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Getting Started

This brief guide will outline several basic processing and analysis methods. For further instruction, please refer to the NIH’s ImageJ user guide and other Fiji documentation.

Downloading Fiji:

To download Fiji, go to and scroll until you see the download section. Determine the appropriate download for your operating system. If you are unsure if you have a 64 bit or a 32 bit OS, type “See if you have a 32-bit or 64-bit OS” into your computer’s search bar or go to Computer Settings > About. Look under device specifications and system type.

Opening Images: 

  1. To open a single channel image, go to File > Open and select your desired image. If the image is a life science image file, the bio-formats window will open. Ensure you are viewing all images as a hyperstack. Choose your desired color mode (grayscale or colorized) and autoscale the image.
  2. To open a multi-channel image, open your image the same way as a single channel and you can choose to “Split Channels”. Splitting channels allows you to alter the channel color, if necessary. You can re-merge the channels later if desired.
  3. To open a Z-stacked image, ensure you are splitting channels if a multi-channeled Z-stack.

Adding a scale bar: Go to Analyze > Tools > Scalebar. Since this creates an overlay of the scale bar, you need to combine your image and the overlay by going to Image > Overlay > Flatten.

Changing image type: Some microscopes create a higher quality 16-bit image, but 8-bit images work best to allow you to view them on your computer without any analysis software. To change your image, go to Image > Type and select desired type.

Color Processing: Make sure the selected channel or image is the primary window.

  1. To pseudocolor the image, go to Image > LookUp Table and select desired color.
  2. To merge several images, go to Image > Color > Merge Channels. Determine which image will be which channel (red, green, blue, cyan etc.) and merge using the dropdown menu. Select keep source images to keep original images open.
  3. To split channels, go to Image > Color > Split Channels.