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

Abstract

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.

Biosensing the Presence of Metal Nanoparticles by Spectrally- and Time-Resolved Endogenous Fluorescence in Water Moss Fontinalis antipyretica

“…In this contribution, FLIM recordings showed that most of the fluorescence signal in the Fontinalis antipyretica was situated inside the cell chloroplasts of the moss leaves. In control conditions, moss leafs exhibited short fluorescence lifetimes (200-350 ps), which is in agreement with others [36,[45][46][47], as well as with our findings in algae [19,20]. In pine wood middle lamella, lignin was also shown to have a 300 ns lifetime.…”

“…This finding is in agreement with previous observations that the main fluorescence recorded naturally in all plants is derived from chlorophyll a, a lightabsorbing pigment [15]. Chlorophyll fluorescence is highly sensitive to changes in the functional state and we employed it previously as a biosensing tool for tracking algae responsiveness to environmental modulators [20]. In the presented study, heavy metal NPs Co and Cu significantly lowered the chlorophyll fluorescence intensity.…”

“…At the same time, most of the fluorescence decayed at tau1. Such fluorescence lifetime situated between 200 and 500 ps (Figure 4B) which corresponds well to the chlorophyll fluorescence [20,36]. In addition to fluorescence lifetime histograms (Figure 4B), we therefore also evaluated amplitudes related to the fixed lifetime of 100 ps (close to low lifetime values recorded in conditions such as Cu) and 350 ps (recorded in most conditions, including the control ones).…”

“…Shortening of the chlorophyll fluorescence lifetime was noted following water stress in plants [48]. Our previous results in algae demonstrated a decrease in the chlorophyll fluorescence with acidification, related to the shortening of the fluorescence lifetimes [20]. Heavy metals are known to affect the chloroplast ultrastructure, causing lipid peroxidation in photosynthetic membranes, degradation of photosynthetic pigments, inhibition of photosystem II (PSII) activity and electron transport, thus restraining the net photosynthetic rate [49].…”

“…In addition to the chlorophyll fluorescence, NADPH dependent blue-green fluorescence (BGF) was also described in isolated chloroplasts [16,17], while most of the green fluorescence in the intercellular space was associated with lignins of the intermediate lamella [18]. We previously demonstrated a sensitivity of endogenous fluorescence to various environmental stressors employing the spectrally and time-resolved microscopy in algae [19,20]. In this contribution, we focus on endogenous fluorescence, derived primarily from chlorophylls, but also from flavonoids and/or lignins, studied by spectrally-and time-resolved imaging in moss.…”