This article was automatically translated from Japanese using AI. The Japanese version is the authoritative version.
In biological research, you will often image samples such as antibody-stained preparations with a confocal laser scanning microscope.
Here I explain how to use ImageJ to turn that imaging data into a stack image (Stack) and to merge images acquired at different wavelengths into a merged image (Merge).
I will finish by showing how to add a scale bar.
First, with a confocal laser scanning microscope you obtain images acquired at different wavelengths (different lasers) for each single slice, and these slices span the thickness of the sample along the z axis.
Here, combining images of different wavelengths taken at the same z position is called merging, and superimposing the 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 will use Fiji in this explanation, but the procedure is largely the same in ImageJ.
That said, Fiji can open confocal microscope image data without any format conversion, which is why I use Fiji here.
For this example I used a confocal microscope image of a fly from flylight.
The image used as an example
As you can see from the files downloadable at that site, confocal microscope images use manufacturer-specific formats such as *.lsm, and these files contain a variety of information.
Scale information is included as well, so you can insert 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.
When it opens, a new window appears showing something blackish or greenish.
This is the image you are currently working on.
You can move along the z axis by dragging 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 the Channel checkboxes.
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 too, so you can view the stacked image for each channel.
You can add a scale bar via Analyze<Tools<Scale Bar.
By changing the value of “Width in 〇〇:”, you can set it to whatever length you like.
To save the processed image, use File<Save as and specify the file format.
For some reason images come out white when saved as Tiff, so I recommend PNG or JPEG.
And here is the finished image.
You can find the link to Fiji here.