Hi, 

do you mean this protocol?

http://www.life.illinois.edu/mcb/253/Semester%20Folders/SP14/6_ImageJ_overlay_protocol.pdf

PROTOCOL FOR IMAGE OVERLAY IN IMAGEJ

1. Make sure each file you want to overlay has a name that you will recognize. For

example the DAPI stained image should have DAPI in the file name. This will be

important to ensure each color is correctly added.

2. Open ImageJ64. Open each image that you want to overlay in the program. To do

this, go to FILE à OPEN and click on the correct file. Do this for each image.

3. Every picture must then be converted to 8-bit. To do this click IMAGE à TYPE

and select 8-bit.

4. Next we will merge the pictures to create the overlay. Click IMAGE à COLOR and

select Merge Colors.

5. A pop-up menu will then allow you to assign a picture to a color. Traditionally,

DAPI is blue, FITC is green, and rhodamine is red. Once assigned, click OK.

6. Your picture will then be displayed.

7. To save your picture you must covert the image type to RBG Color. To do this,

click IMAGE à TYPE and select RGB Color. Next flick FILEàSAVE and you will

be allowed to name your new file and save where you like.

Best Wishes

While, it is possible to create this type of image in ImageJ through threshold masking and overlays or possibly even stacks, I personally find that using ImageJ for line drawing overlays is too complicated, in terms of the number of steps, though I wouldn't be surprised if plug-ins or macros exist to more easily facilitate the process.

The way I would overlay anatomical line drawings onto neural tissue micrographs is by using layers and color transparency in the GNU Image Manipulation Program (GIMP). This is a powerful, free, and open source image editing program you can download at www.gimp.org.

I wrote out simplest form of the procedure:

1. Open your black and white line drawing and scale the dimensions to approximately match those of your histological micrograph. Use the menu command 'Image --> Scale Image.'

2. In the menu select 'Colors --> Color to Alpha' and choose white as the color. Your line drawing should now be a black outline with a checkerboard background pattern.

3. Copy your line drawing and paste it as a new layer onto the histological section image.

4. Using the 'Scale' tool, click the image and then click and drag the middle circle in the grid to move the line drawing overlay into position at the edge of the brain or corner of some identifiable landmark or region, for example caudate putamen in the bottom-most section of the image you uploaded.

5. Pulling on the corners of the scaling grid, adjust the size of the line drawing to match the anatomy of your histological section image. Keep track of the percentage you are scaling your overlay since you can probably reuse the relative adjustment from one coronal plane to others. For example, if you scale the atlas overlay 85% in the bottom-most image, start with an 85% scaled line-drawing overlay for the other two images when you need to demarcate the PFC.

6. You can export a flattened version of the overlay in a variety of formats.

Hope that helps. Tell us if it works! 

Thanks Mikey. I did recently learn of GIMP and its exactly what I needed. So far I have trial-and-error'ed my way through it, but your protocol does fill in some gaps that I hadn't figured out yet.

Definitely helps!

Try this, Puchades MA of the group can be reached by WhatsApp Skype if you need help.

Navigating the Murine Brain: Toward Best Practices for Determining and Documenting Neuroanatomical Locations in Experimental Studies.

Bjerke IE, Øvsthus M, Andersson KA, Blixhavn CH, Kleven H, Yates SC, Puchades MA, Bjaalie JG, Leergaard TB.

Front Neuroanat. 2018 Nov 2;12:82. doi: 10.3389/fnana.2018.00082. eCollection 2018.

PMID: 30450039