > Sent: Friday, November 16, 2018 9:00 AM > To: [hidden email] > Subject: Re: seeking "reliable literature" on correct imaging > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Hi Michael, > > We recently ran a workshop where I gave a 3-hour talk on various kinds of quantification in microscopy. I can send you a PowerPoint file but don't have a text. > > Mike Model > > -----Original Message----- > From: Confocal Microscopy List <[hidden email]> On Behalf Of Cammer, Michael > Sent: Friday, November 16, 2018 11:50 AM > To: [hidden email] > Subject: seeking "reliable literature" on correct imaging > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Increasingly we have found that microscope users do not understand proper imaging. > > A few examples: > Confocal images are saturated and have the offset set to negative numbers intentionally "to reduce background". > Exposures are changed in fluorescent micrographs to make each one "look good". > 16 bit raw data with no saturation or clipping are set to 8 bits with a lot of saturated and clipped pixels at the extremes and these are what are considered raw data. > JPG compression is great because images can be emailed. > People assume (and get angry when this doesn't work) that computers can automatically segment and count anything regardless of experimental design, image quality, etc. > Even if they have saved everything as TIF with no metadata and forgot what magnifications they used. > > A student who recognizes these problems and wants to learn the right way emailed me yesterday asking for "any reliable literature you recommend if I'd like to read more about correct imaging." > > I did Google searches and, after skimming a bunch of web pages, realized that I didn't really have an answer. I could send to this page and to this page, but it's not a coherent narrative. And I don't have a library; most of what I know about this was picked up willy-nilly up in the 1980s and '90s. For instance knowledge of color separation from writing animations for the Apple II in machine code, working in a cable TV studio in the 1980s, and from an art history class in college where we discussed 19th C travel photos and poly/monochromatic film chemistries of the periods. Later I learned fluorescence quantification from a cell biologist intent on making sure we had true linear responses for readouts of f-actin mass and concentration, I was fortunate to attend both a Woods Hole microscopy course and at NCSU John Russ's course on image analysis, and BioRad MRC 600 training course at their offices in Cambridge, MA covered a lot of material on this too. > > So, do people have favorite texts that cover these topics in a manner digestible by new generations of students? Of biology students. Keep in mind many don't know what a byte is; when I teach I ask the class if anyone knows why an image is coded with white as 255 or 4095 or 16383 and typically one or two hands go up but the rest of the class really doesn't know. > Looking forward to suggestions of easy to read definitive texts! > > Thank you. > > Cheers- > > > ------------------------------------------------------------ > This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email. > ================================= > > > > > ------------------------------------------------------------------------- > This message was secured via TLS by MUSC. |
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Karissa Tilbury |
Re: seeking "reliable literature" on correct imaging
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*****
To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Mike, I too would also appreciate this PowerPoint. I think in the context of the ethics thread, this is very interesting and useful conversation. My students are currently in the mandated ethics class and the topics discussed are crazy extremes that really have their roots in 'smaller' less obvious mistakes to most people. Focusing the discussion on what they are doing in the lab to protect the quality of data or understand limitations seems more important to me, than discussing ethics of CRISPR and human life. Karissa On Fri, Nov 16, 2018, 8:44 PM Ganesh Kadasoor <[hidden email] wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Hi > I do visit various institutions to teach basics on microscopy and imaging > always struggle convey the right message in the absence of a proper > material.. > Your presentation may be really a good tool for me to convey correct > message and make the next generation scientist fool proof.. > Kindly provide me the PPT .. > Reg > Ganesh Kadasoor > Bangalore > India > > > Sent from my iPhone > > > On 16-Nov-2018, at 11:06 PM, Lemasters, John J. <[hidden email]> > wrote: > > > > ***** > > To join, leave or search the confocal microscopy listserv, go to: > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > > Post images on http://www.imgur.com and include the link in your > posting. > > ***** > > > > Hi Mike, > > > > I would like a copy of the PowerPoint as well. This is a very important > issue that Michael Cammer raises. Trainees (and even more senior folks) > continue to make these mistakes even as you emphatically tell them not to. > > > > Thanks again, > > > > John > > > > -- > > John J. Lemasters, MD, PhD > > Professor and GlaxoSmithKline Distinguished Endowed Chair > > Director, Center for Cell Death, Injury & Regeneration > > Departments of Drug Discovery & Biomedical Sciences and Biochemistry & > Molecular Biology > > Medical University of South Carolina > > DD504 Drug Discovery Building > > 70 President Street, MSC 139 > > Charleston, SC 29425 > > > > Office: 843-876-2360 > > Lab: 843-876-2354 > > Fax: 843-876-2353 > > Email: [hidden email] > > http://academicdepartments.musc.edu/ccdir > > > > -----Original Message----- > > From: Confocal Microscopy List [mailto:[hidden email]] > On Behalf Of Kathryn Spencer > > Sent: Friday, November 16, 2018 12:32 PM > > To: [hidden email] > > Subject: Re: seeking "reliable literature" on correct imaging > > > > CAUTION: External > > > > ***** > > To join, leave or search the confocal microscopy listserv, go to: > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > > Post images on http://www.imgur.com and include the link in your > posting. > > ***** > > > > Hi Mike; > > Could I get your PowerPoint as well? I have the same issues with > my users. I'm "retraining" all of them with an open-software quiz, to see > if they understand basic concepts. Many are complaining and reluctant, > because they don't see the issues with their images. This after one 6-year > post-doc's data was totally discarded because she saved everything as > JPG....I certainly NEVER taught her to do THAT. > > Your PowerPoint should hold more credibility that I obviously > have.... > > Best; > > Kathy > > > > > > > > -----Original Message----- > > From: Confocal Microscopy List <[hidden email]> On > Behalf Of MODEL, MICHAEL > > Sent: Friday, November 16, 2018 9:00 AM > > To: [hidden email] > > Subject: Re: seeking "reliable literature" on correct imaging > > > > ***** > > To join, leave or search the confocal microscopy listserv, go to: > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > > Post images on http://www.imgur.com and include the link in your > posting. > > ***** > > > > Hi Michael, > > > > We recently ran a workshop where I gave a 3-hour talk on various kinds > of quantification in microscopy. I can send you a PowerPoint file but don't > have a text. > > > > Mike Model > > > > -----Original Message----- > > From: Confocal Microscopy List <[hidden email]> On > Behalf Of Cammer, Michael > > Sent: Friday, November 16, 2018 11:50 AM > > To: [hidden email] > > Subject: seeking "reliable literature" on correct imaging > > > > ***** > > To join, leave or search the confocal microscopy listserv, go to: > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > > Post images on http://www.imgur.com and include the link in your > posting. > > ***** > > > > Increasingly we have found that microscope users do not understand > proper imaging. > > > > A few examples: > > Confocal images are saturated and have the offset set to negative > numbers intentionally "to reduce background". > > Exposures are changed in fluorescent micrographs to make each one "look > good". > > 16 bit raw data with no saturation or clipping are set to 8 bits with a > lot of saturated and clipped pixels at the extremes and these are what are > considered raw data. > > JPG compression is great because images can be emailed. > > People assume (and get angry when this doesn't work) that computers can > automatically segment and count anything regardless of experimental design, > image quality, etc. > > Even if they have saved everything as TIF with no metadata and forgot > what magnifications they used. > > > > A student who recognizes these problems and wants to learn the right way > emailed me yesterday asking for "any reliable literature you recommend if > I'd like to read more about correct imaging." > > > > I did Google searches and, after skimming a bunch of web pages, realized > that I didn't really have an answer. I could send to this page and to this > page, but it's not a coherent narrative. And I don't have a library; most > of what I know about this was picked up willy-nilly up in the 1980s and > '90s. For instance knowledge of color separation from writing animations > for the Apple II in machine code, working in a cable TV studio in the > 1980s, and from an art history class in college where we discussed 19th C > travel photos and poly/monochromatic film chemistries of the periods. > Later I learned fluorescence quantification from a cell biologist intent on > making sure we had true linear responses for readouts of f-actin mass and > concentration, I was fortunate to attend both a Woods Hole microscopy > course and at NCSU John Russ's course on image analysis, and BioRad MRC 600 > training course at their offices in Cambridge, MA covered a lot of material > on this too. > > > > So, do people have favorite texts that cover these topics in a manner > digestible by new generations of students? Of biology students. Keep in > mind many don't know what a byte is; when I teach I ask the class if anyone > knows why an image is coded with white as 255 or 4095 or 16383 and > typically one or two hands go up but the rest of the class really doesn't > know. > > Looking forward to suggestions of easy to read definitive texts! > > > > Thank you. > > > > Cheers- > > > > > > ------------------------------------------------------------ > > This email message, including any attachments, is for the sole use of > the intended recipient(s) and may contain information that is proprietary, > confidential, and exempt from disclosure under applicable law. Any > unauthorized review, use, disclosure, or distribution is prohibited. If you > have received this email in error please notify the sender by return email > and delete the original message. Please note, the recipient should check > this email and any attachments for the presence of viruses. The > organization accepts no liability for any damage caused by any virus > transmitted by this email. > > ================================= > > > > > > > > > > ------------------------------------------------------------------------- > > This message was secured via TLS by MUSC. > |
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Claire Brown |
Re: seeking "reliable literature" on correct imaging
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In reply to this post by Cammer, Michael
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Here are a few resources I use. However, I think a good introductory microscopy course is best. Would be great to see these kinds of courses being built into the undergraduate programs for life scientists but it might be a while before this kind of training is broadly offered. However, there are many great courses offered through core facilities worldwide. Great web resource. They are about to add a module on super resolution imaging. Tests at the end of each module. https://myscope.training/ Other web resources: iBiology microscopy courses: instructive link with a lot of information and videos. Excellent for new users looking to get a solid understanding of microscopy basics. http://www.ibiology.org/ibioeducation/taking-courses.html Good for users with Leica microscopes (somewhat specific to the brand) and some tutorials that are good to understand the basics of fluorescent microscopy. http://www.leica-microsystems.com/science-lab/topics/ Nikon’s Microscopy U.: Lots of articles and tutorials subdivided by application type. A very large amount of information so it’s helpful to know what you are interested in before visiting the site. http://www.microscopyu.com/ Interactive tutorials and educational material on both a basic level (basic primers on microscopy) and an advanced, specialized techniques level. http://www.olympusmicro.com/ Similar to the above sites, but offered through Zeiss with reviews and interactive tutorials from basic concepts up to specialized techniques. http://zeiss-campus.magnet.fsu.edu/ Quantitative Microscopy Swedlow JR. Quantitative fluorescence microscopy and image deconvolution. Methods Cell Biol 2013;114:407–426. Murray JM. Practical aspects of quantitative confocal microscopy. Methods Cell Biol 2007;81:467–478. Waters JC. Accuracy and precision in quantitative fluorescence microscopy. J Cell Biol 2009;185:1135–1148. North, A. J. (2006). Seeing is believing? A beginners' guide to practical pitfalls in image acquisition. J. Cell Biol. 172, 9-18. Methods Cell Biol. 2014;123:113-34. doi: 10.1016/B978-0-12-420138-5.00007-0. Quantitative confocal microscopy: beyond a pretty picture. Jonkman J, Brown CM, Cole RW. Fluorescence microscopy - avoiding the pitfalls (Probably needs to be updated) Claire M. Brown Journal of Cell Science 2007 120: 1703-1705; doi: 10.1242/jcs.03433 Good luck! |
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Rosemary.White |
Re: seeking "reliable literature" on correct imaging
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In reply to this post by mmodel
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi Mike, I have given talks like this in the rather dim past, but would be very interested in a copy of your Powerpoint file. Maybe you might have time to make it into a small (review) paper? One of the microscopy journals might publish it - e.g. Journal of Microscopy(?) thanks, Rosemary Dr Rosemary White CSIRO Black Mountain GPO Box 1700 Canberra, ACT 2601 Australia T 61 2 6246 5475 M 61 468 966 713 ________________________________________ From: Confocal Microscopy List [[hidden email]] on behalf of MODEL, MICHAEL [[hidden email]] Sent: Saturday, 17 November 2018 4:00 a.m. To: [hidden email] Subject: Re: seeking "reliable literature" on correct imaging ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi Michael, We recently ran a workshop where I gave a 3-hour talk on various kinds of quantification in microscopy. I can send you a PowerPoint file but don't have a text. Mike Model -----Original Message----- From: Confocal Microscopy List <[hidden email]> On Behalf Of Cammer, Michael Sent: Friday, November 16, 2018 11:50 AM To: [hidden email] Subject: seeking "reliable literature" on correct imaging ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Increasingly we have found that microscope users do not understand proper imaging. A few examples: Confocal images are saturated and have the offset set to negative numbers intentionally "to reduce background". Exposures are changed in fluorescent micrographs to make each one "look good". 16 bit raw data with no saturation or clipping are set to 8 bits with a lot of saturated and clipped pixels at the extremes and these are what are considered raw data. JPG compression is great because images can be emailed. People assume (and get angry when this doesn't work) that computers can automatically segment and count anything regardless of experimental design, image quality, etc. Even if they have saved everything as TIF with no metadata and forgot what magnifications they used. A student who recognizes these problems and wants to learn the right way emailed me yesterday asking for "any reliable literature you recommend if I'd like to read more about correct imaging." I did Google searches and, after skimming a bunch of web pages, realized that I didn't really have an answer. I could send to this page and to this page, but it's not a coherent narrative. And I don't have a library; most of what I know about this was picked up willy-nilly up in the 1980s and '90s. For instance knowledge of color separation from writing animations for the Apple II in machine code, working in a cable TV studio in the 1980s, and from an art history class in college where we discussed 19th C travel photos and poly/monochromatic film chemistries of the periods. Later I learned fluorescence quantification from a cell biologist intent on making sure we had true linear responses for readouts of f-actin mass and concentration, I was fortunate to attend both a Woods Hole microscopy course and at NCSU John Russ's course on image analysis, and BioRad MRC 600 training course at their offices in Cambridge, MA covered a lot of material on this too. So, do people have favorite texts that cover these topics in a manner digestible by new generations of students? Of biology students. Keep in mind many don't know what a byte is; when I teach I ask the class if anyone knows why an image is coded with white as 255 or 4095 or 16383 and typically one or two hands go up but the rest of the class really doesn't know. Looking forward to suggestions of easy to read definitive texts! Thank you. Cheers- ------------------------------------------------------------ This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email. ================================= |
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Steffen Dietzel |
Re: seeking "reliable literature" on correct imaging
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In reply to this post by Cammer, Michael
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi Michael, I guess we all face similar problems in that respect with our users. I put some of my favorite resources on our web site so that I have something to refer to, when they ask. (https://www.bioimaging.bmc.med.uni-muenchen.de/learn/index.html). Paperbook-wise I often recommend Guy Cox's "optical imaging techniques in cell biology", not because it would be better than the alternatives (Murphy&Davison, The Handbook) but because it is very concise and to the point. For those who'd rather listen than read I recommend the iBiology videos. Within Germanbioimaging we developed a list of topics that should be taught to (new) users and provide some teaching materials. The GerBi website is currently being remodeled but my contribution to the materials also can be found here: https://www.bioimaging.bmc.med.uni-muenchen.de/learn/materialdownload/index.html These powerpoints do not include my complete lectures, all the copyrighted materials are omitted. And sometimes slides are redundant. It is more a collection of slides that may be useful for those who have to give a lecture on the respective topics. But I guess much of the important bits we teach during the practical introduction (always save the original file format, don't overexpose, etc.) And about once in a year I give a 2x3h lecture on the basics of microscopy and imaging. But in some aspects I guess you always will leave room for surprises. Kathryn's example of the postdoc saving only Jpgs and Ganesh's comment on making the next generation of scientists fool proof remind me of one of my favorite Douglas Adam quotes, though: "The problem with making something completely foolproof is the ingenuity of complete fools." Meaning: we can hope to diminish the problem, but we will never solve it completely. I definitely will have a look into John's list, thank you for that (and all the other interesting sources mentioned) Steffen Am 16.11.2018 um 17:50 schrieb Cammer, Michael: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Increasingly we have found that microscope users do not understand proper imaging. > > A few examples: > Confocal images are saturated and have the offset set to negative numbers intentionally "to reduce background". > Exposures are changed in fluorescent micrographs to make each one "look good". > 16 bit raw data with no saturation or clipping are set to 8 bits with a lot of saturated and clipped pixels at the extremes and these are what are considered raw data. > JPG compression is great because images can be emailed. > People assume (and get angry when this doesn't work) that computers can automatically segment and count anything regardless of experimental design, image quality, etc. > Even if they have saved everything as TIF with no metadata and forgot what magnifications they used. > > A student who recognizes these problems and wants to learn the right way emailed me yesterday asking for "any reliable literature you recommend if I'd like to read more about correct imaging." > > I did Google searches and, after skimming a bunch of web pages, realized that I didn't really have an answer. I could send to this page and to this page, but it's not a coherent narrative. And I don't have a library; most of what I know about this was picked up willy-nilly up in the 1980s and '90s. For instance knowledge of color separation from writing animations for the Apple II in machine code, working in a cable TV studio in the 1980s, and from an art history class in college where we discussed 19th C travel photos and poly/monochromatic film chemistries of the periods. Later I learned fluorescence quantification from a cell biologist intent on making sure we had true linear responses for readouts of f-actin mass and concentration, I was fortunate to attend both a Woods Hole microscopy course and at NCSU John Russ's course on image analysis, and BioRad MRC 600 training course at their offices in Cambridge, MA covered a lot of material on this too. > > So, do people have favorite texts that cover these topics in a manner digestible by new generations of students? Of biology students. Keep in mind many don't know what a byte is; when I teach I ask the class if anyone knows why an image is coded with white as 255 or 4095 or 16383 and typically one or two hands go up but the rest of the class really doesn't know. > Looking forward to suggestions of easy to read definitive texts! > > Thank you. > > Cheers- > > > ------------------------------------------------------------ > This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email. > ================================= > ------------------------------------------------------------ Steffen Dietzel, PD Dr. rer. nat Ludwig-Maximilians-Universität München Biomedical Center (BMC) Head of the Core Facility Bioimaging Großhaderner Straße 9 D-82152 Planegg-Martinsried Germany http://www.bioimaging.bmc.med.uni-muenchen.de |
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