Three dimensional quantification of biological samples using micro-computer aided tomography (microCT)

MicroCT is increasingly being used to observe soft animal and plant tissues. Conventional electron and light microscope staining protocols used to enhance the contrast of soft tissues have the potential to be adapted for use in microCT. This would increase the versatility of the microCT beyond improving qualitative observations to facilitating quantitative analysis of soft tissues. This paper describes the development of a culture system and staining protocol which has successfully been used to obtain three dimensional (3-D) quantitative data of filamentous and zoosporic soil fungi. The fungi were grown in an artificial matrix that was developed to simulate the particulate nature of soil. The combination of high contrast staining protocol and use of an X-ray translucent matrix allowed for 3-D qualitative and quantitative analysis of fungal growth. A salient point raised by this study is that the effectiveness of a protocol is reliant on the tissue or cell culture system which includes the composition of the sample, the sampling vessel, the depth of a sample and the combination of stains used. The potential use of this method extends to other fields where distribution and growth patterns in 3-D need to be quantified.

► OsO4 vapors increased the X-ray attenuation of the soil fungi grown in a polystyrene matrix.
► Soil fungi are able to grow in a polystyrene matrix when sufficient nutrients are available.
► PAS stain enhanced the X-ray attenuation of the extracellular matrix secreted by some fungi.
► The microCT was used to obtain 3-D volume and surface area ratios of soil fungi grown in an artificial matrix.