I didn't know about the BIOP version of JaCOP. That's really useful. I get a few subscribers looking for the JaCOP plugin and I just put a link to your video in my comments section.. I'll definitely switch to this version for my BioImaging classes. Thanks.
Thanks for sharing ! However, my fluorogram result is unlike yours at 2:04, it didn't show the original color in each channel (so images in ch1&2 are black and white), only the merge channel has colors. I want to get the colors back, so do you know how to fix this problem ?
Hi Martha. Yes, JACoP also works with z stacks. You will have to choice whether to threshold the images separately or set the threshold on the stack histogram. Everything else should work like 2d images.
Yes, @David Morales. As long as the foci are distinguishable and perhaps selected as ROIs, you can measure colocalization via this plugin. There is another plugin that you can try. It's called DiAna (for distance analysis & colocalization). If you haven't seen this yet, here's the link: th-cam.com/video/CBwWk7wP2kQ/w-d-xo.html
Hi @cheska6148. The JACoP version of BIOP does not really explain what the fluorogram is, but this is just a 2D Histogram or scatterplot. It is a way to visualise the correlation of pixel intensities obetween 2 sets of data. This description is from Olympus (Colocalization of Fluorophores in Confocal Microscopy): "A scatterplot graphs the intensity of one pseudocolor (or channel) versus another for each pixel in the image, or a selected region of interest, on a two-dimensional histogram (see Figures 3 and 4). One channel (usually green) is graphed along the x-axis, while the other (usually red) is plotted on the y-axis with intensities ranging from 0 to 255 on both the abscissa and ordinate. Thus, every pixel of the composite image is characterized by a pair of intensities distinguished as coordinates in a Cartesian system. Analysis of the distribution pattern generated by the intensity pairs enables identification of fluorophore colocalization, as well as background discrimination, bleed-through, photobleaching, and lack of registration between the individual channels."
Really helpful video, thank you Johanna. Which measurement do you find is the best to use, such as Pearson's, overlap coefficient or area overlap? Thank you.
Hi @JamesGriffin47. It really depends on how you define colocalization. It could be that you are looking for one signal on one channel to be within the bounds of another signal in a different channel, or if the signals from both channels overlap but are still separated by a certain distance. Pearson's correlation involves intensity correlation of pixel-wise colocalizing objects, while area overlap is more object-based.
Hi @Haimon Alves. I haven't tried the GLCM Texture Tool and as far as I know, this plugin hasn't been updated. QuPath might be the way to go, but I'll try to explore more about Haralick measurements with Fiji.
@@johanna.m.dela-cruz I have never used QuPath, although I've heard about it. Do you have any reference (videos or tutorials) where they talk about the haralick / texture measurements?
Hi Johanna. Thanks for this great resource! I'm really new to this field and would like to know if you have an idea on how to go about applying colocalization to an image with several cells.
Hi Valeria. JACoP will work on an image with several cells. The important thing is to be able to select a threshold that will segment your cells and whatever it colocalizes with. You will essentially be comparing images from 2 channels. Colocalization measurements will, however, be more accurate if your images were acquired with a high resolution objective.
Great tutorial! This plugin is exactly what I was looking for! Can I also use it for three-dimensional analysis by letting the analysis run on the z-stack instead of a single slide? And if yes, how is the value calculated? By doing a maximum intensity projection or taking each plane into account?
Hi @Daniela Neugebauer. If "Consider Z slices Separately" and "set auto thresholds on stack histogram" are checked, a single Z-stack image is the output. The analysis is performed on each individual slice. The Results table shows measurements for each slice (comparing channel 1 with channel 2).
@@johanna.m.dela-cruz Thanks for your reply! I actually don't want to calculate the coloc for each single z-stack image, but for the whole cell as a three-dimensional unit (Manders representing the coloc between channel 1 and 2 across the whole cell). So I would be interested in Manders calculated by M1 = sum(Ri, colocal)/sum(Ri) where Ri are the intensity values of Red voxels above the red threshold. Is JACoP doing this automatically when I run the analysis on a z-stack and not check 'consider z slices separately' nor 'set auto thresholds on stack histogram'? I hope my explaination is not too confused :D
@@danielaneugebauer5065 Yes, you will have single measurements of the parameters you want to measure. I don't think the value shown would be a Mean (not exactly sure how this was calculated), but this measurement is supposed to be for the entire z stack.
![FIJI (ImageJ): Spot Detection and Colocalization [ComDet]](http://i.ytimg.com/vi/gbsjjNxunSo/mqdefault.jpg)
![FIJI (ImageJ): Spot Detection and Colocalization [ComDet]](/img/tr.png)
I didn't know about the BIOP version of JaCOP. That's really useful. I get a few subscribers looking for the JaCOP plugin and I just put a link to your video in my comments section.. I'll definitely switch to this version for my BioImaging classes. Thanks.
Thank you, Craig. It is a very handy tool to have.
Thank you so much for sharing, BIOP version is much better than the original JACoP, very useful.
Happy to share this. Thanks for watching.
This is so helpful! Save me TONS of time on image analysis. Thank you !!!!!
@FeelingYTChen, I’m glad to help.
Thanks for sharing ! However, my fluorogram result is unlike yours at 2:04, it didn't show the original color in each channel (so images in ch1&2 are black and white), only the merge channel has colors. I want to get the colors back, so do you know how to fix this problem ?
Hello @stanleyshen4552. You can always use LUT (lookup table) to add color to your images.
@@johanna.m.dela-cruz Thanks for your help. Just solve the problem!
Great to know about the BIOP version of JaCOP. It is very helpful. Thanks !
Happy I could be of help.
Thank you for the tutorial!! I do have a question can i use this plugin with 3D images? And if so how do i go about it?
Hi Martha. Yes, JACoP also works with z stacks. You will have to choice whether to threshold the images separately or set the threshold on the stack histogram. Everything else should work like 2d images.
Amazing content, thanks much, Johanna. Btw, is there any way we can use this plugins to measure colocalization of more than two channels/fluorescence?
Thanks for watching, Ian Timothy. The plugin was written for 2 labels, but perhaps measurements of all 3 (or more) channels can be taken by pairs.
unable to find JACoP BIOP plugin
Hello. In Fiji, you will have to add the PTBIOP site to get the plugin.
Hi Johanna, thanks a lot for this. Would it be possible to use this to look for colocalizing foci from two channels in a 2D multichannel image?
Yes, @David Morales. As long as the foci are distinguishable and perhaps selected as ROIs, you can measure colocalization via this plugin. There is another plugin that you can try. It's called DiAna (for distance analysis & colocalization). If you haven't seen this yet, here's the link: th-cam.com/video/CBwWk7wP2kQ/w-d-xo.html
Thank you for the tutorial!! I do have a question, can I use this plugin with 3D images and if so, how do I go about it?
Please see my reply below. Thanks for watching.
Hi, thanks so much for making this video. Do you have any resource on explaining the Fluorogram output?
Hi @cheska6148. The JACoP version of BIOP does not really explain what the fluorogram is, but this is just a 2D Histogram or scatterplot. It is a way to visualise the correlation of pixel intensities obetween 2 sets of data. This description is from Olympus (Colocalization of Fluorophores in Confocal Microscopy): "A scatterplot graphs the intensity of one pseudocolor (or channel) versus another for each pixel in the image, or a selected region of interest, on a two-dimensional histogram (see Figures 3 and 4). One channel (usually green) is graphed along the x-axis, while the other (usually red) is plotted on the y-axis with intensities ranging from 0 to 255 on both the abscissa and ordinate. Thus, every pixel of the composite image is characterized by a pair of intensities distinguished as coordinates in a Cartesian system. Analysis of the distribution pattern generated by the intensity pairs enables identification of fluorophore colocalization, as well as background discrimination, bleed-through, photobleaching, and lack of registration between the individual channels."
Really helpful video, thank you Johanna. Which measurement do you find is the best to use, such as Pearson's, overlap coefficient or area overlap? Thank you.
Hi @JamesGriffin47. It really depends on how you define colocalization. It could be that you are looking for one signal on one channel to be within the bounds of another signal in a different channel, or if the signals from both channels overlap but are still separated by a certain distance. Pearson's correlation involves intensity correlation of pixel-wise colocalizing objects, while area overlap is more object-based.
Do you know of haralick measurements that can be done with fiji? I would Very much appreciate a vídeo about the plugin, How to measure, etc
Hi @Haimon Alves. I haven't tried the GLCM Texture Tool and as far as I know, this plugin hasn't been updated. QuPath might be the way to go, but I'll try to explore more about Haralick measurements with Fiji.
@@johanna.m.dela-cruz I have never used QuPath, although I've heard about it. Do you have any reference (videos or tutorials) where they talk about the haralick / texture measurements?
@@haimonalves3879 I haven't tried QuPath either, although you can check image.sc for discussions about this.
Hi Johanna. Thanks for this great resource! I'm really new to this field and would like to know if you have an idea on how to go about applying colocalization to an image with several cells.
Hi Valeria. JACoP will work on an image with several cells. The important thing is to be able to select a threshold that will segment your cells and whatever it colocalizes with. You will essentially be comparing images from 2 channels. Colocalization measurements will, however, be more accurate if your images were acquired with a high resolution objective.
Great tutorial! This plugin is exactly what I was looking for! Can I also use it for three-dimensional analysis by letting the analysis run on the z-stack instead of a single slide? And if yes, how is the value calculated? By doing a maximum intensity projection or taking each plane into account?
Hi @Daniela Neugebauer. If "Consider Z slices Separately" and "set auto thresholds on stack histogram" are checked, a single Z-stack image is the output. The analysis is performed on each individual slice. The Results table shows measurements for each slice (comparing channel 1 with channel 2).
@@johanna.m.dela-cruz Thanks for your reply! I actually don't want to calculate the coloc for each single z-stack image, but for the whole cell as a three-dimensional unit (Manders representing the coloc between channel 1 and 2 across the whole cell). So I would be interested in Manders calculated by M1 = sum(Ri, colocal)/sum(Ri) where Ri are the intensity values of Red voxels above the red threshold. Is JACoP doing this automatically when I run the analysis on a z-stack and not check 'consider z slices separately' nor 'set auto thresholds on stack histogram'? I hope my explaination is not too confused :D
@@danielaneugebauer5065 Yes, you will have single measurements of the parameters you want to measure. I don't think the value shown would be a Mean (not exactly sure how this was calculated), but this measurement is supposed to be for the entire z stack.
@@johanna.m.dela-cruz Perfect! Thank you sou much!