Arbuscular mycorrhizal (AM) fungi are microscopic throughout their entire lifecycle and grow concealed within plant roots and soils. This means we cannot easily see them with the naked eye. So how can we make this invisible fungi visible? 

The answer is microscopy, and there are many different techniques. Below is a guide to the techniques used in generating the images and movies on this website:

Method 1: live brightfield microscopy

In the laboratory, we can grow AM fungi with a plant in petri dishes. The petri dishes contain a transparent jelly-like substance called agar. Unlike soil, the agar lets light pass through. This means we can see the parts of the fungus that grow outside of the plant root like the hyphae and spores. To magnify these structures, the entire petri dish can be put under a low magnification 'dissecting' microscope. The sample is illuminated with normal white 'brightfield' light. For even higher magnification, small sections of the agar can be placed on a microscope slide and viewed under a 'compound' brightfield microscope.

Method 2: stains + brightfield microscopy

To see the parts of the fungus inside the plant root, one approach is staining. For this technique, we first need to make the roots transparent. We can do this by boiling in a strong alkali and then soaking in an acid. We then use a stain that highlights the mycorrhizal structures, like 'Trypan Blue' or regular fountain pen ink. Once stained, we mount the roots on a microscope slide and magnify them using a compound microscope.

Method 3: fluorescent dyes + confocal microscopy

To reveal the mycorrhizal structures inside a plant root in even more detail, we can use fluorescent dyes and confocal microscopy. The first step is similar to Method 2, using alkalis and acids to make the root transparent. But instead of ink, we add a special fluorescent dye that binds to the fungal cell walls. Using a confocal microscope, we can make the dye fluoresce by illuminating it with a laser (emitting a specific wavelength of light), and capture the resulting fluorescent signal using detectors. 

Method 4: live confocal microscopy 

Confocal microscopy can also be used to see mycorrhizal structures in live roots. This technique is non-destructive, so we can take images of the living plant and AM fungus over long time periods, revealing their growth and behaviours. The first step is germinating seeds of a 'reporter' plant. Reporter plants contain a fluorescent marker that will outline the AM fungi when they are inside root cells. The reporter plants are grown together with AM fungi in a small chamber with a window in the base that fits into a microscope. The window allows us to view the plant roots, and use confocal microscopy to illuminate and magnify the fluorescent marker.