Confocal Microscopy

Confocal microscopy is regarded as a sophisticated way of analyzing the phenotype of cells. In the field of adult neurogenesis, use of confocal microscopy to analyze the phenotype of newly born cells is now de rigueur. However, as with every technique, confocal microscopy can be misused and provide misleading results.

This movie is a good example of how poorly performed confocal can lead to a false appearance of double labeling: a BrdU-labeled cell appears to be a neuron when in fact it is probably a satellite glia cell.

The movie begins with a series of Z slices (from the top of the section to the bottom). In this part of the movie, all BrdU-positive cells (in green) appear to be NeuN-positive (red).

Seeing is believing, right? However, at the 10-second mark, the "Z stack" of slices rotates to show the three-dimensional arrangement of these cells. As the stack rotates, notice that some of the BrdU-positive cells are actually adjacent to the NeuN-positive cells (look at the bottom two BrdU-positive cells). This shows that these particular BrdU-positive cells are not NeuN-positive. Indeed, in this case, these BrdU-positive cells appear to be satellite glia, as have been reported in other regions of the brain.

So how is it that the Z slice part of the movie suggests BrdU-labeled neurons, but the Z stack rotation part of the movie suggests BrdU-labeled satellite glia? The answer is in the way the confocal microscopy was performed. Such misleading information can result from a variety of sources, including:

  • Z slices that are too thick or too thin for the objective and/or sample.
  • Z slices that overlap too little or too much.
  • Inappropriate insertion of Z section thickness in 3D reconstruction programs.
  • Single or simultaneous laser scanning instead of multiple or sequential scanning (very common).
  • A laser configuration that allows bleedthrough of fluorophores.

This is just a short list of things that can provide misleading results in confocal microscopy. In the movie, the slices were taken with an Airy disk unit greater than 1, leading to a wide pinhole and a thick section scanning. Therefore, viewed top-to-bottom, the cells appear double-labeled. Only after 3D reconstruction and rotation is the cells' relationship apparent. This movie is good evidence that in addition to doing confocal microscopy, researchers would be wise to also make 3D reconstruction (or at least orthogonal slice analysis) a necessary part of phenotypic analysis.

In sum, confocal microscopy is only as good as the knowledge of the microscopist. As this movie reminds us, don't trust what you see – unless you understand how the images were captured and processed.

We recommend Jim Pawley's Handbook of Biological Confocal Microscopy. We also recommend these websites: