Z-stack Processing

Maximum Intensity Projection is an algorithm that selects pixels of the highest intensity from every slice throughout the volume to construct a 2D image.

  1. Open Z-stack in Fiji, under Color Options/color mode  choose “Colorize”. Select OK.
  2. Go to Image > Stacks > Z Project. Choose your “Start” and “Stop” slice and type of projection (Maximum). Select Ok.
  3. If you would like to  change the color, go to Image > Look Up Table > and select desired color.
  4. Save MIP as a .tif.

3D Reconstruction/Volume Rendering: Mathematical reconstruction of Z-stacks to produce a three-dimensional volume. This includes linear interpolation between the actual data planes and then transparent volume rendering for visualization.

  1. Open Z-stack in Fiji with hyperstack selected.
  2. Adjust image contrast with histogram stretching.
  3. To make the 360-degree video of the projection, go to Image > Stacks > 3D Project
  4. Set the project method (start with brightest point) and the axis of rotation (your preference)
  5. Try the project without and with interpolate checked to see if gaps need to be filled.
  6. Save as an .avi with no compression- and then click on it to watch your movie!

Geometric Surface Rendering: The surface is defined by a series of connected triangles. This rendering can be used as a polygon mesh for a 3D model. A polygon mesh is a collection of triangles (vertices, edges and faces) that defines the shape of an object in 3D computer graphics.

  1. Open Z-stack in Fiji with hyperstack selected.
  2. Ensure you are using an 8-bit image by selecting Image > Type > 8-bit.
  3. Go to Plugins > 3D Viewer.
  4. Select the image and write in file name.
  5. Change “Display As” from Volume to Surface.
  6. Select color (your choice).
  7. Select Threshold. Try 20. Leave Resampling factor at 2. Select Ok.
  8. In the Image Viewer window, export File > Export > STL (ASCII).

Ideally, you want your reconstruction to be one piece without “holes”, so lower the threshold if you need to fill in the holes. Increase the resampling factor to “smooth” the image. This may take a fair amount of trial and error and works best on a computer with a good graphics card

Volume Viewer: This plugin allows you to view a 3D volume of a fluorescent Z-stack and has more settings and options allowing you to customize your projection.

  1. Open Z-stack in Fiji with hyperstack selected.
  2. Ensure you are using an 8-bit image. This plugin uses a fair bit of memory, so you may need to scale down your image in order for the process to run.
  3. Go to Plugins > Volume Viewer.
  4. Run the plugin – it may take several minutes to load.
  5. Ensure that the selected “Mode” is set to volume.
  6. Take a snapshot of volume at your desired angle(s) and save.