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In this video I will be covering some different ways that we can use layers to control visibility to overlay images as well as to organize and label different layers and layer groups, so we know what we are seeing and we can move like images into sets. In order to get this image we have used several acquisition systems as well as section stained with different histological stains. Now currently while I have put everything together, we just have everything labeled layer 1 to layer 14. And we are going to initially relabel these and organize them because it's best practice is to be able to identify quickly what each layer is, and then we are going to create layer groups so we can combine related items in a single layer group.
Now to turn Visibility on and off we saw we can turn the eyeball on and off. And we want to label this color look- up table, this image on the left. We can see the graph, which was taken from a screen capture of a program called Image Pro Plus that allowed us to analyze the intensity along a band line profile. This is our red autoradiograph, which we apply the color look-up table onto a gray scale image. This is our elastin stain. We have our Movat's. We also want to rename our lipid stain for oil red-o. Here is our trichrome, and here is our H&E.
Now that's just the first stain we want to do. Now we want to put related stains together, in this case both Movat's and the elastin stain stained for connective tissue. So we want to group them together in a layer group. So we move to the folder. Click and we have created a group. We can rename it by double-clicking and just say Connective Tissue or CT, and now we are going to add by selecting that the two different connective tissues to that one folder, Movat's and our elastin stain. And now we can turn them both on and off with the single click and this is allowing us to present the data as we need and we can tell a story this way.
Another two images that should be linked together are the autoradiographic images. So we will create a layer group, double click call it AR and now we are going to move our color look up table and the red autoradiograph. Another thing that's nice is that if we wanted to send this image out and maybe it contained proprietary information, we don't have to rework it, we can just turn off specific portions of the image and then send it as is. The last thing I want to demonstrate with this image is the ability to see colocalization by using bright field images. In this case, we are going to want to convert some of these images to gray scale. We can simply use an adjustment layer to do that, but we also have to think a little ahead and move the group that we need to stain color higher and now we will put an adjustment layer above the Line Profile, go to this half filled circle, black and white and this works out very well, we hit OK and we can open up our group, select the red AR and now we are just going to use the Move tool to move this over and while this completely obscures the one beneath, we can also use the Blend Modes that layers provide and go to the Blend Modes and change from Normal to Multiply where we are just projecting the darker parts of the image on top of the image below and here we can see how well the activity and the autoradiogram aligns in the different tissue sections.
We have it aligning with the connective tissue very well. The activity is not localized where the Oil red-o is and here I can confirm, I can turn that Oil red-o on or off and again I can see how it works with trichrome. And here is the trichrome. So that's one way to do colocalization of a bright field image and you can modify this and you are able to turn on and off the adjustment layer so you can really determine the coloration of a tissue and see how it aligns with the autoradiogram.
Layers is incredibly powerful and you can use the many abilities that the Layers panel provides to interact with your image and create customized ways to present any combination of images.
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