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 Methods Appl. Fluoresc. 8 (2020) 024007 https://doi.org/10.1088/2050-6120/ab77f4

PAPER

Time-resolved endogenous chlorophyll fluorescence sensitivity to

pH: study on Chlorella sp. algae
OPEN ACCESS

RECEIVED
12 April 2019
A Marcek Chorvatova1,2 , M Uherek1, A Mateasik1 and D Chorvat Jr1
REVISED 1
9 January 2020
Department of Biophotonics, International Laser Centre, Ilkovicova 3, 84104 Bratislava, Slovakia
2
Department of Biophysics, Faculty of Natural Sciences, University of Ss. Cyril and Methodius, nam. J Herdu 2, 91701 Trnava, Slovakia
ACCEPTED FOR PUBLICATION
19 February 2020 E-mail: [email protected]
PUBLISHED
Keywords: FLIM, chlorophyll, Chlorella sp. algae, endogenous fluorescence, pH
2 March 2020
Supplementary material for this article is available online
Original content from this
work may be used under
the terms of the Creative
Commons Attribution 4.0 Abstract
licence. To better understand pH-dependence of endogenous fluorescence of algae, we employed spectrosc-
Any further distribution of
this work must maintain
opy and microscopy methods, including advanced time-resolved fluorescence imaging microscopy
attribution to the (FLIM), using green algae Chlorella sp. as a model system. Absorption spectra confirmed two peaks, at
author(s) and the title of
the work, journal citation 400–420 nm and 670 nm. Emission was maximal at 680 nm, with smaller peaks between 520 and 540
and DOI.
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.

Introduction significantly affect algae functioning. Namely, acid

rain is becoming a rising concern, as the dissolution of
Natural sensing of changes in variable environmental carbon dioxide in water creates H2CO3 molecules, a
conditions can provide a very effective tool for weak acid that is causing the rain to be slightly acidic.
monitoring of potential environmental risks, also Previous studies show that environmental pH is sig-
including changes in the pH. Use of endogenous nificantly affecting different algae [1]. Change in the
fluorophores requires no colouring and can easily be external pH affects growth, photosynthesis, as well as
employed in living cells and organisms. However, photosynthetic electron transport [2]. pH has effects
while monitoring fluorescence intensity is often non- on the growth of Chlorella vulgaris [3] and also affects
specific, fluorescence lifetimes were shown to respond chloroplast photosynthesis [4]. The most of algae
well to changes in the fluorophore environment. For function in alkaline lakes and reach optimum growth
that reason, we opted to test the sensitivity of and the photosynthetic rate at a neutral or alkaline pH.
fluorescence lifetimes to environmental modifications Only a limited number of algae species is acid-resistant
in Chlorella sp. algae. [5]. pH variations were shown to induce significant
Algae are aquatic, plant-like organisms ranging changes in the chlorophyll state of the photosystem II
from single-cell phytoplankton to large seaweeds that (PSII) membrane [6]. Changes in pH also affect the
can be found in both freshwater and marine environ- chlorophyll quenching [7] and chlorophyll states [6].
ment, even in snow, anywhere on Earth. The best During photosynthesis, algae employ various Light
known are green algae that produce about half of Harvesting Complexes (LHC) to capture photons
Earth’s oxygen. Chlorella sp. belongs to green algae from light and transfer energy to Photosystem I and II
species. Changes in the acidic environment can (reviewed in [8, 9]). LHC antennas bind chlorophylls.

© 2020 The Author(s). Published by IOP Publishing Ltd

Methods Appl. Fluoresc. 8 (2020) 024007 A Marcek Chorvatova et al

Under stress conditions, a sub-group of LHCs is the conditions of environmental stress, rather than
expressed [10] and has been shown to trigger the pro- precisely understand the provenance of each of the
cess of nonphotochemical quenching of the chlor- resolved lifetime. This approach will be of utmost
ophyll fluorescence [11]. These proteins are mainly practical importance for potential in-field biosensing
activated by high light exposure [12] to tackle the applications.
potential oxidative stress, but they were also proposed
to sense pH changes in the thylakoid lumen and, at low
pH [13–15], switching conformation to activate the
Material and methods
quenching process.
Samples
Monitoring changes in pH by studying its effect on
Chlorella sp. was gathered from the University of Ss.
the algae endogenous fluorescence can provide useful
Cyril and Methodius in Trnava, Faculty of Natural
information for natural biosensing. Chlorophyll a, the
Sciences collection of green algae. The green algae of
primary source of endogenous fluorescence, is
genus Chlorella sp. were previously isolated from the
responsible for photosynthesis [16]. There is a rela-
main drinking water supply. Green algae were culti-
tionship between pH, CO2 and photosynthesis [17]. In
vated in the Hoagland cultivation medium [28], as
sunlight, CO2 decreases and the water becomes more
described below.
alkaline, while in the dark the opposite occurs [17].
Endogenous fluorescence of chlorophylls, emitted in
red and far-red when excited by light, allows gathering Solutions
information on the health state of the photosynthetic The medium was prepared according to [28]; all
organisms. Sensing systems, based on non-invasive chemicals needed for the preparation of Hoagland
and often remote monitoring of chlorophyll fluores- cultivation medium were from Lachema (Czech
cence [18] can, therefore, provide helpful information Republic); the medium contained: NaNO3, CaCl2 . 2
about the effect of stress induced by the environmental H2O; MgSO4 . 7H2O; K2HPO4 . 3 H2O, KH2PO4,
factors, such as a change in the pH. NaCl, H3BO3, FeSO4 . 7H2O, H2SO4, ZnSO4 . 7H2O,
Time-resolved fluorescence imaging presents a MnCl2 . 4H2O; (NH4)6Mo7O24 . 4H2O; CuSO4 .
novel tool for evaluation of the sensitivity of the endo- 5H2O, Co(NO3)2. 6H2O, EDTA, KOH.
genous fluorophores to their environment in living Other chemicals included NaCl, H2O2 and urea -
cells and thus brings potentially very useful informa- NH2CONH2 (all from Centralchem), citric acid,
tion for the examination of changes in response to sodium bicarbonate - NaHCO− 3 and dimethylsufoxide

stress conditions [19, 20]. Mean fluorescence lifetime - DMSO (all from Sigma-Aldrich).
of chlorophyll was found between 0.22–0.32 ns in sor-
ghum to 0.55–0.64 ns in maize [21], while two fluores- Instrumentation
cence lifetimes, 0.262 and 0.728 ns, were found in pH adjustment and measurement
algae [22]. Fluorescence lifetimes for in vivo chlor- The pH was verified by the pH meter (Mettler Toledo
ophyll a using TCSPC were described to be almost MP220, USA) and adjusted to different values between
monoexponential with 0.490 ns in dark-adapted 2 and 9 using citric acid and/or sodium bicarbonate -
chlorella, while chloroplast showed a two-component NaHCO3. In control, algae reached between pH 7.8
decay of 0.410 ns and at around 1.4 ns [23]. We have and 8.8. At least 10 images were studied in each
previously demonstrated the application of fluores- condition, with the summed number of cells in each
cence spectroscopy and time-resolved microscopy condition ranging between 500 (low pH) and 1000
tools to record endogenous fluorescence in the Chlor- (high pH). Environmental modulators capable to
ella sp. algae [24]. We also discussed the potential change pH were employed, namely 10% H2O2
usability of the endogenous fluorescence as a biosen- (pH 5.68, n = 12), 10 mM urea–NH2CONH2
sing tool for tracking algae responsiveness to mod- (pH 7.95, n = 12), or 3.5 M NaCl (pH 8.20, n = 6).
ulators [25]. Recently, we have tested the cell response Using modulators, control cells were at pH 7.80
of marine algae Dunalliela tertiolecta to laboratory- (n = 36), while citric acid was used to adjust pH 2.06
induced stress with cadmium heavy metal [26]. (n = 9) and NaHCO3 to pH 8.21 (n = 6). Before
In the present study, our goal was to evaluate the recording, cells were in solution with variable pH for
sensitivity of the endogenous fluorescence to pH using 20–60 min. All experiments were done at the room
time- and spectrally-resolved microscopy methods in temperature.
a model organism—the sweet water green algae Chlor-
ella sp. Application of the measurement of endogenous Absorption and emission spectrofluorimetry
fluorescence of algae combined with advanced spec- Absorption and fluorescence spectra were recorded on
tral pattern-recognition approach [27] can help to cre- Shimadzu UV-2100 and Horiba-Jobin Yvon SPEX
ate natural biosensors for environmental changes (as Fluorolog 3–11 spectrometers respectively, with the
described in [25]). Employing time- and spectral reso- system’s wavelength response correction. The fluores-
lution, we focused on identifying representative pat- cence spectra were corrected for the wavelength
terns of fluorescence lifetime and spectral changes in response of the system. Excitation at 375 nm, 488 nm

2
Methods Appl. Fluoresc. 8 (2020) 024007 A Marcek Chorvatova et al

and 633 nm, and emission at 560 nm and 670 nm was org/MAF/8/024007/mmedia). The photobleaching
employed for measurement of emission and excitation was induced by consecutive scanning using 100% laser
spectra, respectively. power (as opposed to 10% under classical exper-
imental conditions). Following such irradiation, pho-
Confocal imaging tobleaching induced a clear decrease in the red
Confocal autofluorescence images were gathered with fluorescence after the 3rd scan (about 1.5 min of expo-
the laser scanning confocal microscope Axiovert 200 sure). Longer scanning (for up to 10 min) led to an
using a 16 channel LSM 510 META detector (Carl increase in the green fluorescence with longer (around
Zeiss, Germany) equipped with C-Apochromat 40×, 2 ns) lifetime (supplement figure S1(B)). To prevent
1.2 NA lens. Single cells were excited with the 450 nm such situation, we used the laser only at 10% of its
single-mode laser diode (Kvant, Slovakia). Channel 1 maximal power and we did not perform more than 3
recorded light with BP 500–550 nm bandpass filter experimental protocols on the same sample. We were
with the pinhole opening of 2.28 AU, while channel 2 therefore convinced that there was a minimum of the
detection ranges were 650–710 nm with the pinhole effect of irradiation and thus photodamage in the pre-
opening of 1.02 AU. Maximal laser power density used sented results.
for sample excitation was 1580 Wm−2 for 450 nm Mean power density of the 475 nm laser used in
laser line. FLIM experiments was comparable to that of CLSM
Spectrally-resolved images were taken with 11 nm imaging, with corresponding mean photon fluence
step between 499–573 nm for the green, 638–713 nm 2.2×1019 photons m−2 s−1 (5.2×103 photons/
for the red spectral regions, and 477–633 nm for the pulse). Moreover, due to beam scanning in both
complete spectrum. The spectra were evaluated in 5 CLSM and FLIM imaging regimes, one pixel of the
randomly selected images of cells in each condition. sample was irradiated only for tens of microsecond
Experiments were performed on glass coverslips after long period per each second of the experiment. These
algae immobilisation with the polyethylenimine precautions prevented artefacts related to the effect of
(Sigma-Aldrich, PEI) surfaces (0.2%). irradiation. These results demonstrated that the light
exposure under our experimental conditions is unli-
Fluorescence Lifetime Imaging Microscopy (FLIM) kely to induce significant irradiation artefacts.
FLIM images were recorded using the time-correlated
single-photon counting (TCSPC) technique [20]. In Data analysis
these experiments, a 475 nm picosecond laser diode Confocal data were visualized by ZEN 2011 software
(BDL-475, Becker&Hickl, Germany) was used. The (Zeiss, Germany). Fluorescence intensity from con-
laser beam was reflected in the sample through an focal images was analysed using image segmentation
epifluorescence path of the Axiovert 200 LSM 510 method, where only fluorescence intensities recorded
Meta (Zeiss, Germany) inverted microscope with from fluorescing algae (without surrounding back-
C-Apochromat 40×, 1.2 NA lens. The emitted fluores- ground), was measured.
cence was separated from laser excitation using LP 500 FLIM images were processed using commercially
nm or BP 650–750 filter and detected by HPM 100–40 available software package SPCImage (Becker&Hickl,
photomultiplier and SPC-830 TCSPC board (both Germany) or using a custom-made software. Results
Becker&Hickl, Germany). Mean power density at the were visualized as a map and as a distribution of calcu-
sample of 475 nm laser used for FLIM experiments lated fluorescence lifetimes. Intensities are presented
was 230 Wm−2, with 20 MHz repetition rate and as the mean and the standard error of the mean. Decay
approx. 60 ps pulse length with<0.1 pJ/pulse curves were fitted with a two-exponential fitting
energy. model with plausible χ2 using image segmentation
and home-made lifetime analysis, as specified in the
Result section.
Irradiation conditions
Statistical comparison was done using one-way
Illumination conditions were chosen to minimize the
Anova, with p < 0.05 considered as significant.
effect of laser-induced irradiation on algae. The CLSM
experiments were done using the mean local power
density of the lasers lower than the power density of Results
solar radiation during daylight at visible wavelengths
(400–500 Wm−2). Photon fluence in our conditions Our aim is to discern pattern behaviour of time- and
was 1.4×1019 photons m−2 s−1 for the blue (450 nm) spectrally-resolved characteristics of the endogenous
laser running at 10% of its maximal power, which fluorescence of algae to environmental stress to design,
corresponds to 160 Wm−2 power density at the sample in the future, biosensors sensing environmental
plane of 40×/1.2 objective. changes directly in living organisms. In this contrib-
We also studied the irradiation during imaging by ution, we specifically focus on the effect of acidifica-
laser-induced photobleaching (see supplementary tion on behaviour of single cells of green algae
material, figure S1(A) is available online at stacks.iop. Chlorella sp.

3
Methods Appl. Fluoresc. 8 (2020) 024007 A Marcek Chorvatova et al

Figure 1. Fluorescence spectroscopy of endogenous fluorescence of algae Chlorella sp. A) Absorption spectrum at 300–700 nm (grey),
compared do excitation spectrum recorded at emission 670 nm (black). B) Emission spectra measured following excitation 375 nm
(black), 450 nm (light grey) and 630 nm (dark grey).

Absorption and emission properties of Chlorella sp. nm, possibly linked to NADPH-related flavins, flavo-
endogenous fluorescence noids, carotenoids and/or phenolic plant sub-
Evaluation of absorption properties, recorded between stances [32].
300–700 nm revealed the main absorption at 400–450
nm, with a maximum between 400–420 nm and a Recording of endogenous fluorescence intensity in
second peak at 670 nm (figure 1(A) grey). Excitation Chlorella sp
spectrum was measured at emission 670 nm and To record endogenous fluorescence in Chlorella sp.
recorded between 300–650 nm (figure 1(A) black). It algae, we have employed confocal imaging following
confirmed the absorption measurements, with max- excitation at 450 nm at two emission windows based
imum excitation at wavelengths ranging from ultra- on absorption and emission properties recorded at
violet (UV) to 450 nm and a second increase above 560 figure 1. The emission window of 500–550 nm of the
nm. Gathered data were in good agreement with the channel 1 was designed to record the green fluores-
expected properties of chlorophylls [16]. cence, while the emission window of 650–710 nm of
Emission spectra were recorded at various excita- the channel 2 was chosen to record the red chlorophyll
tions: 375 nm, 450 and 630 nm (figure 1(B)). Measure- fluorescence (figure 2(A)). It is important to precise
ments revealed maximum emission of algae at 680 nm, that the intensity in the green channel was very low;
in accordance with the major fluorescence band for consequently, the pinhole for the green channel was
the chlorophyll a [29, 30]. Excitation at 375 nm also set to 2.28 AU (Versus 1.02 AU for the red channel), to
uncovered a second peak at 420 nm, most likely rela- read comparable intensities on both channels. In order
ted to NADPH [31]. In addition, blue excitation at 450 to take into consideration the fluorescence from
nm stimulated weak emission with a maximum at 540 individual algae only, an image segmentation method

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Methods Appl. Fluoresc. 8 (2020) 024007 A Marcek Chorvatova et al

Figure 2. Fluorescence microscopy of endogenous fluorescence of algae Chlorella sp. A) Image recorded by laser scanning confocal
microscopy in control conditions, excitation 450 nm. Emission: Aa) channel 1: 500–550 nm, Ab) channel 2: 650–710 nm, Ac) channel
3: transmission image, Ad) channel 4: overlay of channels 1–3; scale 20 μm. B, C) pH dependence of endogenous fluorescence of
Chlorella sp. algae gathered by excitation at 450 nm, analysed using image segmentation method at the emission of B) 650–710 nm
(channel 2), C) 500–550 nm (channel 1).

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Methods Appl. Fluoresc. 8 (2020) 024007 A Marcek Chorvatova et al

Figure 3. Spectrally-resolved images of endogenous fluorescence of Chlorella sp. algae. (A) Original images gathered by excitation at
450 nm with 11 nm step between 638–713 nm. (B) pH dependence of spectrally-resolved images of the red endogenous fluorescence
of Chlorella sp. algae between 638–713 nm (composite image, scale: 20 μm). (C) Spectra of the red fluorescence recorded at various
pH (bcg corresponds to the background, shift to cells with observed shifted spectra). Not corrected for background. (D)
pH dependence of intensity at 680 nm, gathered from spectra recorded at C.

was employed. Channel 3 served to monitor algae Recordings and measurements of fluorescence
appearance in transmission. The contour of the cells, lifetimes of Chlorella sp
estimated by image segmentation, reached between FLIM images, recorded by TCSPC, were employed to
9–10 μm, with the cell surface averaging between 9–11 gather fluorescence lifetimes following excitation by
μm2 (supplement figure S2), indicating that the 475 nm picoseconds laser (figures 4Aa). The analysis
diameter of the cells was under 4 μm. Such a small size was performed using a custom-made approach, to
of the algae did not allow demonstration of the spatial allow automatization of the procedure in varying
differences in the distribution of the two sources experimental conditions. Measured FLIM images were
(green and red) of endogenous fluorescence. firstly segmented to delineate boundaries and pixels
To evaluate the spectrum of the red fluorescence, belonging to each chlorella body in the image,
comparably to approach employed for fluorescence
we recorded confocal images at separate fluorescence
evaluation. Segmentation was done using simple Otsu
wavelength between 638 nm and 713 nm using an 11
thresholding approach [33] on the fluorescence inten-
nm step (figure 3(A)). The red fluorescence peaked at
sity image that was built by summation of fluorescence
680 nm (figure 3(C)), corresponding well to the fluor-
signal across all time channels of corresponding FLIM
escence of chlorophyll a [30]. An attempt to record the image. Segmented intensity image was further used to
green fluorescence separately from the red one (sup- construct a binary image mask that enables to sum
plement figure S3(A)–(C)), unmasked fluorescence decay signals corresponding to pixels of individual
peaking between 520–540 nm. These data were in chlorella body for each chlorella object in the image.
agreement with our previous recordings of endogen- Final summed decay signals were then analysed with
ous fluorescence in the algae Chlorella sp. [24, 25]. In two-component multi-exponential model separately
the latter study, we showed that the green fluorescence for each chlorella object in image and results were
increases in the presence of stress conditions, such as presented as average values from all the chlorella
chemical bleaching by sodium hypochlorite (SAVO). bodies found in image. Data fitting was done using

6
Methods Appl. Fluoresc. 8 (2020) 024007 A Marcek Chorvatova et al

Figure 4. Fluorescence lifetime imaging microscopy (FLIM) of endogenous fluorescence of Chlorella sp. (A) FLIM image showed in
the range 200–1200 ps in control condition recorded using Aa) LP500 filter or Ab) BP650-750 filter (b) following excitation at 475 nm.
(B) Example of fitting of the measured fluorescence decay by a custom-made software (original data: black points, fitted curve: red
line) (a). Comparison of fluorescence decays in chosen individual cells at different pH (b).

custom software exploiting open source NLOPT 2-exponential decay to analyse data gathered in this
library [34]. As an instrument response function in study.
data fitting procedure, the synthetic IRF function To record FLIM images (figures 4Aa), we
produced by SPCImage software (Becker Hickl, Ger- employed an LP500 filter. The choice of the filter was
many) was used. Example of the employed custom- done to block the excitation laser of 475 nm and, at the
made fit (figures 4Ba), as opposed to a fit performed same time, to accumulate a maximum of photons. We
using commercially available SPCImage software tested also the use of BP 650–750 (figures 4Ab), which
(Becker Hickl, Germany, supplement figure S4 and lead to a decrease in the number of photon counts,
table S1) shows a good correlation between the two without a change in the a1/a2 and slight, but not a sig-
approaches; main difference concerns the fitting of a nificant increase in the two fluorescence lifetimes.
very low number of photons with long lifetimes. Such lifetime increase could be related to many effects,
To analyse gathered data, the double-exponential including blocked green emission, better suppression
analysis was used. The choice of the number of fluor- of the excitation light and lowering of the fluorescence
escence lifetimes was based on the best χ2 in most of intensity when recordings were performed in the pre-
the recorded experimental conditions. Such choice sence of the red filter. The number of photons in the
was not an easy issue. When our approach was com- trace of the presented images reached 32–42 000 when
pared to a commercially available one in a randomly using the green filter (figures 4Aa), but when using the
selected Chlorella sp. cell, we reached good agreement red filter, the number of photons in the trace was only
with resolved tau/a (see Supplement table S1). We tes- 22–32 000 (figures 4Ab). This result, together with the
ted also the possibility to resolve data using analysis by one gathered by measurements of spectral character-
3 exponentials. However, the number of photons was istics (figure 3(C)) indicates that 2/3 of the fluores-
not sufficient for using 3 exponential analysis in all cence recorded in control FLIM images were derived
samples and also, in most cells, 3 exponentials did not from photons with a wavelength between 650–750
give better χ2. That is why we have decided to employ a nm, which correspond to chlorophylls, namely

7
Methods Appl. Fluoresc. 8 (2020) 024007 A Marcek Chorvatova et al

chlorophyll a. However, in conditions when the red was also shown [37, 38]. When recorded at a max-
fluorescence was inhibited and rise in the green fluor- imum of 680 nm, we observed a gradual decrease in
escence occurred, the green fluorescence could also the fluorescence intensity with the pH (figure 3(D)).
contribute to the resolved lifetimes in FLIM images Observed fluorescence thus suggest a gradual shut
recorded using LP500. down of the PSII at low pH.
At the same time, it is important to reiterate that Green fluorescence spectra (supplement figure
we aimed to find the behavioural pattern of the endo- S3(C)), measured between 499–573 nm, showed two
genous fluorescence lifetimes in the presence of stres- peaks: first at 510–520 nm and second at 540 nm. We
sors, not to resolve individual components related to noted little effect under acidification; only at pH 2 a
specific pigments. Presented lifetime analysis was significant increase in the green fluorescence was
therefore mainly intended to obtain ‘pattern-like’ recorded at all studied wavelength, suggesting a possi-
information on the potential change of the lifetime ble damage to the cell at very acidic pH.
map in different environmental conditions than to
precisely discern individual pigments based on their pH-dependence of Chlorella sp. endogenous
specific lifetime. fluorescence lifetimes
When FLIM data were sorted as a function of pH in
pH-dependence of Chlorella sp. endogenous individual conditions, we have observed a decrease in
fluorescence intensity the fluorescence intensity following acidification
We have then evaluated changes in the acidic and basic (figures 4(A) and 5(A)). Tau 1 fluorescence lifetime
environments. Sodium bicarbonate is crucial for the presented shortening (figure 5(B) black) in a quasi-
functioning of PSII, but not PSI [35]. On the other linear way. At pH 2, a possible contamination with
hand, a very acidic environment shuts down the PSII another fluorescence species (most likely bound
[36]. The pH was adjusted to specific values between 2 flavoproteins) can be assumed. For tau 2, the values
and 9, with a step of 1, using citric acid and/or sodium did not show the significant change (figure 5(C)); only
bicarbonate. For statistical comparisons, fluorescence at pH 3 and 2, we have recorded a significant rise in the
intensity from confocal images was analysed using an tau 2 (possibly due to appearance of free flavopro-
image segmentation method, as mentioned in the Data teins). This effect was accompanied by a little effect on
analysis. We recorded a gradual decrease in the red the amplitudes (figures 5(D), (E)), although an
fluorescence (figure 2(B)). Although gathered mea- increase in a1 and a decrease in a2 can be noted
surements point to a quasi-linear relationship of the at pH 3.
red fluorescence decrease with pH, with only n = 8 We wanted also to see whether this effect could be
different pH studied there were not enough points for induced by other pH regulators, such as H2O2, urea or
more advanced analysis. It was therefore not possible NaCl (as specified in Material and Method section),
to conclude on the curve’s precise shape. In this regard, capable of inducing variations of ionic environment,
it is also important to take into consideration that such as active oxygen and nitrogen. We have observed
pH scale itself is logarithmic. Green fluorescence was good correlation for the data for a1, a2, as well as tau2
stable (figure 2(C)); significant increase could only be (figures 5(C)–(E)) grey). In the case of tau1 (figure 5(B)
observed at very acidic pH of 2. Neither the contour grey), we assumed that a faster decrease in the fluores-
nor the area of the cells was significantly affected by the cence lifetime in the presence of urea and NaCl at
short-term exposure of cells to the pH change (supple- pH 7.95 and pH 8.2 indicated either the effect of
ment figure S2(A) and S2(B). We previously described sodium bicarbonate and/or that other factors (such as
a differential sensitivity of the two components of oxidative changes in the presence of H2O2, etc) addi-
endogenous fluorescence to severe stress conditions, tionally affect the cells.
namely active sodium hypochlorite [25]. The lack of In conclusion, taking into consideration that 1) the
increase in the green fluorescence at acidic pH down to major contribution to the recorded fluorescence
pH 3 indicates a good resistance of the algae to the comes from the red spectral region, as well as that 2)
short-term exposure to low pH. the gradual decrease in this fluorescence was observed
Red fluorescence spectra, recorded between 638 with pH and that 3) it was accompanied by shortening
nm and 713 nm (figure 3(C)), showed a gradual of the tau 1, all suggested that the tau 1 fluorescence
decrease in the fluorescence at 680 nm, suggesting an lifetime could be most likely attributed to the chlor-
involvement of the same molecular species in the acid- ophylls of the PSII. Shortening of the tau1 in the pre-
ification process. Interestingly, at pH 2 and 3 we recor- sence of acidification was in agreement with gradual
ded also cells in which the red fluorescence peaked at shutting down of the PSII in this condition [36]. On
690–700 nm (named as ‘shift’ at figure 3(C)), instead the other hand, prolongation of the longer lifetime in
of 680 nm. Although it is well demonstrated that most severe acidification can, at least in part, explain the rise
of the chlorophyll a fluorescence come from PSII in the green fluorescence observed in this condition.
emitting at 685 nm [30], the existence of the weak However, the shift of the red spectra towards 690 nm
fluorescence at 693–695 nm arising from PSI system in pH 2 and 3 suggests that the contribution of

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Methods Appl. Fluoresc. 8 (2020) 024007 A Marcek Chorvatova et al

Figure 5. FLIM images of endogenous fluorescence of Chlorella sp. by excitation 475 nm, LP 500 nm. (A) FLIM images showed in the
range 200–1200ps in different pH. Distribution of fluorescence lifetime (B) tau 1, (C) tau2 and fluorescence amplitudes (D) a1, (E) a2
in different pH. Modulation in the presence of variable pH (black squares) and different pH modulators (grey circles) is presented.

9
Methods Appl. Fluoresc. 8 (2020) 024007 A Marcek Chorvatova et al

photons of the chlorophyll a with longer lifetimes can- recorded in living Chlorella sp. algae cells is in agree-
not be excluded [39, 40]. ment with these observations. Using silica gel chroma-
tography measurements we demonstrated previously
[24] that main endogenous pigment in the Chlorella
Discussion sp. algae is related to the chlorophyll a; other bands
corresponding to chlorophyll b and carotenoids were
Data presented in this paper demonstrated the sensi- also found. As a result, we conclude that the main
tivity of the endogenous fluorescence intensity and its fluorescence recorded in Chlorella sp. under our
fluorescence lifetimes to acidification in Chlorella sp. experimental conditions was derived from that of the
algae, naturally presented in drinking water that is chlorophyll a.
capable of photosynthesis. The main aim of the study Fluorescence lifetime recording is a very powerful
was to test the possible use of the endogenous measurement of molecular parameters at the biologi-
fluorescence of living algae to create natural biosensors cal level. Fluorescence lifetime depends on multiple
for environmental changes (a concept described in factors: pH, temperature, oxygenation, density, pre-
[25]). We therefore mainly aimed to identify a ‘pattern’ sence or absence of quenchers, etc [reviewed in 43].
of a spectral and time-resolved fluorescence changes Our previous results demonstrated that time-resolved
in the conditions of environmental stress, rather than emission micro-spectroscopy allows fast and repro-
precisely understand the provenance of each of the ducible measurements of complex patterns of spec-
resolved lifetime. We demonstrated that short-term trally-resolved fluorescence decays directly in living
exposure to acidification resulted in lowering of the cells [44]. Monitoring fluorescence lifetimes is parti-
red fluorescence associated with shortening of the cularly advantageous when studying the responsive-
fluorescence lifetime tau1. In severe acidification ness of the system to changes in its environment while
conditions, increase in the green fluorescence, a shift remaining independent of fluorescence intensity and/
of the red fluorescence and prolongation of the or photobleaching [19]. In living organisms, using
fluorescence lifetime tau 2 was also present. picosecond excitation, the fluorescence lifetime of
Fluorescence recorded under our experimental chlorophyll a was described under normal conditions
condition was derived mainly from pigments of the in a vast range of values, often including a, short life-
photosystem II (PSII), namely chlorophylls. Chlor- time‘ between 250–600 ps and a, long lifetime‘
ophylls are light-absorbing pigments present naturally between 700–1600 ps [22, 39, 40, 45]. In the in vitro
in all plants [16]. The main forms of chlorophyll in conditions, fluorescence lifetimes of chlorophyll a/b
plants and algae are chlorophyll a and chlorophyll b, proteins were shown to have much longer lifetimes
other pigments include xanthophylls and beta-car- than in living cells and responded to the aggregation of
otenes. These highly conjugated compounds are cap- monomeric forms [46]. We have demonstrated that
able to capture light energy in the form of radiation the fluorescence lifetime of chlorophyll a band, iso-
and subsequently, in a series of transfers to other lated by chromatography from the Chlorella sp. algae,
molecules and complexes, to convert it into chemical reached monoexponential fluorescence decay of 4 ns
energy in the form of ATP. Chlorophyll fluorescence following excitation by 633 nm ps laser, which is much
is, therefore, a highly valuable non-destructive intrin- longer than that recorded directly in living cells [47].
sic probe of several aspects of oxygenic photosynthesis For that reason, only measurements performed
sensitive to change in the functional state of the algae, directly in living cells were considered.
as well as plants [30]. This knowledge can be useful for The choice of the right analysis to evaluate
evaluation of water pollution, allowing reliably mon- corresponding lifetimes in living organisms is not an
itoring of water quality, as well as for better compre- easy issue. We decided to employ a double exponential
hension of the efficient solar energy capture decay to study patterns of the fluorescence lifetime
mechanisms. Chlorophyll a fluorescence is hetero- change and we recorded shortening of the tau 1 fluor-
geneous, but its major fluorescence emission band at escence lifetime, corresponding to the decrease in the
680–685 nm and its vibrational satellite at 720–735 nm red fluorescence, as well as, in severe acidification, the
is considered to be originating mostly in the PSII prolongation of the tau2, which corresponded to the
antenna complexes [29, 30]. When PSII reaction cen- rise in the green fluorescence and/or in the red fluor-
tres are closed, the existence of the weak fluorescence escence shifted towards 690 nm. Under our exper-
at 693–695 nm was shown by Krey and Govindjee imental conditions, using 2 exponential decays, tau1
[37, 38], arising from the PSI system, related to a trans- turned out to be the most sensitive parameter, corre-
fer between chlorophyll a molecules in PSI and LHC lating with a decrease in the fluorescence intensity.
[41]. These longer emission wavelengths were pro- The amplitude a1 corresponding to this lifetime repre-
posed to originate from a long-wavelength form of sented a major (80%–90%) contribution. The tau 2
chlorophyll a rather than from vibrational bands [42]. had lower contribution and was often close to noise
Consequently, most of the chlorophyll a fluorescence values. We, therefore, consider the main result of our
(around 90%) comes from PSII, with the emission study the pH-sensitivity of the tau1 fluorescence life-
bands at 680–685 nm [30]. Endogenous fluorescence time. At the same time, it is important to note that

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Methods Appl. Fluoresc. 8 (2020) 024007 A Marcek Chorvatova et al

these changes were relatively small and their possible denature, stop working, or slow down - they can no
use for biosensing still needs to be further tested, parti- longer carry out photosynthesis in the cell to their full
cularly by also exploring long-term exposure of algae potential.
to different pH conditions. Temporal characteristics of chlorophyll fluores-
In addition to the chlorophyll fluorescence, we cence were proposed to be a biomarker for the chan-
observed also the presence of much lower, but sus- ging environment, such as Cd toxicity [55], in algal
tained green/yellow fluorescence. Previously, the cells and we thus aimed to test the potential use of the
blue-green fluorescence (BGF) was described in iso- algae endogenous fluorescence lifetimes in biosensing.
lated chloroplasts, where it showed a reversible Following water stress in plants, shortening of the
increase of BGF when illuminated with light, and it has chlorophyll fluorescence lifetime was noted from 1 ns
been proposed that this light-induced variation of to 0.45 ns [56]. On the other hand, the lifetime pro-
chloroplast BGF is completely NADPH dependent longation is in agreement with the reaction of chlor-
[48, 49]. Energy transfer from proteins to NADPH in ophyll a to stress conditions [22, 31], namely UV-
chloroplasts was proposed to affect fluorescence life- induced stress. Our results pointed to shortening of
times [50, 51]. In our experiments, green/yellow the fluorescence lifetime tau1 with pH. In photosynth-
fluorescence stimulated by excitation at 450 nm was esis, the light energy absorbed by chlorophyll is uti-
most pronounces between 510–550 nm. Observed lised in the oxidation-reduction process against a
green/yellow fluorescence can, therefore, be derived gradient of chemical potential. Change in the
from NADPH-related flavins and flavonoids, presence pH affects this homeostasis by shutting down the PSII
of carotenoids [52], or lipid accumulation [53], but [36]. The decrease in red fluorescence at low pH is
can also result from degradation products. This fluor- most likely resulting from inhibition of the PSII and/
escence can affect the measurement of chlorophyll or decrease in the photosynthesis rate. This conclusion
fluorescence only under conditions of a very low red is also supported by the observed red-spectral shift
fluorescence, either due to bleaching or in conditions from 680 nm to 690–700 nm in the conditions of
where chlorophyll fluorescence is inhibited. We pre- severe acidification, which also points to shut down of
viously observed an increase in this fluorescence under PSII. Other stressors leading to modulation of
conditions of severe stress during chemical photo- pH revealed comparable effects on a1, a2 and tau 2. In
bleaching in the Chlorella sp. algae [25]. Also, laser- the case of tau1, their effect was stronger, possibly due
induced photobleaching leads to an increase in the to their other action on the oxidation of cells, etc.
green fluorescence associated with the prolongation of We hypothesize that quenching due to shutting
the tau2 fluorescence lifetime (supplement figure S1). down of the PSII during acidification is the possible
For this reason, we assume that the green fluorescence mechanism involved. Stress-related LHCs, which
could contribute to the tau 2 lifetime and, namely in serves as feedback mechanisms that dissipate excess
conditions when the red fluorescence is quenched, photoexcitation and avoid detrimental oxidative
also partially to the tau 1. In this study, we also con- stress, are also capable of sensing pH variations and
firmed a rise in this fluorescence, but only under reversibly tune its conformation from light-harvesting
severe acidification at pH 2. state to a dissipative one. For example, in green algae
Testing algae responsiveness to acidification arises Chlamydomonas reinhardtii, stress-related LHCs were
from the fact that endogenous fluorescence in algae shown to sense pH variations - the conformational
and lower plants is sensitive to environmental pollu- change is induced only by acidification of the environ-
tion that includes pH changes. Most algae thrive and ment and the size of the quenching correlates with the
multiply in water with high pH levels ranging between degree of acidification [14]. Stress-related LHCs are
7 and 9, with optimum pH for most algae species at 8.2 present in green algae and we previously demonstrated
to 8.7. Neutral or lower water pH decreases the growth their role in cadmium-induced stress responses in sea
of algae. The photosynthetic rates of marine algae were algae Dunaliella [26]. We, therefore, believe that a
estimated to be highest above 8; however, in a large comparable mechanism can also take place under our
number of red, brown and green algae the rate experimental conditions in Chlorella sp. In the Chla-
decreased at pH 9.5 and higher [54]. At such pH, the mydomonas, a sensor of the luminal pH was found in
HCO3- ions were unable to penetrate the cells and C-terminal subdomain, capable of tuning the quench-
were therefore unavailable for photosynthesis. When ing level of the complex [14]. In this case, the quench-
measured at various pH, PSII electron flow was inhib- ing was accompanied by a shortening of the
ited at pH 6.5 at a high light intensity, while at low light fluorescence lifetime. The comparable situation can
the inhibition occurred at pH 9.5 [36]. This observa- also occur under our experimental conditions. Gath-
tion was explained by substrate (CO2) limitation at ered results support the idea of the quenching mech-
alkaline pH. In the process of photosynthesis, carbon- anism taking place during the acidification, resulting
fixing enzymes function optimally at pH 8; conse- in the shortening of the fluorescence lifetimes and thus
quently, raising or lowering the pH from 8 negatively the decrease in the fluorescence amplitude. This pro-
influences the rate of photosynthesis. At too high or cess is quasi-linear until pH 4, most likely due to a gra-
too low pH levels, the enzymes in the plant can dual change in the number of molecules affected.

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Methods Appl. Fluoresc. 8 (2020) 024007 A Marcek Chorvatova et al

Under pH 4, another process is also u nmasked, as with the cultivation of Chlorella sp, M. Danisova for
demonstrated by the spectral shift in the red fluores- helping with the original experimentation.
cence, as well as the rise in the green fluorescence,
accompanied by the prolongation of tau 2. In this case,
Conflict of interest
as most of the molecules responsible for the red fluor-
escence are quenched, the presence of the green or the
None declared, Ethical approval: none applicable.
shifted red fluorescence prevails.
Performed experimentation acknowledges the
employment of the time-resolved chlorophyll fluores- ORCID iDs
cence in biosensing. Our results also revealed good
resistance of the Chlorella sp. algae to short-term expo- A Marcek Chorvatova https://orcid.org/0000-
sure to acidification. In the future, long-term effects 0002-8798-0624
(in terms of days or weeks) need also to be tested. The
sensitivity of the fluorescence lifetimes to various References
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