PACMan: A software package for automated single‐cell chlorophyll fluorometry

Abstract

AbstractMicroalgae, small photosynthetic unicells, are of great interest to ecology, ecotoxicology and biotechnology and there is a growing need to investigate the ability of cells to photosynthesize under variable conditions. Current strategies involve hand‐operated pulse‐amplitude‐modulated (PAM) chlorophyll fluorimeters, which can provide detailed insights into the photophysiology of entire populations‐ or individual cells of microalgae but are typically limited in their throughput. To increase the throughput of a commercially available MICROSCOPY‐PAM system, we present the PAM Automation Control Manager (‘PACMan’), an open‐source Python software package that automates image acquisition, microscopy stage control and the triggering of external hardware components. PACMan comes with a user‐friendly graphical user interface and is released together with a stand‐alone tool (PAMalysis) for the automated calculation of per‐cell maximum quantum efficiencies (=Fv/Fm). Using these two software packages, we successfully tracked the photophysiology of >1000 individual cells of green algae (Chlamydomonas reinhardtii) and dinoflagellates (genusSymbiodiniaceae) within custom‐made microfluidic devices. Compared to the manual operation of MICROSCOPY‐PAM systems, this represents a 10‐fold increase in throughput. During experiments, PACMan coordinated the movement of the microscope stage and triggered the MICROSCOPY‐PAM system to repeatedly capture high‐quality image data across multiple positions. Finally, we analyzed single‐cellFv/Fmwith the manufacturer‐supplied software and PAMalysis, demonstrating a median difference <0.5% between both methods. We foresee that PACMan, and its auxiliary software package will help increase the experimental throughput in a range of microalgae studies currently relying on hand‐operated MICROSCOPY‐PAM technologies.