A novel dual-excitation pulse-amplitude-modulation fluorometer for investigating photosynthesis of plants

A modular instrument was developed to measure the fluorescence yield in plants subject to a combination of two harmonically-oscillating blue lights with independently controlled frequencies and phases.It uses the pulse-amplitude-modulation (PAM) method to measure the fluorescence yield independently of the plant irradiance. Compared to existing commercial instruments, it uses a higher measuring frequency (≈ 60 kHz) and higher measuring flash irradiance. This enables averaging over a number of subsequent measurement data points to achieve a higher signal-to-noise ratio. The manuscript describes the design, testing, and characterization of the operational limits of the instrument. It identifies its current weaknesses and makes recommendations for improvements. It is accompanied by supplementary materials containing the electronic schematics and the source code. The instrument was used to study a plant response to a mixture of two oscillating lights. It resulted in an excellent signal-to-noise ratio of the measured fluorescent yield. The measurements clearly demonstrated that the fluorescence yield of a plant subject to a combination of two harmonically-oscillating lights is not the same as the sum of the responses to the two oscillating lights applied independently. The observed non-linearity leads to the important conclusion that the time- and frequency-domain cannot be connected by a Fourier transform. Therefore, the frequency-domain approach will yield novel information that is not redundant to the well-established time-domain measurements.