In biological research, samples such as antibody-stained preparations are often imaged with a confocal laser scanning microscope.
Here I explain how to use ImageJ to turn that imaging data into a stack, and how to merge images acquired at different wavelengths.
I’ll finish by showing how to add a scale bar.
First, in confocal laser scanning microscopy you obtain, for each single slice, images acquired at different wavelengths (i.e., with different lasers), and these slices span the thickness of the sample along the z axis.
Combining images of different wavelengths taken at the same z position is called merging here, while combining images along the z axis is called stacking.
ImageJ uses these terms in the same sense, so keep in mind what you actually want to do as you work.
I’ll be using Fiji here, but the procedure is essentially the same in ImageJ.
I use Fiji because it can open confocal microscope image data without any format conversion.
For this example I used a confocal image of a fly from flylight.
The image used as an example
As you can see from the files available on that site, confocal images are saved in manufacturer-specific formats such as *.lsm, and these files contain a variety of metadata.
This includes scale information, so you can add an accurate scale bar without having to look up the microscope’s scale yourself.
First, open Fiji and drag your imaging data onto the menu-bar-like window that appears.
Once it opens, a new window will appear showing something black or green.
This is your current working window.
You can move along the z axis using the scroll bar at the bottom.
Merge the images via Image<Color<Make Composite in the top menu.
Then open the Channels Tool via Image<Color<Channels Tool.
In the Channels Tool, you can toggle the display of each channel using its checkbox.
To stack the images, click Image<Stacks<Z project and the images will be stacked.
Unless you have a particular preference, set Projection Type to Sum Slices.
A new window will then open showing the stacked image.
The Channels Tool works here as well, so you can view the stacked image for each channel.
You can add a scale bar via Analyze<Tools<Scale Bar.
Change the “Width in ○○:” value to set the length you want.
To save the processed image, use File<Save as and choose the file format.
For some reason the image comes out washed out in Tiff, so I recommend PNG or JPEG.
And here is the finished image.
You can find Fiji here.