ChloroSpec: A new in vivo chlorophyll fluorescence spectrometer for simultaneous wavelength‐ and time‐resolved detection

Chlorophyll fluorescence is a ubiquitous tool in basic and applied plant science research. Various standard commercial instruments are available for characterization of photosynthetic material like leaves or microalgae, most of which integrate the overall fluorescence signals above a certain cut‐off wavelength. However, wavelength‐resolved (fluorescence signals appearing at different wavelengths having different time dependent decay) signals contain vast information required to decompose complex signals and processes into their underlying components that can untangle the photo‐physiological process of photosynthesis. Hence, to address this we describe an advanced chlorophyll fluorescence spectrometer ‐ ChloroSpec ‐ allowing three‐dimensional simultaneous detection of fluorescence intensities at different wavelengths in a time‐resolved manner. We demonstrate for a variety of typical examples that most of the generally used fluorescence parameters are strongly wavelength dependent. This indicates a pronounced heterogeneity and a highly dynamic nature of the thylakoid and the photosynthetic apparatus under actinic illumination. Furthermore, we provide examples of advanced global analysis procedures integrating this three‐dimensional signal and relevant information extracted from them that relate to the physiological properties of the organism. This conveniently obtained broad range of data can make ChloroSpec a new standard tool in photosynthesis research.