Plate-Based Imaging Assay for Quantifying Photoperiodic Responses in Neurospora crassa Using Protoperithecia...

Neurospora crassa is a well-established model organism for studying biological timing and light-regulated development. The Protoperithecia Assay (PPA) is a plate-based imaging method for quantifying photoperiodic responses by measuring protoperithecia formation as a developmental output. The goal of this protocol is to provide a reproducible and scalable workflow for assessing how defined light-dark cycles influence fungal sexual development. In this assay, fungal cultures are grown on synthetic crossing medium under controlled photoperiod conditions, followed by standardized removal of surface conidia to improve visualization. Protoperithecia are quantified by manual image-based counting, and average counts per plate are used for statistical comparisons across photoperiod conditions. Unlike prior approaches, the PPA integrates standardized culture conditions, controlled photoperiod exposure, and quadrant-based imaging to enable reproducible quantitative comparisons across strains. This workflow extends beyond a basic protocol by establishing a consistent and scalable framework for measuring photoperiod-dependent developmental output under defined experimental conditions. The method requires approximately one week from inoculation to analysis and can be applied across multiple strains under defined environmental conditions. The PPA provides a simple and accessible platform for comparative analysis of photoperiod-dependent development in fungi under controlled laboratory conditions.