Explant migration assays
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Hello,
I have an assay that seems simple enough to describe, but I cannot find the ImageJ/FIJI tool that quite does what we need. We have tissue explants transferred to a thin collagen gel, and we are measuring migration of cells in um from the edge of the explant. We would automatically detect these nuclei and use, e.g. CellProfiler, but the throughput is not very high and manually marking cells should be faster than troubleshooting a segmenter. This seems like it could be addressed with FISH tools - treat the explant as a "nucleus" or simply as a drawn oval and manually mark the nucleus of each migrating cell; the tool would measure the distance of each marked nucleus from the nearest edge of the explant. Can anyone recommend a tool for this job? Many thanks, Tim Timothy Feinstein, Ph.D. Research Scientist University of Pittsburgh Department of Developmental Biology -- ImageJ mailing list: http://imagej.nih.gov/ij/list.html |
overlay single image on stack?
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I have an application where I need to overlay a single monochrome image (rendered, say, in Blue), over a monochrome STACK (rendered, say, in Green). All images are the same size in X and Y. I would like to scroll through the stack, and optionally have the ability to modify the blending of the Green and Blue (even simply on/off for the single image would be useful).
Is this straightforward? Does anyone have a code snippet to share? Just to make it interesting, I *might* want to annotate the single image (what’s left? - in Red) - but this is not yet absolutely necessary, and I can do without it if it’s difficult. -- Kenneth Sloan [hidden email] Vision is the art of seeing what is invisible to others. > On Jun 1, 2015, at 15:08 , Feinstein, Timothy N <[hidden email]> wrote: > > Hello, > > I have an assay that seems simple enough to describe, but I cannot find the ImageJ/FIJI tool that quite does what we need. We have tissue explants transferred to a thin collagen gel, and we are measuring migration of cells in um from the edge of the explant. We would automatically detect these nuclei and use, e.g. CellProfiler, but the throughput is not very high and manually marking cells should be faster than troubleshooting a segmenter. > > This seems like it could be addressed with FISH tools - treat the explant as a "nucleus" or simply as a drawn oval and manually mark the nucleus of each migrating cell; the tool would measure the distance of each marked nucleus from the nearest edge of the explant. Can anyone recommend a tool for this job? > > Many thanks, > > > Tim > > Timothy Feinstein, Ph.D. > Research Scientist > University of Pittsburgh Department of Developmental Biology > > > -- > ImageJ mailing list: http://imagej.nih.gov/ij/list.html -- ImageJ mailing list: http://imagej.nih.gov/ij/list.html |
Re: overlay single image on stack?
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Hi Kenneth,
probably the easiest way to have the same overlay for all stack slices is Image>Overlay>Add Image. http://rsb.info.nih.gov/ij/docs/guide/146-28.html#toc-Subsubsection-28.14.2 The image used as an overlay can have a lookup table to get a specific color. Also the stack can have a LUT. To create e.g. a 'red only' lut, you can use the LUT editor. http://rsb.info.nih.gov/ij/plugins/lut-editor.html Michael ________________________________________________________________ On Jun 1, 2015, at 22:48, Kenneth R Sloan wrote: > I have an application where I need to overlay a single monochrome image (rendered, say, in Blue), over a monochrome STACK (rendered, say, in Green). All images are the same size in X and Y. I would like to scroll through the stack, and optionally have the ability to modify the blending of the Green and Blue (even simply on/off for the single image would be useful). > > Is this straightforward? Does anyone have a code snippet to share? > > Just to make it interesting, I *might* want to annotate the single image (what’s left? - in Red) - but this is not yet absolutely necessary, and I can do without it if it’s difficult. > > -- > Kenneth Sloan > [hidden email] > Vision is the art of seeing what is invisible to others. > > > > >> On Jun 1, 2015, at 15:08 , Feinstein, Timothy N <[hidden email]> wrote: >> >> Hello, >> >> I have an assay that seems simple enough to describe, but I cannot find the ImageJ/FIJI tool that quite does what we need. We have tissue explants transferred to a thin collagen gel, and we are measuring migration of cells in um from the edge of the explant. We would automatically detect these nuclei and use, e.g. CellProfiler, but the throughput is not very high and manually marking cells should be faster than troubleshooting a segmenter. >> >> This seems like it could be addressed with FISH tools - treat the explant as a "nucleus" or simply as a drawn oval and manually mark the nucleus of each migrating cell; the tool would measure the distance of each marked nucleus from the nearest edge of the explant. Can anyone recommend a tool for this job? >> >> Many thanks, >> >> >> Tim >> >> Timothy Feinstein, Ph.D. >> Research Scientist >> University of Pittsburgh Department of Developmental Biology >> >> >> -- >> ImageJ mailing list: http://imagej.nih.gov/ij/list.html > > -- > ImageJ mailing list: http://imagej.nih.gov/ij/list.html -- ImageJ mailing list: http://imagej.nih.gov/ij/list.html |
Re: overlay single image on stack?
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Thanks. I did manage to find that, and it works reasonably well. Right now, I’m struggling with specifying how the overlay and the underlying stack should interact.
Briefly, I want to allow someone to assess co-localization of two signals. One (the stack, conventionally presented as green) has features which are possible CAUSES of certain effects seen in the other (the single image, conventionally presented as blue). My first, naïve attempt gives me images which are fairly dark - presumably because the blue single image is very dark (but not zero) over most of the image. I probably need to process the blue image into something more like a mask (a simple threshold might do) and present the green stack signal ONLY in the part of the image where the blue signal also exists. Or, perhaps better, present the green signal as BRIGHT when it co-localizes with the blue signal and DIM when it does not. So…turn the blue signal into WHITE above a threshold and GRAY below the threshold and then overlay this 2-level mask on the green stack? [obviously, I want the observer to be able to scroll through the green stack, filtered by the blue signal mask. This looks promising. That will work when the question is: does the green signal CAUSE the blue signal. A second question is: are there features revealed in the green signal that BLOCK the blue signal. That may require more thought. I guess I need to talk more to the customer... Thank you very much for the reply - it was useful. -- Kenneth Sloan [hidden email] Vision is the art of seeing what is invisible to others. > On Jun 3, 2015, at 03:34 , Michael Schmid <[hidden email]> wrote: > > Hi Kenneth, > > probably the easiest way to have the same overlay for all stack slices is Image>Overlay>Add Image. > http://rsb.info.nih.gov/ij/docs/guide/146-28.html#toc-Subsubsection-28.14.2 > > The image used as an overlay can have a lookup table to get a specific color. > Also the stack can have a LUT. To create e.g. a 'red only' lut, you can use the LUT editor. > http://rsb.info.nih.gov/ij/plugins/lut-editor.html > > Michael > ________________________________________________________________ > On Jun 1, 2015, at 22:48, Kenneth R Sloan wrote: > >> I have an application where I need to overlay a single monochrome image (rendered, say, in Blue), over a monochrome STACK (rendered, say, in Green). All images are the same size in X and Y. I would like to scroll through the stack, and optionally have the ability to modify the blending of the Green and Blue (even simply on/off for the single image would be useful). >> >> Is this straightforward? Does anyone have a code snippet to share? >> >> Just to make it interesting, I *might* want to annotate the single image (what’s left? - in Red) - but this is not yet absolutely necessary, and I can do without it if it’s difficult. >> >> -- >> Kenneth Sloan >> [hidden email] >> Vision is the art of seeing what is invisible to others. >> >> >> >> >>> On Jun 1, 2015, at 15:08 , Feinstein, Timothy N <[hidden email]> wrote: >>> >>> Hello, >>> >>> I have an assay that seems simple enough to describe, but I cannot find the ImageJ/FIJI tool that quite does what we need. We have tissue explants transferred to a thin collagen gel, and we are measuring migration of cells in um from the edge of the explant. We would automatically detect these nuclei and use, e.g. CellProfiler, but the throughput is not very high and manually marking cells should be faster than troubleshooting a segmenter. >>> >>> This seems like it could be addressed with FISH tools - treat the explant as a "nucleus" or simply as a drawn oval and manually mark the nucleus of each migrating cell; the tool would measure the distance of each marked nucleus from the nearest edge of the explant. Can anyone recommend a tool for this job? >>> >>> Many thanks, >>> >>> >>> Tim >>> >>> Timothy Feinstein, Ph.D. >>> Research Scientist >>> University of Pittsburgh Department of Developmental Biology >>> >>> >>> -- >>> ImageJ mailing list: http://imagej.nih.gov/ij/list.html >> >> -- >> ImageJ mailing list: http://imagej.nih.gov/ij/list.html > > -- > ImageJ mailing list: http://imagej.nih.gov/ij/list.html -- ImageJ mailing list: http://imagej.nih.gov/ij/list.html |
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Dear Kenneth,
Maybe you could make a stack from your blue image so it matches the number of images in your green stack and merge them via Image > Color > Merge Channels. If you create a composite you can use Image > Color > Channel Tool to switch on and off channels. You could also use now one of the colocalization plugins (Colocalization or maybe RG2B Colocalization) to find the green stack signal in the part of the image where the blue signal also exists. Using the Channels Tool you can after colocalization recolor the channels to green and blue (the colocalization plugin will merge your individual stacks but will make your channels red and green) what will result in a cyan coloc signal. At least it might help with your first question and does not cause darkening of your images. Best wishes Kees Dr Ir K.R. Straatman Senior Experimental Officer Advanced Imaging Facility Centre for Core Biotechnology Services University of Leicester http://www2.le.ac.uk/colleges/medbiopsych/facilities-and-services/cbs/lite/aif -----Original Message----- From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of Kenneth R Sloan Sent: 03 June 2015 16:22 To: [hidden email] Subject: Re: overlay single image on stack? Thanks. I did manage to find that, and it works reasonably well. Right now, I'm struggling with specifying how the overlay and the underlying stack should interact. Briefly, I want to allow someone to assess co-localization of two signals. One (the stack, conventionally presented as green) has features which are possible CAUSES of certain effects seen in the other (the single image, conventionally presented as blue). My first, naïve attempt gives me images which are fairly dark - presumably because the blue single image is very dark (but not zero) over most of the image. I probably need to process the blue image into something more like a mask (a simple threshold might do) and present the green stack signal ONLY in the part of the image where the blue signal also exists. Or, perhaps better, present the green signal as BRIGHT when it co-localizes with the blue signal and DIM when it does not. So.turn the blue signal into WHITE above a threshold and GRAY below the threshold and then overlay this 2-level mask on the green stack? [obviously, I want the observer to be able to scroll through the green stack, filtered by the blue signal mask. This looks promising. That will work when the question is: does the green signal CAUSE the blue signal. A second question is: are there features revealed in the green signal that BLOCK the blue signal. That may require more thought. I guess I need to talk more to the customer... Thank you very much for the reply - it was useful. -- Kenneth Sloan [hidden email] Vision is the art of seeing what is invisible to others. > On Jun 3, 2015, at 03:34 , Michael Schmid <[hidden email]> wrote: > > Hi Kenneth, > > probably the easiest way to have the same overlay for all stack slices is Image>Overlay>Add Image. > http://rsb.info.nih.gov/ij/docs/guide/146-28.html#toc-Subsubsection-28.14.2 > > The image used as an overlay can have a lookup table to get a specific color. > Also the stack can have a LUT. To create e.g. a 'red only' lut, you can use the LUT editor. > http://rsb.info.nih.gov/ij/plugins/lut-editor.html > > Michael > ________________________________________________________________ > On Jun 1, 2015, at 22:48, Kenneth R Sloan wrote: > >> I have an application where I need to overlay a single monochrome image (rendered, say, in Blue), over a monochrome STACK (rendered, say, in Green). All images are the same size in X and Y. I would like to scroll through the stack, and optionally have the ability to modify the blending of the Green and Blue (even simply on/off for the single image would be useful). >> >> Is this straightforward? Does anyone have a code snippet to share? >> >> Just to make it interesting, I *might* want to annotate the single image (what's left? - in Red) - but this is not yet absolutely necessary, and I can do without it if it's difficult. >> >> -- >> Kenneth Sloan >> [hidden email] >> Vision is the art of seeing what is invisible to others. >> >> >> >> >>> On Jun 1, 2015, at 15:08 , Feinstein, Timothy N <[hidden email]> wrote: >>> >>> Hello, >>> >>> I have an assay that seems simple enough to describe, but I cannot find the ImageJ/FIJI tool that quite does what we need. We have tissue explants transferred to a thin collagen gel, and we are measuring migration of cells in um from the edge of the explant. We would automatically detect these nuclei and use, e.g. CellProfiler, but the throughput is not very high and manually marking cells should be faster than troubleshooting a segmenter. >>> >>> This seems like it could be addressed with FISH tools - treat the explant as a "nucleus" or simply as a drawn oval and manually mark the nucleus of each migrating cell; the tool would measure the distance of each marked nucleus from the nearest edge of the explant. Can anyone recommend a tool for this job? >>> >>> Many thanks, >>> >>> >>> Tim >>> >>> Timothy Feinstein, Ph.D. >>> Research Scientist >>> University of Pittsburgh Department of Developmental Biology >>> >>> >>> -- >>> ImageJ mailing list: http://imagej.nih.gov/ij/list.html >> >> -- >> ImageJ mailing list: http://imagej.nih.gov/ij/list.html > > -- > ImageJ mailing list: http://imagej.nih.gov/ij/list.html -- ImageJ mailing list: http://imagej.nih.gov/ij/list.html -- ImageJ mailing list: http://imagej.nih.gov/ij/list.html |
Re: overlay single image on stack?
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It looks like the key is to adjust the brightness and contrast of both the overlay image and the stack, in anticipation of the loss of both caused by the overlay mixing (essentially a NEGATIVE mixing).
I’d love to see some sort of ADDITIVE overlay - but this seems adequate, for now. If I need something more complicated, I can always write my own. Thanks, very much, for the replies - they were helpful. -- Kenneth Sloan [hidden email] Vision is the art of seeing what is invisible to others. > On Jun 4, 2015, at 03:39 , Straatman, Kees (Dr.) <[hidden email]> wrote: > > Dear Kenneth, > > Maybe you could make a stack from your blue image so it matches the number of images in your green stack and merge them via Image > Color > Merge Channels. If you create a composite you can use Image > Color > Channel Tool to switch on and off channels. You could also use now one of the colocalization plugins (Colocalization or maybe RG2B Colocalization) to find the green stack signal in the part of the image where the blue signal also exists. Using the Channels Tool you can after colocalization recolor the channels to green and blue (the colocalization plugin will merge your individual stacks but will make your channels red and green) what will result in a cyan coloc signal. At least it might help with your first question and does not cause darkening of your images. > > Best wishes > > Kees > > > Dr Ir K.R. Straatman > Senior Experimental Officer > Advanced Imaging Facility > Centre for Core Biotechnology Services > University of Leicester > http://www2.le.ac.uk/colleges/medbiopsych/facilities-and-services/cbs/lite/aif > > > -----Original Message----- > From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of Kenneth R Sloan > Sent: 03 June 2015 16:22 > To: [hidden email] > Subject: Re: overlay single image on stack? > > Thanks. I did manage to find that, and it works reasonably well. Right now, I'm struggling with specifying how the overlay and the underlying stack should interact. > > Briefly, I want to allow someone to assess co-localization of two signals. One (the stack, conventionally presented as green) has features which are possible CAUSES of certain effects seen in the other (the single image, conventionally presented as blue). > > My first, naïve attempt gives me images which are fairly dark - presumably because the blue single image is very dark (but not zero) over most of the image. I probably need to process the blue image into something more like a mask (a simple threshold might do) and present the green stack signal ONLY in the part of the image where the blue signal also exists. Or, perhaps better, present the green signal as BRIGHT when it co-localizes with the blue signal and DIM when it does not. So.turn the blue signal into WHITE above a threshold and GRAY below the threshold and then overlay this 2-level mask on the green stack? [obviously, I want the observer to be able to scroll through the green stack, filtered by the blue signal mask. This looks promising. > > That will work when the question is: does the green signal CAUSE the blue signal. > > A second question is: are there features revealed in the green signal that BLOCK the blue signal. That may require more thought. I guess I need to talk more to the customer... > > Thank you very much for the reply - it was useful. > > -- > Kenneth Sloan > [hidden email] > Vision is the art of seeing what is invisible to others. > > > > >> On Jun 3, 2015, at 03:34 , Michael Schmid <[hidden email]> wrote: >> >> Hi Kenneth, >> >> probably the easiest way to have the same overlay for all stack slices is Image>Overlay>Add Image. >> http://rsb.info.nih.gov/ij/docs/guide/146-28.html#toc-Subsubsection-28.14.2 >> >> The image used as an overlay can have a lookup table to get a specific color. >> Also the stack can have a LUT. To create e.g. a 'red only' lut, you can use the LUT editor. >> http://rsb.info.nih.gov/ij/plugins/lut-editor.html >> >> Michael >> ________________________________________________________________ >> On Jun 1, 2015, at 22:48, Kenneth R Sloan wrote: >> >>> I have an application where I need to overlay a single monochrome image (rendered, say, in Blue), over a monochrome STACK (rendered, say, in Green). All images are the same size in X and Y. I would like to scroll through the stack, and optionally have the ability to modify the blending of the Green and Blue (even simply on/off for the single image would be useful). >>> >>> Is this straightforward? Does anyone have a code snippet to share? >>> >>> Just to make it interesting, I *might* want to annotate the single image (what's left? - in Red) - but this is not yet absolutely necessary, and I can do without it if it's difficult. >>> >>> -- >>> Kenneth Sloan >>> [hidden email] >>> Vision is the art of seeing what is invisible to others. >>> >>> >>> >>> >>>> On Jun 1, 2015, at 15:08 , Feinstein, Timothy N <[hidden email]> wrote: >>>> >>>> Hello, >>>> >>>> I have an assay that seems simple enough to describe, but I cannot find the ImageJ/FIJI tool that quite does what we need. We have tissue explants transferred to a thin collagen gel, and we are measuring migration of cells in um from the edge of the explant. We would automatically detect these nuclei and use, e.g. CellProfiler, but the throughput is not very high and manually marking cells should be faster than troubleshooting a segmenter. >>>> >>>> This seems like it could be addressed with FISH tools - treat the explant as a "nucleus" or simply as a drawn oval and manually mark the nucleus of each migrating cell; the tool would measure the distance of each marked nucleus from the nearest edge of the explant. Can anyone recommend a tool for this job? >>>> >>>> Many thanks, >>>> >>>> >>>> Tim >>>> >>>> Timothy Feinstein, Ph.D. >>>> Research Scientist >>>> University of Pittsburgh Department of Developmental Biology >>>> >>>> >>>> -- >>>> ImageJ mailing list: http://imagej.nih.gov/ij/list.html >>> >>> -- >>> ImageJ mailing list: http://imagej.nih.gov/ij/list.html >> >> -- >> ImageJ mailing list: http://imagej.nih.gov/ij/list.html > > -- > ImageJ mailing list: http://imagej.nih.gov/ij/list.html > > -- > ImageJ mailing list: http://imagej.nih.gov/ij/list.html -- ImageJ mailing list: http://imagej.nih.gov/ij/list.html |
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