Perithecial development in Neurospora crassa, as observed by thick sectioned correlative microscopy

The development of sexual fruiting bodies is a critical stage in completing the life cycle of many filamentous fungi. During the sexual phase of Neurospora crassa development, the immature fruiting body (protoperithecium) significantly increases in size to eventually form the pear-shaped melanised structure comprised of layered cell wall material engulfing a centrum, in which ascogenous tissue develops. Mature ascospores are shot from the ostiloe (an opening at the top of the perithecial neck). While light and electron microscopy are commonly used to analyse perithecial structure, each have their limitations in providing detailed analysis of these structures. Using a thick sectioning (20-30 µM) procedure, we have established a protocol that provides the possibility of combining light and scanning electron microscopy to improve the visualization of developing perithecia.

We used cryostat sections with mild sample preparation. Tissue samples were either pre- or post-stained with fluorescent markers. Following light microscopy imaging tissue samples were further prepared for high resolution scanning electron microscopy (SEM) imaging. The combination of these methods allows the assessment of the integrity of the layered cell wall, ascospore development and orientation and provides the option for detailed structural analysis of normal and impaired fruiting body development. This simple correlative microscopy approach can be widely applied to the study of samples of other tissues and can also be combined with gene/protein localisation for high resolution tissue morphology analyses.