ImageJ is one of my favourite image-analysis programs. ImageJ is free, it is compatible with all major operating systems (it is written in Java) and it is very powerful. Here I am showing how to use ImageJ for the analysis of an immunofluorescence (IF) experiment. The source file (LSM file) was produced using a ZEISS microscope and the ZEN digital imaging software. This file includes a 2-channel IF scan of some cells grown on a coverslip and then stained with an anti-DDB2 (Abcam) antibody and counterstained with DAPI. A copy of the file used in this tutorial is available at the following URL. Note: ImageJ doesn’t require installation, as it is a Java-based tool. To start using ImageJ it is sufficient to download the last version of the software from https://imagej.nih.gov/ij/download.html and extract the compressed files on your local drive.
Step-by-step protocol
- Start JmageJ
- Open the lsm file (just use the file >> open command. Locate the file and click OK)
- Split the channels (Image >> colors >> split channels). This will generate an individual image for each channel
- Select each image and convert it to a 8-bit (grayscale) image (Image >> type >> 8-bit)
- Now we have 2 or more grayscale images open in the ImageJ workspace (it is important not to have other images open in the workspace before proceeding to the next step). Generate a stack from the open images using the Images to stack command (Image >> stacks >> images to stack). The user is required to provide a name for the new stack (use a name that is consistent with the current sample). Also, make sure that both check-boxes (use title as labels; keep source images) are checked. Click OK
The stack opens in a new window (overlied images retrieved from the LSM file). Right now each channel is displayed as scale of grays. We will now generate a composite (merged) image and replace the default palettes with colors of our choice.
- First, generate a composite visualization (Image >> colors >> make composite). Select composite as default visualization and click OK
- Open the Channels Tool by clicking Image >> color >> channels tool
- Click on the top text-box and select color instead of composite
- Adjust the false colors applied to each channel (if needed). For example, channel #1 may be set as red while it was supposed to be blue. To fix this, (with channel #1 selected in the Channels tool window) click on more >> blue. To switch to the next channel, click on the slide tab at the bottom of the stack or on the channel #2 check box in the Channels tool Window. Adjust colors of the other channels if needed
- In the Channels Tool Window select the composite check box. Chances are you may want to adjust the brightness or contrast of individual channels to get a better looking visualization. Please, be aware that tweaking B&C in a biased way will generate artifacts. Please, DO NOT generate artifacts. Science DOES NOT need them. We are not artists, we are scientists!!
- Play with the bars if you need to adjust
To adjust the other channel, just click on the slide tab at the bottom of the stack or select the challes from the channels tool (if still open). The brigthness and contrast window will change color according to the color of the channel
Once you are done click apply
for the merge (here called composite) image, open the channel tool again (image >> color >> channels tool)
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You can adjust the single channel brightness and contrast from the composite as well. In this case make sure you are adjusting the correct channel by looking at the color of the plot in the B&C window. You can change channel by clicking on the slide tab at the bottom of the stack
Export
You can save as tiff or a much faster way of dealing with the images is the Edit >> copy to system.
This will copy to the system clipboard the current selection. You can paste the resulting image anywhere you want (power point, Illustrator and so on) with bitmap quality. Use the channels tool to change what the image is displaying and copy to system each time. Save as tiff will save a 2-channel TIFF image (if I remember correctly). Save a TIFF as a backu
