Quantification of subaerial algae on building materials by a new standardised imaging Pulse-Amplitude-Modulation Fluorometry (PAM-F) method

Imaging Pulse-Amplitude-Modulation Fluorometry (PAM-F) is a quantitative, sensitive, and non-destructive tool for detecting early biofilm establishment, yet its broader use has been limited by the lack of standardised protocols and by uncertainties arising from material-specific effects. In this study, the first comprehensive protocol is presented considering substrate properties and achieving reproducible dark fluorescence yield (F 0 ) measurements even for low algal cell densities. Our workflow combines standardised sample preparation with an algorithm-driven evaluation to generate calibration curves, detection limits, and optimal device settings. By applying two defined inoculation ranges of the subaerial green alga Jaagichlorella sp. AB13.021D5 to marble and concrete pellets, and polycarbonate membranes, we show that F 0 intensity reflects not only algal abundance and device settings but also physical and mineralogical substrate properties. Particularly the high reflectance of marble increased noise and oversaturation effects, demonstrating the necessity of substrate-specific calibration. This transferable protocol establishes Jaagichlorella sp. AB13.021D5 as a test organism for comparative and application-oriented bioreceptivity studies (such as evaluating material susceptibility, comparing protective treatments, and monitoring early colonisation). By delivering validated measurement settings as well as substrate-specific calibration curves and detection limits for Imaging PAM-F studies, this work closes a critical methodological gap and supports the development of evidence-based biodeterioration management strategies.