Time-resolved endogenous chlorophyll fluorescence sensitivity to pH: study on Chlorella sp. algae.

To better understand pH-dependence of endogenous fluorescence of algae, we employed spectroscopy and microscopy methods, including advanced time-resolved fluorescence imaging microscopy (FLIM), using green algae Chlorella sp. as a model system. Absorption spectra confirmed two peaks, at 400-420 nm and 670 nm. Emission was maximal at 680 nm, with smaller peaks between 520 and 540 nm. Acidification led to a gradual decrease in the red fluorescence intensity with the maximum at 680 nm when excited by 450 nm laser. FLIM measurements, performed using 475 nm picoseconds excitation, uncovered that this effect is accompanied by a shortening of the tau1 fluorescence lifetime. Under severe acidification, we also noted an increase in the green fluorescence with a maximum between 520-540 nm and a shift toward 690-700 nm of the red fluorescence, accompanied by prolongation of the tau2 fluorescence lifetime. Gathered data increase our knowledge on the responsiveness of algae to acidification and indicate that endogenous fluorescence derived from chlorophylls can potentially serve as a biosensing tool for monitoring pH change in its natural environment.