\documentclass[10pt]{article}

\usepackage{fullpage}
\usepackage{setspace}
\usepackage{parskip}
\usepackage{titlesec}
\usepackage[section]{placeins}
\usepackage{xcolor}
\usepackage{breakcites}
\usepackage{lineno}
\usepackage{hyphenat}





\PassOptionsToPackage{hyphens}{url}
\usepackage[colorlinks = true,
            linkcolor = blue,
            urlcolor  = blue,
            citecolor = blue,
            anchorcolor = blue]{hyperref}
\usepackage{etoolbox}
\makeatletter
\patchcmd\@combinedblfloats{\box\@outputbox}{\unvbox\@outputbox}{}{%
  \errmessage{\noexpand\@combinedblfloats could not be patched}%
}%
\makeatother


\usepackage{natbib}




\renewenvironment{abstract}
  {{\bfseries\noindent{\abstractname}\par\nobreak}\footnotesize}
  {\bigskip}

\titlespacing{\section}{0pt}{*3}{*1}
\titlespacing{\subsection}{0pt}{*2}{*0.5}
\titlespacing{\subsubsection}{0pt}{*1.5}{0pt}


\usepackage{authblk}


\usepackage{graphicx}
\usepackage[space]{grffile}
\usepackage{latexsym}
\usepackage{textcomp}
\usepackage{longtable}
\usepackage{tabulary}
\usepackage{booktabs,array,multirow}
\usepackage{amsfonts,amsmath,amssymb}
\providecommand\citet{\cite}
\providecommand\citep{\cite}
\providecommand\citealt{\cite}
% You can conditionalize code for latexml or normal latex using this.
\newif\iflatexml\latexmlfalse
\providecommand{\tightlist}{\setlength{\itemsep}{0pt}\setlength{\parskip}{0pt}}%

\AtBeginDocument{\DeclareGraphicsExtensions{.pdf,.PDF,.eps,.EPS,.png,.PNG,.tif,.TIF,.jpg,.JPG,.jpeg,.JPEG}}

\usepackage[utf8]{inputenc}
\usepackage[ngerman,greek,english]{babel}



\usepackage{float}








\begin{document}

\title{Multi-modal imaging reveals a small subpopulation of Deinococcus
radiodurans exhibiting correlated metabolic activities}



\author[1]{Weinan Zhu}%
\author[1]{Xinyu He}%
\author[1]{Haoran Li}%
\author[1]{Bo Jiang}%
\author[1]{Ruo-Chen Xie}%
\author[1]{Wei Wang}%
\affil[1]{Nanjing University}%


\vspace{-1em}



  
  \date{May 25, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
Since a polyvalent strategy has recently been assumed to be adopted by
Deinococcus radiodurans that can generate various resistance against
many different detrimental sources of oxidative damage (e.g. reactive
oxygen species, heavy metal ions and ionising radiation), investigating
more than one restorative metabolic activities and their interrelation
of the very same entities of Deinococcus radiodurans is of great
significance for exploring its polyextremophile nature, which will be
insightful for obtaining fundamental generic insights into life
sustainability. Herein, we apply mainly fluorescence microscopy and back
reflection microscopy to visibly assess the respective activities of
superoxide radical generation and silver ion metabolism for individual
Deinococcus radiodurans. Strikingly, only a minority (\textless{}20\%)
of the bacteria which show low superoxide radical levels is revealed to
exhibit considerable formation of silver nanoparticles whilst those
containing more superoxide radicals all show minimum silver ion
metabolism. The discovery of the strong negative correlation for the
small subpopulation between the two visualised different metabolic
activities not only provides direct experimental evidence in terms of
bacterial functionality for the inferred survival regime of the extreme
microbe, but also suggests a new way of chemically examining biology
from the perspective of inter-functional relationship.%
\end{abstract}%



\sloppy


\textbf{Multi-modal imaging reveals a small subpopulation of}

\emph{Deinococcus radiodurans} \textbf{exhibiting correlated metabolic
activities}

Weinan Zhu\textsuperscript{1}, Xinyu He\textsuperscript{1}, Haoran
Li\textsuperscript{1}, Bo Jiang\textsuperscript{1}, Ruo-Chen
Xie\textsuperscript{1}, Wei Wang\textsuperscript{1}

\textsuperscript{1}State Key Laboratory of Analytical Chemistry for Life
Science, School of Chemistry and Chemical Engineering, Chemistry and
Biomedicine Innovation Center (ChemBIC), Nanjing University, Nanjing,
China.

\textbf{Correspondence}

Ruo-Chen Xie and Wei Wang

Email: \emph{crxie@nju.edu.cn} (R.C. Xie); \emph{wei.wang@nju.edu.cn}
(W. Wang)

\textbf{Funding information}

National Natural Science Foundation of China, Grant Numbers: 21925403
and 21874070;

Excellent Research Program of Nanjing University, Grant Numbers:
ZYJH004.

\textbf{Abstract}

Since a polyvalent strategy has recently been assumed to be adopted
by\emph{Deinococcus radiodurans} that can generate various resistance
against many different detrimental sources of oxidative damage (e.g.
reactive oxygen species, heavy metal ions and ionising radiation),
investigating more than one restorative metabolic activities and their
interrelation of the very same entities of \emph{Deinococcus
radiodurans} is of great significance for exploring its polyextremophile
nature, which will be insightful for obtaining fundamental generic
insights into life sustainability. Herein, we apply mainly fluorescence
microscopy and back reflection microscopy to visibly assess the
respective activities of superoxide radical generation and silver ion
metabolism for individual \emph{Deinococcus radiodurans} . Strikingly,
only a minority (\textless{}20\%) of the bacteria which show low
superoxide radical levels is revealed to exhibit considerable formation
of silver nanoparticles whilst those containing more superoxide radicals
all show minimum silver ion metabolism. The discovery of the strong
negative correlation for the small subpopulation between the two
visualised different metabolic activities not only provides direct
experimental evidence in terms of bacterial functionality for the
inferred survival regime of the extreme microbe, but also suggests a new
way of chemically examining biology from the perspective of
inter-functional relationship.

\textbf{Key points}

\begin{itemize}
\tightlist
\item
  The use of back reflection microscopy allows the discovery of a small
  subpopulation of \emph{Deinococcus radiodurans} exhibiting
  extraordinary silver ion metabolism.
\item
  Combining fluorescence microscopy and bright field microscopy we
  further show the subpopulation all contain correlated relatively low
  levels of superoxide radicals.
\item
  The findings from the angle of exploring inter-functional relationship
  directly supports the polyvalent strategy currently proposed for the
  polyextremophile for survivals against various sources of oxidative
  damage.
\end{itemize}

\textbf{KEYWORDS}

optical microscopy, subpopulation, extremophile, \emph{Deinococcus
radiodurans} , oxidative damage, heavy metal metabolism, reactive oxygen
species.

\textbf{1. Introduction}

The survival of the polyextremophile \emph{Deinococcus
radiodurans}(\emph{D.r.} ) under a variety of extreme conditions raises
the question of the underlying mechanism. Unravelling the mechanism of
this particular microbe is expected to greatly improve the understanding
of the principles of life sustainability in general. The current
mainstream view is that the fact is likely based on a single polyvalent,
rather than multiple parallel (each for a specific condition), strategy
generating various resistances.\textsuperscript{1} The key of the
strategy lies in the presence of a strong proteome protection system
that consists of an antioxidant fraction of the \emph{D.r.} cytosol with
a small molecular weight of less than 3 kDa,\textsuperscript{2, 3} and
otherwise protein carbonylation can easily occur and cause cell
death\emph{via} irreversible and progressive degeneration of most vital
cellular functions.\textsuperscript{4, 5} Such a constitutive protection
is thus considered to possibly cope with many different sources causing
oxidative damages including reactive oxygen
species,\textsuperscript{2}heavy metal ions\textsuperscript{6-9} and
ionising radiation\textsuperscript{10-12}. While the identity of the
antioxidant system is largely revealed, direct experimental evidence of
the bacterial multifunction and inter-function relationship is needed to
testify or consolidate the inferred regime for the unusual resilience of
the creature.

The functionality of a bacterium is, however, of a complex origin. Even
within a bacterial culture of a same genome, phenotypic heterogeneity
would occur and contribute the functional diversity to microbial
populations.\textsuperscript{13, 14} Statistical fluctuation, for
example, leads to a random generation of subpopulations with distinct
gene expressions.\textsuperscript{15-17} Alternatively, heterogeneity
can possibly be driven by environmental changes\textsuperscript{18} and
results in a small subpopulation with phenotypic differences from the
rest of the culture.\textsuperscript{19-21} Quorum sensing of pathogenic
bacteria can yield such type of heterogeneity to protect themselves
against host attack.\textsuperscript{22, 23} These discoveries imply
that the major body of current research on the biochemical properties of
bacteria, which is mainly based on the bulk analysis (e.g. ion
chromatography,\textsuperscript{6} UV/vis and infrared
spectroscopy\textsuperscript{7, 8, 24}) of the extracts of intra- or
extracellular contents, can hardly resolve the function profile of a
particular bacterium on a colony basis. Therefore, studying more than
one biological activities and their interrelation at the single
bacterium level is crucial to provide unambiguous fundamental insights
into the complex behaviour of a bacterial strain.

Given the desired spatial resolutions and the non-invasive nature of
optical microscopic techniques for studying single living cells under
various conditions,\textsuperscript{25-29} in this work we apply optical
microscopy under suitable imaging modes for different species as a major
means to visibly characterise two different biological activities of
superoxide radical (SR) generation and silver ion metabolism of
individual bacteria. Although metal ion metabolism can increase the
production of reactive oxygen species,\textsuperscript{30} the metabolic
pathways of the silver ion reduction and superoxide radical generation
have not shown any relation in the literature. We discover that these
two seemingly independent capacities of the bacteria are clearly
correlated and this occurs only within a small subgroup
(\textless{}20\%) of a clonal population. Note that we clarify here as
the \emph{D.r.} bacteria predominantly appear as four linked bodies
while the latter is distinctly separate from all others, the term
`individual bacteria' used in this work actually means the entity of the
tetracocci form.

\textbf{2. Experimental}

\textbf{2.1. Chemicals and bacterial cultures}

AgNO\textsubscript{3}, HCl, NaOH, and sodium alginate were purchased
from Sigma Aldrich, USA, dihydroethidium (DHE) from Yeasen
Biotechnology, China and both PBS buffer solution (135 mM NaCl, 4.7 mM
KCl, 10 mM Na\textsubscript{2}HPO\textsubscript{4}, 2 mM
NaH\textsubscript{2}PO\textsubscript{4}, pH=7.3\selectlanguage{ngerman}±0.1) and
polydimethylsiloxane (PDMS) were from Beyotime Biotechnology, China. All
these reagents were analytical-grade and used as received. All solutions
were prepared with Milli-Q ultrapure water (18 M\selectlanguage{greek}Ω\selectlanguage{ngerman}·cm, Thermo Fisher).

\emph{Deinococcus radiodurans} Strain R1 (ATCC 13939) was grown from a
single bacterium aerobically (under atmospheric conditions) at 30°C in
peptone yeast glucose (PYG) broth (containing 2\% peptone, 0.5\%
glucose, 1\% yeast extract, pH 7.2) with agitation at 300 rpm. The pH of
the broth was adjusted to 7 by adding hydrochloric acid or sodium
hydroxide. The bacterial growth was assessed by measuring optical
density (OD) \emph{via} a microplate reader (see below Section 2.2.3) at
the wavelength of 600 nm of the strain. The OD value stayed stable since
reaching 1.0, and the \emph{D.r.} strain at this stage was considered to
have a stable population and morphology, and therefore selected to be
used throughout the experiment. For obtaining the bacteria containing
silver particles, the grown culture above was cultivated in a fresh PYG
broth initially containing 2.0 mM AgNO\textsubscript{3} with a pH of 7
and at 30 \selectlanguage{english} for another 9 hours. The bacteria after incubation in
AgNO\textsubscript{3} solutions were centrifuged at 8000xg to collect
the microbes and washed with PBS for further analysis.

\textbf{2.2. \emph{Apparatus and measurements}}

\textbf{2.2.1. Optical set-up and the microscopic measurements}

The microscopic imaging and measurements were performed on a commercial
inverted microscope (Nikon, Ti-E), which was equipped with a 60X
objective lens with a numerical aperture (NA) of 1.49. Depending on the
studied species, the microscope was operated under three different
modes: fluorescent, bright field, back-reflection. In the fluorescence
mode, a white-light diode (X-cite\textsuperscript{(r)} 110LED, Excelitas
Technologies) coupled with a bandpass filter (450--490~nm) was used to
excite DHE-induced fluorophores. A digital CMOS camera (C11440,
Hamamastu) was used to collect the fluorescence signals, and a long-pass
filter (\selectlanguage{greek}λ \selectlanguage{english}\textgreater{} 520~nm) to prevent the excitation light from
entering the camera. The back-reflection mode using the same light path
as that of the fluorescent mode was achieved by using a 520-560 nm
bandpass filter for the illumination light (to avoid fluorescent
excitation when the solution contained DHE molecules), and also removing
the long-pass filter. As such, only the back reflected green light
signals were collected. The two imaging modes were switched by
converting the filter cube manually. In the bright field mode, the light
source was a quartz tungsten halogen lamp (LV-LH50PC, Nikon) and used
without any optical filters. The three modes shared the same camera as
above.

For fluorescence analysis, the bacteria containing Ag particles were
prepared as a bacterial solution with a concentration of
2\selectlanguage{ngerman}×10\textsuperscript{8} mL\textsuperscript{-1} using PBS buffer, and DHE
was added into the latter with a final concentration of 100 \selectlanguage{greek}μ\selectlanguage{english}M. The
mixture was incubated at 30  for 1 hour and then washed using PBS
buffer to remove the excess DHE molecules. A 100 \selectlanguage{greek}μ\selectlanguage{english}L of the washed
mixture was added into a PDMS chamber with a sodium alginate-modified
glass substrate and let it stand still for 10 minutes. Consequently, the
bacterial cells were observed to adsorb strongly onto the glass wall
forming a monolayer. PBS was used to gently rinse the mixture in the
chamber for the removal of suspended bacteria. As such, the adhered
bacteria were studied using the fluorescence microscopy described above.
The sample preparation for the bright field and back reflection imaging
was all the same but the DHE molecules were not added in the experiments
described in Section 3.2.

\textbf{2.2.2. Flow cytometry}

Scattering properties of silver-containing bacteria were measured using
a CytoFlex S flow cytometer (Beckman Coulter). The bacterial mixture was
transferred to the sheath fluid with a bacterial concentration of
10\textsuperscript{5} mL\textsuperscript{-1} for flow cytometry. Next,
with the laser beam passing the bacteria in the fluid, both forward
scattering and side scattering signals of those bacteria were detected
and analysed. In addition, specific concentrations of bacteria in a
solution were measured by flow cytometry.

\textbf{2.2.3. Microplate reader}

The monitoring of OD values and the UV-vis spectra of the bacterial
mixtures used in this work were conducted using a multifunctional
microplate reader (SynergyLX, Biotek/Agilen). The OD measurements were
done on the original mixture of the bacteria with the broth, whilst the
UV-vis spectra were measured for the silver-containing bacteria that
were re-suspended in PBS buffer solutions.

\textbf{2.2.4. Electrochemical measurements}

The electrochemical measurements was performed using a CS120
electrochemical work station (CorrTest, China). A three-electrode system
was used for the measurement, where a gold disc electrode (d=3.0 mm,
Tianjin Aida, China; the same brand for other electrodes below) was used
as the working electrode, an Ag/AgCl (saturated KCl,
E\textsuperscript{o} = 0.190 V vs. standard hydrogen electrode) was used
as the reference electrode and a platinum sheet as the counter
electrode. The electrodes were inserted into a solution of
\emph{D.r.}bacteria (with a concentration of
2\selectlanguage{ngerman}×10\textsuperscript{8}mL\textsuperscript{-1}) which was directly taken
from the incubation mixture with the PYG broth. Next, anodic stripping
voltammetry was applied every 1.5 hours to monitor the silver ion
concentration of the bulk solution within a total period of 9 hours.
Specifically, the stripping voltammetry was conducted by applying in
sequence a surface-refreshing potential of 0.6 V vs. Ag/AgCl for 60
seconds, a pre-concentration potential of -0.45 V for 60 seconds again,
and a potential cycle between 0.0 and 0.6 V at a scan rate of 0.1 V
s\textsuperscript{-1}.

\textbf{2.2.5. Scanning electron microscopy}

Electron microscopic imaging was performed using a JEOL JSM-7800F
Scanning Electron Microscope (SEM) equipped with Energy Dispersive
Spectroscopy (EDS). The silver particle-containing bacteria were
dropcast on to a cover glass and let dry overnight at room temperature,
and after gold sputtering the immbolised bacteria were imaged using the
electron microscope at an acceleration voltage of 15.0 kV. In addition,
EDS was conducted on the selected areas of the sputtered glass for
elemental analysis.

\textbf{3. Results and discussion}

\textbf{3.1. Fluorescence analysis of the superoxide radical level in
individual bacteria}

The SR levels of individual bacteria were analysed using fluorescence
microcopy. Experimentally, the mature bacteria (OD=1.0) were first
resuspended in PBS buffer solutions and imaged using bright field
microscopy. Figure 1a shows a total number of nearly 200 separate
entities of bacteria in the view. Next, dihydroethidium (DHE) was added
into the bacterial solution for use as a specific fluorescence probe to
uncover the SR levels with a positive response. The DHE-uptaken bacteria
were then immoblised on to a sodium alginate-modified coverglass and
imaged using fluorescence microscopy at an excitation light wavelength
of 510 nm. Consequently, Figure 1b presents all the \emph{ca.} 200
bacteria observed in Figure 1a which now appear red, suggesting the
superoxide radical generation is active all over the bacterial colony.
The inset with a much better contrast than that in Figure 1a further
resolves the structure of each entity of the bacteria and shows their
predominant existence as tetracocci. Importantly, it is seen that within
the bacterial culture, their fluorescence (FL) intensities differ from
each other. The FL intensity distribution as shown in the Figure 1c was
obtained by measuring the integral intensity of the 2D area of each
bacteria. It is therefore clear that individual bacteria exhibit
different SR levels inside them, indicating the differences of their
antioxidant capacities within the colony.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1.} (a) Bright field and (b) fluorescence images of
individual bacteria, while the insets enlarge a same single entity of a
D.r.tetracocci. (c) FL intensity distribution.

\textbf{3.2. Assessment of silver ion metabolism of
individual\emph{D.r.} bacteria}

While the dissimilar SR levels of individual \emph{D.r.} are clear,
their abilities to reduce silver ions and to form less toxic Ag metal
particles are carefully characterised both at the bulk level and the
single bacterium level. First, anodic stripping voltammetry was
conducted as a function of time to a gold macrodisc electrode inserted
in a grown bacterial solution initially containing 2 mM silver ions and
the broth compositions. Figure 2a shows throughout the experiment an
oxidation peak at the potentials of 0.2\textasciitilde{}0.3 V in
contrast to the blank voltammgram obtained with the bacterial solution
without Ag\textsuperscript{+} added. This indicates that the peak
corresponds to the oxidation of the reduced silver from the bacterial
solutions in the pre-concentration step. Importantly, the charge
transferred during the voltammetric peak was observed to gradually
decrease to nearly none (\textless{}5\%), suggesting an almost complete
consumption of silver ions by the bacteria in the bulk solution over the
9 hours. This inference is on the basis that since the stripping of a
monolayer deposition of silver is estimated to correspond to an
oxidative charge of 14 \selectlanguage{greek}μ\selectlanguage{english}C (see Supporting Information Section 1 for the
calculation), which is far more than the largest amount
(\textasciitilde{} 3 \selectlanguage{greek}μ\selectlanguage{english}C) measured in our experiment, the electrode used
is thus thought to be large enough to minimise inhomogeneous surface
redox reactions,\textsuperscript{31} and the measured charge can be
quantitatively related to the silver ion concentration. Besides, it is
noted that the negative shift of the peak potential over time, despite
the inferred decrease in the silver ion concentration, is probably due
to an increased level of halide anions in the extracellular solution
that results from the microbial metabolism. Next, the resulting
bacterial mixture with the minimum Ag\textsuperscript{+} content was
briefly examined by UV-vis spectroscopy. In comparison with the bacteria
incubated without silver ions, a distinct absorption band at around 430
nm was seen in Figure 2b, which coincides with the well-known
characteristic wavelength of the plasmonic absorption of silver
nanoparticles, suggesting the product of the silver ion uptake by the
bacteria is metallic nanosilver.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 2.} Consumption of Ag\textsuperscript{+} ions and Ag
nanoparticle formation of \emph{D.r.} bacteria. (a) Anodic stripping
voltammograms of bacterial solutions initially containing 2 mM
AgNO\textsubscript{3} as a function of time within 9 hours. (b) UV-vis
spectra of the bacteria with (red) and without (blue)
Ag\textsuperscript{+} incubation. (c) SEM image and (d) Ag-element
mapping.

The product of the bacterial silver consumption is further investigated
by looking into a single bacterium using scanning electron microscopy.
Figure 2c shows a representative SEM image of a silver
particle-containing bacterium. A total number of 106 bright features
with an average diameter of 95\selectlanguage{ngerman}±22 nm within the bacterial cell is
evident (see Figure S1). Furthermore, elemental mapping based on EDS
(Figure 2d) shows the presence of silver and importantly the spatial
distribution of the Ag element is well consistent with that in the
electron microscopic image. These observations strongly confirm that
inside the bacteria silver nanoparticles form as a result of the silver
ion uptake. As such, the characterisations above clearly demonstrate the
ability of individual \emph{D.r.} bacteria to reduce the silver metal
ions.

On the basis of the strongly scattering properties of silver
nanoparticles, we next turn to optically explore the differences in the
silver ion reducing ability of bacteria within their cultures. To this
end, the high-throughput method of flow cytometry was conducted for
detecting the bacteria after the incubation with
Ag\textsuperscript{+}and both the forward scattering and side scattering
signals were collected for individual bacteria. It is recognised that
forward scatter detection is mainly related to the size of a cell and
thus for a certain bacterial strain a larger cell gives higher
scattering intensity, whilst side scatter detection is relatively
sensitive to the strongly scattering particles inside the
cell.\textsuperscript{32, 33} The two kinds of scatter signals are
plotted for each detected bacteria in form of a two dimensional
intensity distribution graph as shown in Figure 3a\& 3b. As a result,
the scattering signals of bacteria after incubation with
Ag\textsuperscript{+} exhibit a bimodal distribution (Figure 3b) where
the first contour peak at the SSC intensity of\emph{ca.}
10\textsuperscript{5} becomes larger than that for the bacteria before
Ag\textsuperscript{+} incubation (i.e. some 10\textsuperscript{4}) as
shown in Figure 3a, which is consistent with the formation of the
strongly scattering silver nanoparticles for most bacteria. Strikingly,
a second, though smaller, peak emerges at a much higher SSC intensity of
nearly 10\textsuperscript{6}, indicating a unusually considerable number
of silver nanoparticles formed in those bacteria. In contrast, the FSC
intensity of the bacteria before and after Ag\textsuperscript{+}
incubation stays almost constant. Nevertheless, the bacteria
synthesising large quantities of silver nanoparticles show a relatively
higher FSC intensity than most bacteria on average, to which the
scattering silver particles possibly contribute. Figure 3c shows the
number distributions of bacteria with different SSC intensity before and
after the Ag\textsuperscript{+}incubation. While the total number of
bacteria seems unchanged despite being incubated, the after-incubation
bacteria with the SSC values within the second peak account for a small
portion of nearly 10\% by integral. These observations implies the
emergence of a minority of `strongly reducing' bacteria that are
distinct from the others in silver metal metabolism when exposed to
silver ions. The phenomenon points to the interesting fact that when
facing environmental threats, bacteria owning the same genome can evolve
to a subpopulation of a very different phenotype -- in this case, those
that can reduce silver ions much more greatly than the rest. This
natural strategy is consistent with the extraordinary adapting ability
of the polyextremophile.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 3.} Flow cytometric detection of the bacterial
populations in terms of side scattering and forward scattering without
(a) and with (b) Ag\textsuperscript{+} incubation. (c) Side scattering
intensity distribution of the bacteria with and without
Ag\textsuperscript{+} incubation. (d) Schematics of back-illumination
microscopy. (e) Bright-field image and (f) back-illumination image of
bacteria in a same region of interest.

We further seek to distinguish the individual `strongly reducing'
bacteria visibly. Bright field microscopy is first applied to the
bacteria mixture after Ag\textsuperscript{+} incubation and washing with
PBS buffers. Figure 3e shows that in the view over 100 bacteria is seen
which yet exhibit very different contrasts. While most bacteria appear
to be greyer than the background, a small portion shows somewhat even
darker which is not observed in the control group (see Figure S2).
Although bright field microscopy allows the observations of all the
bacteria in the optical field, the difference in the contrasts between
different bacteria is sometimes not very distinct (e.g. the upper left
inset in Figure 3e). To improve the situation, we have developed an
alternative imaging mode utilising the back reflection of a light beam
that is able to presumably better distinguish between different types of
bacterial cells. As shown in Figure 3d, the technique basically uses the
same light path as that in fluorescent microscopy where a light beam
illuminates the bacteria and reflect back to the camera, but in this
back-reflection mode the light is not restricted to the narrow range of
wavelength for fluorophore excitation. Consequently, Figure 3f insets
show a more significant contrast between these bacteria -- much greater
optical intensity for the darker bacteria seen using the bright field
mode (Figure 3e insets), while far lower intensity for the rest
population. Moreover, the former type of bacteria is observed to be
apparently fewer than the latter in the back reflection imaging mode,
which is consistent with the results of the flow cytometric
measurements. We thus assume the imaging mode is more sensitive to the
internal silver nanoparticles. This imaging mode is therefore considered
to be a well-suited approach to discern the bacteria of a strong
capacity of silver metal metabolism from those of an average ability.

\textbf{3.3. Correlated superoxide radical levels and silver
nanoparticle levels of individual bacteria}

Having evidenced the differing SR levels of bacteria using fluorescence
microscopy and varying silver nanoparticle concentration inside a
bacterium using back reflection imaging, we finally investigate the
possible correlation between the levels of two biologically redox active
products at the single bacterium level. Experimentally, the DHE
fluorescence probes were added to a bacterial mixture after the
incubation with Ag\textsuperscript{+} to create DHE-labelled Ag
particle-containing bacteria. In turn, bright field microscopy, back
reflection imaging and fluorescence microscopy were applied to the
immoblised bacteria in a same optical field. Consequently, the optical
intensity of respective images are compared for each single entity.

Surprisingly, as the bright field image in Figure 4a presents all the
adhered bacteria in the optical field, a small fraction
(\textless{}20\%) of the bacteria exhibiting an exceptional back
reflection intensity (Figure 4b) are shown to be exactly complementary
to those exhibiting a high fluorescent intensity (Figure 4c).
Ultimately, the fluorescent intensity and the back reflection intensity
of a total number of 1626 bacteria were individually measured and
plotted in the correlation chart (Figure 4d). Overall, a distinct
negative correlation between the two kinds of optical signals is
evident. This strongly suggests that a bacterium which has a potent
ability to reduce silver ions tends to possess a low superoxide radical
level. This conclusion further implies that the two different kinds of
biological activities -- superoxide radical reduction and silver ion
reduction -- may share to some extent the same reducing system
within\emph{D.r.} bacteria: in this case, the pool of antioxidant
molecules in the bacterial cytosol as discussed in Introduction. On the
ground that most single entity research focuses on structure-activity
relationship, our work suggests an interesting perspective of looking
into the relationship between different activities of a same microbe,
which may gain fundamental implications for chemical aspects of biology.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Figure 4.} (a) A bright field, (b) back reflection and (c)
fluorescent image of a same optical field of the DHE-labelled Ag
particle-containing bacteria immbolised on a glass substrate in PBS
buffer solutions. (d) Correlation chart between the back reflection (BR)
intensity and fluorescence (FL) intensity of a considerable number of
individual bacteria.

\textbf{4. Conclusions}

This work applies optical microscopy under varying imaging modes
depending on the analyte species to visualise both metabolic activities
of silver ion reduction and superoxide radical generation of
the\emph{D.r.} colony. It is discovered that a distinct negative
correlation between the two biochemical processes surfaces yet only for
a minor subpopulation of the bacteria. Interestingly, this finding
directly supports the current view of the single polyvalent strategy
against various conditions lethal to most species for explaining the
polyextremophile nature of \emph{D.r.} bacteria.

\textbf{Acknowledgements}

This research is funded by National Natural Science Foundation of China
(Grant No. 21925403 and 21874070) and Excellent Research Program of
Nanjing University (Grant No. ZYJH004).

\textbf{Competing interest}

The authors declare no competing financial interest.

\textbf{References}

1. Krisko, A.; Radman, M., Biology of extreme radiation resistance: the
way of Deinococcus radiodurans. \emph{Cold Spring Harbor perspectives in
biology} \textbf{2013,} \emph{5} (7), a012765.

2. Krisko, A.; Radman, M., Protein damage and death by radiation in
Escherichia coli and Deinococcus radiodurans. \emph{Proceedings of the
National Academy of Sciences} \textbf{2010,} \emph{107} (32),
14373-14377.

3. Daly, M. J.; Gaidamakova, E. K.; Matrosova, V. Y.; Kiang, J. G.;
Fukumoto, R.; Lee, D.-Y.; Wehr, N. B.; Viteri, G. A.; Berlett, B. S.;
Levine, R. L., Small-molecule antioxidant proteome-shields in
Deinococcus radiodurans. \emph{PloS one} \textbf{2010,} \emph{5} (9),
e12570.

4. Krisko, A.; Radman, M., Phenotypic and genetic consequences of
protein damage. \emph{PLoS genetics} \textbf{2013,} \emph{9} (9),
e1003810.

5. Po, K. H. L.; Chow, H. Y.; Cheng, Q.; Chan, B. K. w.; Deng, X.; Wang,
S.; Chan, E. W. C.; Kong, H. K.; Chan, K. F.; Li, X., Daptomycin exerts
bactericidal effect through induction of excessive ROS production and
blocking the function of stress response protein Usp2. \emph{Natural
Sciences} \textbf{2021,} \emph{1} (2), e10023.

6. Fredrickson, J. K.; Kostandarithes, H. M.; Li, S.; Plymale, A. E.;
Daly, M., Reduction of fe (III), cr (VI), U (VI), and tc (VII) by
Deinococcus radiodurans R1. \emph{Applied and Environmental
Microbiology} \textbf{2000,} \emph{66} (5), 2006-2011.

7. Kulkarni, R. R.; Shaiwale, N. S.; Deobagkar, D. N.; Deobagkar, D. D.,
Synthesis and extracellular accumulation of silver nanoparticles by
employing radiation-resistant Deinococcus radiodurans, their
characterization, and determination of bioactivity. \emph{International
journal of nanomedicine} \textbf{2015,} \emph{10} , 963.

8. Li, J.; Webster, T. J.; Tian, B., Functionalized nanomaterial
assembling and biosynthesis using the extremophile Deinococcus
radiodurans for multifunctional applications. \emph{Small}\textbf{2019,}
\emph{15} (20), 1900600.

9. Ge, C.; Huang, D.; Wang, D.; Zhang, E.; Li, M.; Zhu, F.; Zhu, C.;
Chen, N.; Wu, S.; Zhou, D., Biotic Process Dominated the Uptake and
Transformation of Ag+ by Shewanella oneidensis MR-1. \emph{Environmental
Science \& Technology} \textbf{2022,} \emph{56} (4), 2366-2377.

10. Daly, M. J.; Gaidamakova, E. K.; Matrosova, V. Y.; Vasilenko, A.;
Zhai, M.; Leapman, R. D.; Lai, B.; Ravel, B.; Li, S.-M. W.; Kemner, K.
M., Protein oxidation implicated as the primary determinant of bacterial
radioresistance. \emph{PLoS biology} \textbf{2007,} \emph{5} (4), e92.

11. Daly, M. J., A new perspective on radiation resistance based on
Deinococcus radiodurans. \emph{Nature Reviews
Microbiology}\textbf{2009,} \emph{7} (3), 237-245.

12. Brim, H.; McFarlan, S. C.; Fredrickson, J. K.; Minton, K. W.; Zhai,
M.; Wackett, L. P.; Daly, M. J., Engineering Deinococcus radiodurans for
metal remediation in radioactive mixed waste environments. \emph{Nature
Biotechnology} \textbf{2000,} \emph{18} (1), 85-90.

13. Davis, K. M.; Isberg, R. R., Defining heterogeneity within bacterial
populations via single cell approaches. \emph{Bioessays}
\textbf{2016,}\emph{38} (8), 782-790.

14. Ackermann, M., A functional perspective on phenotypic heterogeneity
in microorganisms. \emph{Nature Reviews Microbiology}
\textbf{2015,}\emph{13} (8), 497-508.

15. Ackermann, M.; Stecher, B.; Freed, N. E.; Songhet, P.; Hardt, W.-D.;
Doebeli, M., Self-destructive cooperation mediated by phenotypic
noise.\emph{Nature} \textbf{2008,} \emph{454} (7207), 987-990.

16. Sandoz, K. M.; Mitzimberg, S. M.; Schuster, M., Social cheating in
Pseudomonas aeruginosa quorum sensing. \emph{Proceedings of the National
Academy of Sciences} \textbf{2007,} \emph{104} (40), 15876-15881.

17. Dandekar, A. A.; Chugani, S.; Greenberg, E. P., Bacterial quorum
sensing and metabolic incentives to cooperate.
\emph{Science}\textbf{2012,} \emph{338} (6104), 264-266.

18. Rangarajan, A. A.; Koropatkin, N. M.; Biteen, J. S.,
Nutrient-dependent morphological variability of Bacteroides
thetaiotaomicron. \emph{Microbiology} \textbf{2020,} \emph{166} (7),
624-628.

19. Helaine, S.; Thompson, J. A.; Watson, K. G.; Liu, M.; Boyle, C.;
Holden, D. W., Dynamics of intracellular bacterial replication at the
single cell level. \emph{Proceedings of the National Academy of
Sciences} \textbf{2010,} \emph{107} (8), 3746-3751.

20. Eldar, A.; Elowitz, M. B., Functional roles for noise in genetic
circuits. \emph{Nature} \textbf{2010,} \emph{467} (7312), 167-173.

21. Avery, S. V., Microbial cell individuality and the underlying
sources of heterogeneity. \emph{Nature Reviews
Microbiology}\textbf{2006,} \emph{4} (8), 577-587.

22. Davis, K. M.; Mohammadi, S.; Isberg, R. R., Community behavior and
spatial regulation within a bacterial microcolony in deep tissue sites
serves to protect against host attack. \emph{Cell host \&
microbe}\textbf{2015,} \emph{17} (1), 21-31.

23. S\selectlanguage{ngerman}ánchez-Romero, M. A.; Casadesús, J., Contribution of phenotypic
heterogeneity to adaptive antibiotic resistance. \emph{Proceedings of
the National Academy of Sciences} \textbf{2014,} \emph{111} (1),
355-360.

24. Tian, B.; Li, J.; Pang, R.; Dai, S.; Li, T.; Weng, Y.; Jin, Y.; Hua,
Y., Gold nanoparticles biosynthesized and functionalized using a
hydroxylated tetraterpenoid trigger gene expression changes and
apoptosis in cancer cells. \emph{ACS applied materials \&
interfaces}\textbf{2018,} \emph{10} (43), 37353-37363.

25. Chia, H. E.; Marsh, E. N. G.; Biteen, J. S., Extending fluorescence
microscopy into anaerobic environments. \emph{Current Opinion in
Chemical Biology} \textbf{2019,} \emph{51} , 98-104.

26. Chia, H. E.; Zuo, T.; Koropatkin, N. M.; Marsh, E. N. G.; Biteen, J.
S., Imaging living obligate anaerobic bacteria with bilin-binding
fluorescent proteins. \emph{Current research in microbial
sciences}\textbf{2020,} \emph{1} , 1-6.

27. Xiong, B.; Zhou, R.; Hao, J.; Jia, Y.; He, Y.; Yeung, E. S., Highly
sensitive sulphide mapping in live cells by kinetic spectral analysis of
single Au-Ag core-shell nanoparticles. \emph{Nature
communications}\textbf{2013,} \emph{4} (1), 1-9.

28. Xiong, B.; Huang, Z.; Zou, H.; Qiao, C.; He, Y.; Yeung, E. S.,
Single plasmonic nanosprings for visualizing
reactive-oxygen-species-activated localized mechanical force
transduction in live cells. \emph{ACS nano} \textbf{2017,} \emph{11}(1),
541-548.

29. Nikel, P. I.; Silva\selectlanguage{english}-Rocha, R.; Benedetti, I.; de Lorenzo, V., The
private life of environmental bacteria: pollutant biodegradation at the
single cell level. \emph{Environmental Microbiology}
\textbf{2014,}\emph{16} (3), 628-642.

30. Ojuederie, O. B.; Babalola, O. O., Microbial and plant-assisted
bioremediation of heavy metal polluted environments: a
review.\emph{International journal of environmental research and public
health}\textbf{2017,} \emph{14} (12), 1504.

31. Hyde, M. E.; Banks, C. E.; Compton, R. G., Anodic stripping
voltammetry: an AFM study of some problems and
limitations.\emph{Electroanalysis} \textbf{2004,} \emph{16} (5),
345-354.

32. Van Der Vlist, E. J.; Stoorvogel, W.; Arkesteijn, G. J.; Wauben, M.
H., Fluorescent labeling of nano-sized vesicles released by cells and
subsequent quantitative and qualitative analysis by high-resolution flow
cytometry. \emph{Nature protocols} \textbf{2012,} \emph{7} (7),
1311-1326.

33. Robinson, J. P., Flow cytometry. \emph{Encyclopedia of biomaterials
and biomedical engineering} \textbf{2004,} \emph{3} , 630-642.

\selectlanguage{english}
\FloatBarrier
\end{document}

