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Confocal Microscopy Imaging of the Biofilm Matrix
Sebastian Schlafer, Rikke L. Meyer
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S0167-7012(16)30036-7
doi: 10.1016/j.mimet.2016.03.002
MIMET 4851

To appear in:

Journal of Microbiological Methods

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Accepted date:

16 October 2015
29 February 2016
2 March 2016

Please cite this article as: Schlafer, Sebastian, Meyer, Rikke L., Confocal Microscopy Imaging of the Biolm Matrix, Journal of Microbiological Methods (2016), doi:
10.1016/j.mimet.2016.03.002

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Confocal Microscopy Imaging of the Biofilm Matrix

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REVISED

Department of Dentistry, HEALTH, Aarhus University, Vennelyst Boulevard 9, 8000 Aarhus C, Denmark

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Sebastian Schlafera*, Rikke L. Meyerb,c

Interdisciplinary Nanoscience Center (iNANO), SCIENCE AND TECHNOLOGY, Aarhus University, Gustav

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Wieds Vej 14, 8000 Aarhus C, Denmark

Department of Bioscience, SCIENCE AND TECHNOLOGY, Aarhus University, Ny Munkegade 114, 8000

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Aarhus C, Denmark

E-mail addresses: SS: sebastians@microbiology.au.dk; RLM: rikke.meyer@inano.au.dk

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*Corresponding author

Sponsor: Robert S. Burlage

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Abstract

The extracellular matrix is an integral part of microbial biofilms and an important field of research. Confocal

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laser scanning microscopy is a valuable tool for the study of biofilms, and in particular of the biofilm matrix,

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as it allows real-time visualization of fully hydrated, living specimens. Confocal microscopes are held by
many research groups, and a number of methods for qualitative and quantitative imaging of the matrix
have emerged in recent years. This review provides an overview and a critical discussion of techniques used

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to visualize different matrix compounds, to determine the concentration of solutes and the diffusive

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properties of the biofilm matrix.

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Keywords: Biofilm; CLSM; confocal microscopy; extracellular matrix; EPS; fluorescent stains

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Abbreviations

CBM: carbohydrate-binding modules; CLSM: confocal laser scanning microscopy; DDAO: 1,3-dichloro-7-

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hydroxy-9,9-dimethyl-2(9H)-acridinone; ECM: extracellular matrix; eDNA: extracellular DNA; EPS:

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extracellular polymeric substances; FLIM: fluorescence lifetime imaging; FRET: Frster resonance energy
transfer; GFP: green fluorescent protein; MALDI: matrix-assisted laser desorption ionization; PI: propidium
iodide; SECM: scanning electrochemical microscopy; SIMS: secondary ion mass spectrometry; SPT: single

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particle tracking; STED: stimulated emission depletion microscopy; ThT: thioflavin T; WGA: wheat germ

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agglutinin

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1 Introduction

The extracellular matrix of microbial biofilms is a highly complex scaffold, characterized by a multitude of

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structurally and chemically heterogeneous microenvironments. Its functions are manifold: It provides

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mechanical stability to the biofilm and protects the microorganisms from desiccation. It can act as a barrier
against adverse chemical and biological influences, such as osmotic stress, acid/base challenges, oxygen,
antibiotics and antiseptics, the host immune defense, and grazing protozoa. Moreover, it contributes to the

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sorption and storage of nutrients and trace elements, it is the location of numerous extracellular enzymatic
reactions, and it keeps the microorganisms in tight contact to each other to facilitate genetic exchange and

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bacterial communication. If the biofilm is a microbial city, then the matrix is its infrastructure.

Polysaccharides were long believed to be the main macromolecular constituent of the extracellular matrix,

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and the abbreviation EPS, today used for extracellular polymeric substances, originally designated
extracellular polysaccharides. Today it is well-known that a multitude of different biopolymers, including

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DNA, proteins, and lipids, i.e. in outer membrane vesicles, contribute to matrix structure and function.
For decades, the cellular components of biofilms held the center of research attention, as the

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microorganisms are the driving force behind both detrimental and beneficial effects of biofilms. The past
ten years witnessed an increased focus on the matrix and its functional interplay with the microbiota. An
integrated view on both compartments is necessary to attain in-depth understanding of biofilms and to
develop target-oriented strategies for the control of biofilm-related problems.
Confocal laser scanning microscopy (CLSM) is a valuable tool for the study of biofilms, and in particular of
the biofilm matrix, as it allows real-time visualization of fully hydrated, living specimens. The past years
have brought about several new imaging technologies that improve the spatial resolution of light
microscopy, and the Nobel Prize in Chemistry was in 2014 awarded to Eric Betzig, Stefan W Hell and
William E. Moerner for their development of super-resolution optical microscopy. Confocal laser scanning

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microscopes are available in many research laboratories, and consequently, methods based on CLSM have
evolved considerably in the past decade to retrieve information about the composition and the properties

of the biofilm matrix. The aim of this review is to provide an overview and to discuss the opportunities and

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challenges of fluorescence labelling techniques that can be used to acquire either qualitative or

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quantitative information about the biofilm matrix.

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2 Qualitative confocal microscopy imaging of matrix components

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The functionality of bacterial biofilms is entwined with its microscale structure, as mass transport by
diffusion and convection affects chemical gradients that dictate the limits of metabolic activity and the

conditions in the microenvironment experienced by individual cells. The physical structure also affects the

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mechanical stability of the biofilm, and the protective properties of the matrix towards host immune cells

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and antimicrobial agents. There is currently no fluorescence labeling method available which visualizes the
biofilm matrix in general, and this is due to the complex and highly variable composition of the matrix
produced by different bacteria and under different environmental conditions. Each matrix component must

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therefore be stained individually.


2.1 Polysaccharide staining

Polysaccharides are often an important part of the biofilm matrix where they contribute to cohesion,
retention of water, sorption of organic and inorganic compounds, and protection against biocides and
grazing protozoa (for recent reviews, see Aricola et al. (Arciola et al., 2015) or Flemming and Wingender
(Flemming and Wingender, 2010)). Unfortunately a general stain for polysaccharides does not exist, as the
chemical structure of matrix polysaccharides differs among different bacteria. Calcofluor white has been
used for polysaccharide staining, but it binds only -1,3 and -1,4 glucans (Rasconi et al., 2009), which are
found in cellulose and chitin but not in the more common matrix polysaccharide poly--1,6-N-acetyl

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glucosamine (Sadovskaya et al., 2005). A better approach is therefore the use of fluorescently labelled
lectins, which was pioneered by Neu et al. (Neu et al., 2001). Lectins typically recognize specific di- or tri-

saccharides. Such oligosaccharides can be present both in the matrix and as glycoconjugates on the cell

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surface e.g. in the teichoic acids of Gram-positive bacteria and the lipopolysaccharides of Gram-negative

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bacteria. Glycoconjugates are also highly diverse in structure (Messner et al., 2013), and lectin staining
therefore always starts with a large screening of commercial lectins to identify which are able to bind.

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A new approach to carbohydrate staining was recently introduced by Nguyen et al. (Nguyen et al., 2014),
exploiting the high affinity of carbohydrate-binding modules (CBM): the non-catalytic carbohydrate-binding

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domain of polysaccharide-degrading enzymes. The authors constructed a green fluorescent (GFP) fusion
protein with the carbohydrate-binding module 3 (GFP-CBM3) which has high affinity for cellulose. As a

proof of concept, they showed that this new polysaccharide label did not bind planktonic cells, but only to

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E. coli biofilms and flocs induced by overexpression of the cellulose synthase BscB (Nguyen et al., 2014).

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The same authors elegantly turned this concept into a quantitative and non-destructive assay for
exopolysaccharides, as GFP was cleaved off the GFP-CBM3 fusion protein by a site-specific protease and
subsequently quantified in solution (Ojima et al., 2015b). With 64 families of CBM, there is a wide scope for

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using this novel approach to visualize and quantify other polysaccharides in the biofilm matrix.
2.2 Staining of extracellular DNA (eDNA)
The significance of eDNA in the biofilm matrix was discovered when Whitchurch et al. added DNase to a
Pseudomonas aeruginosa biofilm and watched the biofilm disappear (Whitchurch et al., 2002). Since then,
it has become evident that eDNA plays an important role for bacterial attachment and the early stages of
biofilm formation in many species from across the phylogenetic tree. Despite the apparent ubiquity of
eDNA as an important matrix component, it remains unclear what it interacts with in the matrix. Colocalization of eDNA with other elements of the matrix has led to the suggestion that eDNA interacts with a
variety of different components, such as DNA-binding integration host factor (Goodman et al., 2011),

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pyocyanines (Das et al., 2013), Staphylococcus -toxin (Huseby et al., 2010) and polysaccharides (Hu et al.,
2012; Jennings et al., 2015). These findings suggests a variety of ligands and ways for DNA to interact. The

desire to address eDNAs role in biofilm formation and antibiotic resistance has motivated the analysis of

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eDNA in the biofilm using cell-impermeant DNA-binding fluorescent stains, such as propidium iodide (PI),

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1,3-dichloro-7-hydroxy-9,9-dimethyl-2(9H)-acridinone (DDAO), TOTO-1, TO-PRO 3, PicoGreen and


SYTOX stains. Most reports have used DDAO for staining eDNA in biofilms after the initial publication by

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Allesen-Holm et al. (Allesen-Holm et al., 2006; Conover et al., 2011; Schooling et al., 2009). However, a
recent evaluation of eDNA stains in biofilms of Pseudomonas, Staphylococcus and Bacillus species showed

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that TOTO-1, SYTOX Green and PI provide the most reliable results, whereas TO-PRO-3 and DDAO
were not completely cell impermeant (Okshevsky and Meyer, 2014). PicoGreen also becomes cell

permeant after 10-15 minutes of incubation, but by keeping incubation times short, Tang et al (Tang et al.,

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2013) used this sensitive and quantitative stain to quantify eDNA in biofilms of environmental isolates. They
demonstrated a transient or a gradually increasing accumulation of eDNA over time in the biofilm matrix of

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the different isolates, suggesting that the role of eDNA in the biofilm matrix is highly dynamic. Suprisingly,
eDNA strongly affected the initiation of biofilms, even when present in concentration below the detection

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limit. The amount of eDNA accumulating in biofilms may therefore not necessarily reflect its importances,
as it can exert its adhesive effect at very low concentrations.
eDNA is often stained in combination with another cell-permeant DNA-binding stain, such as the
combination of PI with SYTO 9 in the LIVE/DEAD BacLightTM kit for viability. However, using two stains
binding to the same target molecule requires optimization of the concentrations used to ensure accurate
identification of eDNA, which is exposed to both stains. One has to consider the relative concentration of
the two stains as well as stain concentration relative to the amount of DNA in the sample. Simultaneous
intercalation of the two stains can lead to Frster resonance energy transfer (FRET) if the emission
spectrum of one stain overlaps with the excitation spectrum of the other, and it turns out that this effect
works to ones advantage in SYTO 9/ PI staining. PI cannot completely displace SYTO 9, but when used

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in the right concentration, the FRET effect quenches emitted light from SYTO 9 and leads to enhanced
emission by PI. However, if not used at the right concentration, the high quantum yield of SYTO 9

compared to PI will lead to simultaneous emission from both stains (Stocks, 2004). Due to these

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complications, it is recommended to stain eDNA with TOTO-1 in combination with e.g. SYTO 60, as

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TOTO-1 has low intrinsic fluorescence and a high quantum yield (twice that of SYTO 60). Furthermore,
fast one-track scanning can be performed with microscopes containing two photomultipliers, as the

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emission spectra of the two stains are easily separated. As an example of TOTO-1/SYTO 60 staining,
Figure 1 shows the increasing accumulation of eDNA over time in S. epidermidis biofilms. The eDNA is

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tightly associated with the cell surface, and the inhomogeneous distribution demonstrates the stark cell to
cell variations in the ability to bind eDNA to the cell surface.

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2.3 Protein staining

In recent years, it has become evident that proteins also can be important for the biofilm matrix, and

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proteins are in some cases even more predominant than polysaccharides. For example, cell wall anchored
proteins in e.g. Staphylococcus aureus and Staphylococcus epidermidis contribute to aggregation through

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homophilic interactions (Geoghegan et al., 2010; Schaeffer et al., 2015), or by interacting with matrix
components originating from the host, such as collagen, fibrin and fibronectin (Bttner et al., 2015; Foster
and Hk, 1998). The protein-component of the biofilm matrix can be visualized with non-specific stains,
such as the FilmTracerTM SyPro stains (Frank and Patel, 2007; Lawrence et al., 2003), but specific protein
labeling is also possible through monoclonal antibodies. So far, antibody labeling in the biofilm matrix has
mostly been used for imaging the localization of proteins by electron microscopy (Webster et al., 2006),
but the technique is easily transferrable to fluorescence microscopy by using fluorescently labeled primary
or secondary antibodies (Greiner et al., 2005). Berk et al. (2012) elegantly showed how the genetic
insertion of FLAG tags in specific matrix proteins allowed following the production and location of key
matrix proteins in real time during initiation and development of biofilms in Vibrio cholera, demonstrating

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complementary architectural roles of the three proteins Bap1, RbmA and RbmC. This powerful approach
will undoubtedly reveal new insights into the specific roles of different matrix proteins, which at first glance

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appear to have overlapping roles in the biofilm formation of e.g. staphylococci (Christner et al., 2010).

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Proteins that fold into a cross-beta structure and polymerize into insoluble fibers are called amyloids.
Amyloids initially received attention due to their role in neurodegenerative diseases where they occur due
to a misfolding of proteins, but it turns out that bacteria purposely produce amyloids, and Larsen et al.

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(2007) changed our perception of amyloids when showing that they are abundant in bacterial biofilms from
a variety of different habitats. Amyloid fibers are resistant to degradation by proteases and they contribute

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to the structural integrity of biofilms by e.g. Bacillus subtilis (Romero et al., 2010) and Staphylococcus
aureus (Schwartz et al., 2012). A recent study showed that over-expression of amyloids in the matrix of

Pseudomonas fluorescens biofilms led to a 20-fold increase in the biofilm stiffness (Zeng et al., 2015). As

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methods for amyloid detection improve, we will learn more about the role of amyloid production in the

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ecology of biofilms.

Larsen et al. (2007) compared the specificity of thioflavin T (ThT) and Congo red staining with detection by
amyloid-specific antibodies and showed that ThT was highly sensitive and more easily penetrated biofilms

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compared to the antibodies, but it was less specific, probably due to its ability to bind DNA (Ilanchelian and
Ramaraj, 2004). Antibodies are thus more appropriate for amyloid visualization in complex samples, but the
cumbersome procedure with multiple incubation steps at different temperatures to allow the primary and
secondary antibodies to bind, does not allow time-resolved imaging. However, a recent study presents a
novel fluorescent probe, CDy11, with specificity for amyloid and looks like a promising new approach that is
even suitable for in vivo detection of bacterial amyloid in e.g. Pseudomonas aeruginosa biofilm infections
(Kim et al., 2016).
2.4 Lipid staining

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Production of biosurfactants is an important part of the biofilm life cycle in many bacteria. Biosurfactants
include polysaccharides, proteins, lipoproteins, glycolipids and lipopeptides, and in the context of biofilms

they can have very diverse functions in the production, maturation and dispersal of biofilms (Raaijmakers et

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al., 2010). Lipids in general (including membranes) can be stained with Nile red, which also binds to

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hydrophobic domains of proteins. Nile red is not confined to the extracellular matrix and will therefore
stain the cell membranes as well as intracellular lipids. It has been used extensively to visualise intracellular

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lipophilic storage compounds, such as polyhydroxyalkanoates (Zuriani et al., 2013). The Nile red emission
peak differs according to whether it binds to polar or nonpolar lipids (Diaz et al., 2008), and this was

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exploited to show that Rhodococcus strain RC291 with a hydrophobic cell surface associated closely with
both polar and non-polar lipids in the biofilm matrix, suggesting an important role of lipids in the biofilm

architecture of this strain (Andrews et al., 2010). Alternatives to Nile Red are the hydrophobic BODIPY

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dyes and carbocyanine DiD, which will stain lipids, membranes and other hydrophobic compounds (Baird et
al., 2012; Rumin et al., 2015). In contrast to Nile Red and BODIPY dyes, FM stains, which brightly stain

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lipids in membranes, do not enter the cytoplasm. They are extensively used to study endocytosis and
exocytosis in plants (Bolte et al., 2004), and would be ideal to study the formation of outer membrane

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vesicles, which can be a significant part of the biofilm matrix (Ojima et al., 2015a; Schooling and Beveridge,
2006) and have been suggested to interact with or contribute to release of extracellular DNA (Sahu et al.,
2012; Schooling et al., 2009).
2.5 Monitoring of enzyme reactions
Various enzymatic reactions are carried out in the extracellular matrix of biofilms, and a multitude of
enzyme activity assays based on the detection of fluorescent products have been developed (Barizuddin et
al., 2015; Ju et al., 2015; Ko et al., 2015; Walther et al., 2015). While diffusion of the reaction product
renders the visualization of enzymatic activities difficult, a fluorogenic phosphatase substrate that forms
precipitates at the site of reaction (ELF 97) is commercially available. Van Ommen Kloeke and Geesey

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used the assay in activated sludge, combining it with fluorescence in situ hybridization, albeit with an
epifluorescence microscope (Van Ommen Kloeke, F. and Geesey, 1999). They were able to demonstrate

that the cytophaga-flavobacteria group makes an important contribution to the removal of phosphorus

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from wastewater.

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3 Confocal microscopy approaches to quantitative analysis of the biofilm matrix


In addition to the many qualitative descriptions of the ECM that have been performed, a number of studies

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have used CLSM based approaches that investigate different properties of the biofilm matrix quantitatively.
These require the combination of confocal microscopy with digital image analysis and/or mathematical

modelling. Due to the intense development of specialized image processing software with user-friendly

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interfaces and the implementation of techniques such as fluorescence lifetime imaging (FLIM) or

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fluorescence recovery after photobleaching (FRAP) in commercially available confocal microscopes,


quantitative methods have seen a rise in recent years.

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3.1 Geometric measurements

Geometric measurements performed on fluorescently labelled structural components of the ECM


contribute in many ways to a better understanding of matrix functionality. Area and biovolume calculations
permit to determine the predominant matrix components and their spatial distribution at different time
points during biofilm formation. Measuring of distances and colocalization patterns between different
matrix components allows drawing conclusions about the interplay between different molecules, and
analysing colocalization patterns between matrix components and microbial cells can help to identify e.g.
EPS producers in multispecies biofilms.
While these kinds of geometric measurements are routinely carried out on the cellular components of
biofilms (Bridier et al., 2010; Dige et al., 2012; Guo et al., 2013a; Lupini et al., 2011), until now most studies

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investigating the ECM have been limited to qualitative or semi-quantitative descriptions. Only a few reports
performed quantitative geometric analyses of the biofilm matrix and the relationship between matrix

components and cells (Fish et al., 2015; Houari et al., 2013; Kuehn et al., 2001; Lawrence et al., 1998;

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Sweity et al., 2011). The comprehensive work of Koo and collaborators who investigated the amount of

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fluorescently labelled polysaccharides in the ECM of Streptococcus mutans biofilms should be noted
(Falsetta et al., 2012; Klein et al., 2009; Klein et al., 2011; Xiao and Koo, 2010). They showed that starch in

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the growth medium leads to increased amounts of EPS in the presence of sucrose, and that a combination
treatment of biofilms with myricetin, farnesol and fluoride targets genes involved in matrix production and

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reduces EPS production. As extracellular polysaccharides in dental biofilm play an important role for biofilm
stability and the conservation of low pH at the tooth surface, quantitative studies of the effect of different

treatments on EPS production are particularly valuable to identify new therapeutic approaches to caries

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control. In general, geometric measurements of structural matrix components should be performed more
frequently to facilitate comparisons between different reports, and to reduce the bias that might arise if

entire sample.

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phenomena are described on the basis of subjective observations that might not be representative for the

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Digital image analysis tools for geometric analyses of confocal microscopy images are readily available:
Specially designed programs, such as Comstat, daime, CMEIAS (all freeware), Imaris, Amira, Volocity, Arivis
and the open source software environments ImageJ, Icy and BioImageXD provide a number of possibilities
for quantitative structural analysis of fluorescence images.
When geometric measurements in biofilms are performed, microscope settings must be chosen carefully,
ideally by calibration with fluorescent beads of known size (Lawrence et al., 1998). Pinhole size, detector
gain and amplifier gain/offset have been shown to influence area measurements based on confocal images
(Sekar et al., 2010). Overexposure must be avoided and microscope settings should be kept constant
throughout a series of experiments.

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The most critical step in the subsequent image analysis is the segmentation process, the differentiation
between stained objects and background fluorescence. A number of different algorithms, e.g. based on

intensity thresholding, edge detection or region-growing, can be employed to identify objects. The ideal

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algorithm and segmentation parameters have to be determined individually for a particular set of biofilm

image data are mandatory to ascertain proper calculations.

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samples. In any case, meticulous visual inspection of the segmented images and comparison to the original

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Not only the area or volume covered by different structures of the ECM, but also their spatial arrangement
can be investigated quantitatively. Colocalization analyses were first performed to determine the spatial

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interaction of different bacterial populations in activated sludge flocs (Rodenacker et al., 2000). This kind of
analysis has become more widely used for bacterial cells after its incorporation into the capability of the

image analysis software daime (Augspurger et al., 2010; Kara et al., 2007; Maixner et al., 2006; Schillinger

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et al., 2012). The program employs a linear dipole algorithm (Reed and Howard, 1999) to determine if two

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different populations of fluorescently labelled objects co-localize (Daims et al., 2006). Images subjected to
colocalization analysis must be cleared carefully for artifacts (i.e. by using algorithms that remove objects
up to a certain pixel size), as the presence of fluorescent objects other than the investigated populations,

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irrespective of their size, will lead to erroneous results. Moreover, the physical properties of the samples
must be taken into account. Areas without biofilm formation, including carrier materials, need to be
excluded from the image analysis. So far, colocalization analyses of ECM components have been employed
in laboratory biofilms to determine the spatial relationship between dental bacteria and fluorescently
labelled dextrans (Xiao et al., 2012), and to study the colocalization of eDNA and wheat germ agglutinin
(WGA) targeted exopolysaccharides in Myxococcus xanthus biofilms (Hu et al., 2012). The software Duostat
(www.imageanalysis.dk) and the ImageJ plugin JACoP (Schneider et al., 2012) were used for calculations in
the respective studies. An increased focus on quantitative analyses of spatial arrangements in the biofilm
matrix is highly desirable, as it can expand our knowledge on the production and function of different ECM
components, especially in multispecies biofilms.

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3.2 Measuring concentrations of diffusing molecules
Many dissolved molecules are important for biofilm development, maintenance and virulence. Oxygen and

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carbon dioxide concentrations, pH, and metal ions such as Ca2+, Mg2+, Zn2+ and Fe3+ have dramatic effects

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on metabolic processes carried out in biofilms. For all of these molecules, different fluorescent probes are
commercially available, and within a certain range, the fluorescence intensity correlates with the molecule
concentration. At first glance, it might seem straightforward to use fluorescence intensity measurements to

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determine local concentrations of small solutes. However, a number of factors other than bulk
concentration of the dye affect the fluorescence. Microscope parameters such as laser power, detector

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gain and amplifier offset/gain are crucial and difficult to standardize, and fluorescent probes are bleached
to a different extent during measurements, depending on the sample processing. Moreover, the local

probe concentration in a biofilm is unknown. All molecules face differences in penetration, reaction-

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diffusion limitations and compartmentalization in biofilms, which makes it impossible to use calibration

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data obtained from homogenous buffer solutions. Finally, interactions with biopolymers or other,
simultaneously employed dyes might alter the fluorescence properties of a probe. Consequently, only
semiquantitative comparisons can be made, even if specimens are handled in identical ways and imaged

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with identical settings (Epstein et al., 2011; Guo et al., 2013b).


3.2.1 Immobilization of fluorescent dyes in particles
Several strategies can be employed to circumvent these problems. A fluorescent probe for a particular
solute might be applied along with a reference dye that emits light in a different part of the spectrum and
does not show a spectral response to the solute (Barker et al., 1998; de los Rios, Asuncion et al., 2003).
Calculating the ratio between the fluorescence from both dyes would then allow determination of the
solute concentration, but only if the concentration ratio of the dyes is identical in every location of the
biofilm. As the diffusive properties and the binding patterns of two different stains in a biofilm might differ,

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both stains have to be immobilized, tied together with a fixed concentration ratio to enable reliable
measurements.

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To date, only two studies have taken this approach, immobilizing a sensitive fluorescent dye and a

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reference dye on particles. Hidalgo et al. (2009) investigated pH microenvironments in E. coli and mixed
species wastewater biofilms, using core shell silica nanoparticles containing covalently bound pH sensitive
fluorescein isothiocyanate, and Cy5 as the reference stain (Hidalgo et al., 2009). Acosta et al. monitored

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oxygen gradients in S. aureus biofilms using silica microparticles containing Ru(Ph2phen3)Cl2, which is
quenched in an oxygen dependent fashion, and Nile blue chloride as reference (Acosta et al., 2012). Dyes

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immobilized on a particle offer good photostability, and due to the embedding in the silica matrix,
interactions with biopolymers and their potential influence on the emission spectra are reduced. Still, the

effect of biofilm components on fluorescence should be tested, as done by Hidalgo et al. (2009). Moreover,

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calibration should be performed at the same temperature as the actual pH measurements, as the

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fluorescence intensity might be temperature dependent. The simultaneous use of two dyes brings about
some difficulties. FRET between the two dyes must be excluded, and the emission spectra of the dyes must
not overlap. Visualization of the bacterial biomass with a third stain is complicated, as any overlap between

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the dyes would affect the calculated fluorescence ratios. Acosta et al. (2012) used DyLightTM 488-labelled
antibodies, the emission of which partly overlaps with the one of Ru(Ph2phen3)Cl2. Hidalgo et al. (2009)
refrained from employing a third stain and used bright-field images to visualize bacterial cells, at the cost of
losing three-dimensional information.
The use of particle sensors for solute visualization has some inherent disadvantages. Production of the
sensors requires knowledge foreign to the field of microbiology, and unless the relative concentrations of
both dyes on the particles are stable, calibration must be performed for every new batch of particles.
Furthermore, biofilm penetration and the spatial distribution of the particles pose problems. The
microparticles employed by Acosta et al. did not penetrate established S. aureus biofilms and had to be

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applied before and during biofilm growth. This precludes their use in in situ grown medical biofilms. Hidalgo
et al. tested different sizes of nanoparticles and found that only the smallest, 10 nm particles penetrated

the biofilms sufficiently. Incubation had to be performed for several hours, which makes the rapid

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examination of in situ grown biofilms impossible. The 10 nm particles employed were not distributed

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evenly across the biofilms, but left certain cell-free areas unstained, precluding calculation of pH in the
entire extracellular matrix. While bigger particles settle in the extracellular matrix, it cannot be excluded

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that small particles are internalized by microbial cells. Interactions with intracellular macromolecules might
change their fluorescence properties, and moreover, the concentration of the investigated solute might

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differ considerably between intracellular and extracellular compartments, due to bacterial homeostasis. In
the case of pH, averaging fluorescence ratios deriving from both intra- and extracellular areas is of little

relevance.

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3.2.2 Fluorescence lifetime imaging (FLIM) and pH ratiometry

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Some of the problems encountered with two dyes immobilized in a particle can be avoided when solutes
are quantified by FLIM of a single probe or when intrinsically ratiometric dyes are employed. For FLIM, dye
molecules are excited by a short light pulse, and the resulting fluorescence is recorded in a time-resolved

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manner. Fluorescence decay over time changes with the surrounding environmental conditions and follows
an exponential function that is independent of the initial fluorescence intensity and thus probe
concentration. Vroom et al. made use of the pH dependent fluorescence lifetime of carboxyfluorescein to
determine pH in a 10-species laboratory dental biofilm (Vroom et al., 1999). The ratio of fluorescence
intensities recorded in two different time gates was calculated and translated into pH values.
FLIM uses the inherent fluorescence properties of a single dye in a concentration independent way to
quantify solutes. Immobilization is thus unnecessary, penetration of the dye into the biofilm is
unproblematic and interactions between different stains can be avoided. The same advantages also apply
to ratiometric dyes, and in addition, their use requires less advanced microscopy equipment. Ratiometric

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dyes are characterized by a shift of their fluorescence spectrum upon binding to an analyte. In the case of
pH-sensitive ratiometric dyes, both the unprotonated and the protonated form emit light when excited, but

with two different emission spectra. Simultaneous recording of the intensity in two different detection

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windows allows determining the fluorescence ratio which is correlated to pH but independent of probe

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concentration. The first reports on ratiometric pH microscopy in microbiology date back to the 1990s
(Hassan et al., 1995a, 1995b), and the technique has been used more frequently in recent years (Franks et

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al., 2009; Hunter and Beveridge, 2005; Schlafer et al., 2011; Schlafer et al., 2015; Schlafer and Dige, 2015;
Xiao et al., 2012).

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It is of central importance for both FLIM and ratiometry whether the fluoroprobe penetrates microbial cells
in the biofilm. If pH in the extracellular matrix is to be determined, inclusion of fluorescence deriving from

intracellular compartments leads to erroneous results. C-SNARF-4, employed by Hunter and Beveridge

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and Franks et al., and carboxyfluorescein, employed by Vroom et al., both penetrate bacterial cells at low

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pH, and the authors report calculations that are based on both intra- and extracellular fluorescence. Xiao et
al., on the other hand, used dextran-coupled LysosensorTM Yellow/Blue, which they report does not target
bacterial cells. As outlined above, the concomitant use of several fluorescent dyes poses problems for

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quantitative microscopy. FLIM and ratiometry both allow quantification of a solute with just one dye, but if
additional stains are used to visualize cells or molecules in the ECM, the fluorescence emission spectra of
the applied dyes must not overlap. Xiao et al. employed Alexa Fluor 647-conjugated dextran and SYTO
60 to visualize bacterial cells and ECM, respectively, both of which do not interfere with Lysosensor TM
Yellow/Blue fluorescence. In contrast, Hunter and Beveridge (2005) used GFP, Franks et al. (2009) mcherry,
both of which overlap with the emission of C-SNARF-4, and Vroom et al. (1999) employed rhodamine B,
which interferes with carboxyfluorescein.
The authors of the present review exploited the cell permeability of C-SNARF-4 to both confine the
recording of pH-dependent fluorescence to the extracellular space and to visualize the microbial biomass.

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They could show that C-SNARF-4 is upconcentrated in bacterial cells at acidic pH, and they used a digital
image analysis procedure based on intensity thresholding to remove the bacterial biomass from the

confocal microscopy images (Figure 2) (Schlafer et al., 2015). As this procedure offers a simple solution to

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the problems described above, the authors recommend digital image post-processing to ascertain

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adequate monitoring of extracellular pH. In dental biofilms, microscale landscaping of extracellular pH


contributes to our understanding of the caries process. It allows studying how pH profiles and mineral loss

controlling agents on pH (Schlafer et al., 2016).

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of the underlying dental tissues correlate and it is a valuable tool to investigate the effect of caries

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For all confocal microscopy approaches to solute quantification in biofilms the importance of the
calibration procedure needs to be stressed. A variety of environmental factors, such as temperature, the

presence of biopolymers and other fluorescent stains, and varying concentrations of other solutes than the

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one in question might affect the fluorescence intensity of the chosen fluoroprobe. Ratiometric calcium-

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sensitive dyes might serve as an example. The emission spectra of probes such as Fura-2 and Indo-1 are
dependent on calcium-binding, but also on temperature and pH (Larsson et al., 1999; Oliver et al., 2000). If
they were to be applied in an acid-producing biofilm, local variations in pH would render an appropriate

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interpretation of the effect of calcium concentration on the observed fluorescence ratios impossible. While
a large number of fluorescent probes for different solutes are commercially available, increased attention
needs to be paid to their specific target-oriented calibration prior to experimental use. Ideally, calibration
of a dye should be performed in the presence of biofilms and all other stains employed concomitantly to
exclude adverse effects on the performed measurements. If the described technical difficulties can be
overcome, confocal microscopy measurements of solute concentrations can make an important
contribution to our understanding of the metabolic processes carried out in different microenvironments of
biofilms.

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Given the cell permeability of some pH-sensitive dyes, it is tempting to employ these fluoroprobes to
monitor intracellular changes of pH at the individual cell level (Shabala et al., 2006). For calibration, trans-

membrane gradients of bacterial cells can be collapsed using permeant acids and bases, but for reliable

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intracellular pH measurements, it must be ascertained that the probe is exclusively located inside the

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bacterial cells and not bound on the outside of the cell wall. It is impossible to determine this based on
confocal images alone. Martinez et al. elegantly circumvented this problem using genetically engineered

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strains of E. coli and Bacillus subtilis that expressed the intracellular ratiometric protein pHluorin (Martinez
et al., 2012). Their approach, however, cannot be extended to in situ grown biofilms.

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3.2.3 Frster resonance energy transfer (FRET)

FRET-based biosensing is another technique that might be exploited for concentration measurements. FRET

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is widely used in cell biology for in situ, real-time monitoring of macromolecule interactions and
concentrations of intracellular metabolites and signaling molecules through FRET-based biosensors (Mohsin

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et al., 2015; Shrestha et al., 2015). FRET-based biosensing involves a fluorescent donor (typically a
fluorescent protein) and an acceptor, which quenches the fluorescence of the donor upon interaction,
resulting in excitation and emission of light from the acceptor fluorophore. Although applications in

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microbiology are less common, FRET-based biosensing is making its way into biofilm research, and a
biosensor was recently developed to detect the intracellular c-di-GMP concentration in Caulobacter
crescentus (Christen et al., 2010) and Salmonella typhimurium (Mills et al., 2015) through conformational
changes in the c-di-GMP binding protein YcgR fused with a donor and acceptor fluorescent protein at either
end. Despite the great potential for characterizing inter-molecular interactions and extracellular
concentrations of e.g. signaling molecules in biofilm, this approach is yet to see its first application in
studies of the biofilm matrix. The main requirement for extracellular detection is of course that the
genetically encoded biosensor must be transported outside of the cell, and secondly that the sensing
molecule then remains in the biofilm. One way to achieve this could be to initially focus the effort on

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detecting interactions of cell wall- or cell membrane anchored proteins, for which fluorescent fusion
proteins can be generated. This was recently done for the membrane bound nuclease Nuc2 in S. aureus,

and a FRET-based assay was used albeit not in combination with microscopy to study the activity of this

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extracellular nuclease in vitro and in vivo (Kiedrowski et al., 2014). Hence FRET-based biosensing in the

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extracellular environment of a biofilm might not be so far into the future.

3.3.1 Time-lapse imaging of fluorescent solutes

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3.3 Measuring diffusion properties

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A number of different confocal microscopy techniques can be used to quantitatively investigate diffusion
properties in biofilms. Time-lapse imaging of fluorescent or fluorescently labelled molecules is a

straightforward approach: A biofilm is exposed to a fluorescent molecule under static or dynamic

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conditions, and repeated confocal microscopy images are taken in a particular location. The fluorescence
intensity is recorded in a semiquantitative way, using the intensity in the bulk fluid or the intensity of a

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stain that already has penetrated as a reference, until equilibrium is reached. The publications of Stewart
and coworkers should be noted, who found that antibiotic-sized tracers, daptomycin and even a variety of

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macromolecular solutes penetrated bacterial clusters (>100 m) in laboratory biofilms within a few
minutes (Rani et al., 2005; Stewart et al., 2009; Takenaka et al., 2009). Their work and a similar study
conducted by Stone et al. (2002) proved the long standing theory wrong that the biofilm matrix constitutes
a significant penetration barrier against antibiotics.
3.3.2 Single particle tracking (SPT) microscopy
Time-lapse imaging can also be employed to track the fate of single nano- or microscale particles in
biofilms. Determining particle trajectories under flow conditions, as performed by Stoodley et al. (1994)
and Kuehn et al. (2001), allows collecting detailed information on the hydrodynamics in different parts of
the biofilm matrix, e.g. in bioreactors for wastewater treatment. Moreover, studies of particle diffusion

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properties are useful to develop suitable carrier particles for controlled drug delivery to biofilms. Two
recent reports investigated the diffusional behavior of particles with different size and surface chemistry in

biofilms under static conditions (Birjiniuk et al., 2014; Forier et al., 2013). Both studies found that neutral

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(PEGylated) particles diffuse more rapidly through biofilms than charged particles and that diffusion is size-

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dependent. Moreover, Birjiniuk et al. (2014) showed that matrix density of E. coli biofilms increased with
age, leading to reduced diffusion, and that charge density increased in deeper layers of the biofilm. By

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comparing the motion paths of particles present during biofilm growth and particles added after biofilm
growth they evidenced the presence of channels permitting rapid diffusion.

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3.3.3 Fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS)
While time-lapse imaging is a valuable tool for diffusion measurements, it has a limited spatial and

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temporal resolution. Diffusion is quantified over a rather large volume of the biofilm, i.e. cell clusters with a
radial dimension of 100 200 m (Stewart et al., 2009), and local differences in diffusivity cannot be

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resolved. Acquisition times for microscopic images are in the order of seconds, and the diffusion and
reactivity of the molecules in question cannot be tracked any longer, once equilibrium is reached. FRAP and
FCS can overcome these problems, and today both techniques can be implemented in commercially

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available confocal microscopes. FRAP measures the increase of fluorescence intensity in a defined volume
of the matrix after irreversible photobleaching of the fluorophore. The resulting fluorescence recovery
curve is dependent on the inward diffusion of the fluorescent molecule into the bleached area. For FCS,
fluorescent dyes or fluorescently labelled molecules are applied in nanomolar concentrations, and the
durations and amplitudes of fluctuations in the fluorescence intensity are recorded in a defined volume of
the matrix. Both techniques allow determining diffusion coefficients in small volumes of the biofilm with a
temporal resolution in the order of milliseconds. Early FRAP approaches date back to the 1990s (Bryers and
Drummond, 1998; Lawrence et al., 1994), but in particular, the effort of Briandet and coworkers should be
mentioned, who have improved both techniques considerably and applied them to study diffusion of

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bacteriophages, dextrans and antibiotics in different biofilms (Briandet et al., 2008; Daddi Oubekka et al.,
2012; Guiot et al., 2002; Lacroix-Gueu et al., 2005; Waharte et al., 2010). For a review, see Bridier et al.

(2011). As biofilms are the causative agents of many diseases and responsible for biofouling in different

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industrial fields, a detailed understanding of the diffusional behavior of macromolecules in biofilms is of the

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utmost importance and contributes to the rational design of anti-biofilm agents.


3.3.4 Rheology measurements

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As outlined above (3.3.2) the behavior of nano- or microscale particles can be used to describe diffusion

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processes in biofilms, but it can also be employed to determine the rheological properties of the matrix.
Galy et al. (2012) embedded 2.8 m sized magnetic particles in E. coli biofilms during growth and applied
defined forces to the particles via magnetic tweezers. Tracking the movements of the particles allowed

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plotting creep curves from which the viscoelastic behavior of the matrix in defined locations could be
extracted. A similar approach was taken by Rogers et al. (2008) who tracked the trajectories of bacteria in

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S. aureus and P. aeruginosa biofilms exposed to different flow regimes, albeit with bright field microscopy.
Mathias and Stoodley (2009) used digital image correlation to quantify the strains deriving from different
flow rates, equally based on bright field microscopy. The latter two approaches might be combined with

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confocal imaging to quantitatively assess the mechanical properties of biofilms.

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4 Conclusions

In recent years, there has been an increased research focus on the extracellular matrix in biofilms, which

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has led to a better understanding of matrix complexity, its structural, chemical and physical organization.

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Confocal microscopy techniques allow preserving the three-dimensional structure of biofilms, investigating
the matrix in fully hydrated state. Different structural matrix components and their spatial arrangement can
be studied, the concentrations of solutes and their role in biofilm physiology and virulence determined, and

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the mechanical and diffusive properties of different microenvironments in the matrix probed. Being a
standard analysis tool in many research laboratories, the use of confocal microscopy evolves constantly and

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will continuously provide deeper insight into the structure and functioning of the extracellular matrix.

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Figure captions

Figure 1. eDNA in Staphylococcus epidermidis biofilm becomes more abundant over time. The biofilm

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and 20 M SYTO 60 for intracellular DNA (red). Bar = 5 m.

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was grown in Tryptic Soy Broth for 24 (A) or 48 hours (B) and stained with 2 m TOTO-1 for eDNA (green),

Figure 2. Ratiometric imaging of pH in in vivo grown dental biofilm exposed to glucose. The ratiometric

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pH-sensitive dye C-SNARF-4 is employed in combination with digital image post-processing to visualize
extracellular pH in real-time. A) C-SNARF-4 is taken up by all bacterial cells in the biofilm, yielding

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different levels of fluorescent intensity in extracellular and intracellular compartments. An overlay of


fluorescent emission in the green and red spectra is shown. B) The bacterial biomass has been removed

from the image in panel A) using digital image analysis. Extracellular pH has been calculated by dividing the

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green by the red fluorescent intensities pixel-wise. A color lookup table was used to visualize pH. Acid

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production in the observed field of view is moderate. With an average extracellular pH of 6.28, 5 min after

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exposure to glucose, levels critical for enamel dissolution (ca. 5.5) have not yet been reached. Bars = 20 m

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References

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CE
P

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MA

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SC
R

IP

Acosta, M.A., Velasquez, M., Williams, K., Ross, J.M., Leach, J.B., 2012. Fluorescent silica particles for
monitoring oxygen levels in three-dimensional heterogeneous cellular structures. Biotechnology and
bioengineering 109 (10), 26632670.
Allesen-Holm, M., Barken, K.B., Yang, L., Klausen, M., Webb, J.S., Kjelleberg, S., Molin, S., Givskov, M.,
Tolker-Nielsen, T., 2006. A characterization of DNA release in Pseudomonas aeruginosa cultures and
biofilms. Molecular microbiology 59 (4), 11141128.
Andrews, J.S., Rolfe, S.A., Huang, W.E., Scholes, J.D., Banwart, S.A., 2010. Biofilm formation in
environmental bacteria is influenced by different macromolecules depending on genus and species.
Environmental microbiology 12 (9), 24962507.
Arciola, C.R., Campoccia, D., Ravaioli, S., Montanaro, L., 2015. Polysaccharide intercellular adhesin in
biofilm: structural and regulatory aspects. Frontiers in cellular and infection microbiology 5, 7.
Augspurger, C., Karwautz, C., Mussmann, M., Daims, H., Battin, T.J., 2010. Drivers of bacterial colonization
patterns in stream biofilms. FEMS microbiology ecology 72 (1), 4757.
Baird, F.J., Wadsworth, M.P., Hill, J.E., 2012. Evaluation and optimization of multiple fluorophore analysis of
a Pseudomonas aeruginosa biofilm. Journal of microbiological methods 90 (3), 192196.
Barizuddin, S., Balakrishnan, B., Stringer, R.C., Dweik, M., 2015. Highly specific and rapid immunofluorescent visualization and detection of E. coli O104:H4 with protein-A coated magnetic beads based
LST-MUG assay. Journal of microbiological methods 115, 2733.
Barker, W.W., Welch, S.A., Chu, S., Banfield, J.F., 1998. Experimental observations of the effects of bacteria
on aluminiosilicate weathering. Am Mineralogist 83, 15511563.
Berk, V., Fong, Jiunn C N, Dempsey, G.T., Develioglu, O.N., Zhuang, X., Liphardt, J., Yildiz, F.H., Chu, S., 2012.
Molecular architecture and assembly principles of Vibrio cholerae biofilms. Science (New York, N.Y.) 337
(6091), 236239.
Birjiniuk, A., Billings, N., Nance, E., Hanes, J., Ribbeck, K., Doyle, P.S., 2014. Single particle tracking reveals
spatial and dynamic organization of the E. coli biofilm matrix. New journal of physics 16 (8), 085014.
Bolte, S., Talbot, C., Boutte, Y., Catrice, O., Read, N.D., Satiat-Jeunemaitre, B., 2004. FM-dyes as
experimental probes for dissecting vesicle trafficking in living plant cells. Journal of Microscopy 214 (Pt
2), 159173.
Briandet, R., Lacroix-Gueu, P., Renault, M., Lecart, S., Meylheuc, T., Bidnenko, E., Steenkeste, K., BellonFontaine, M.-N., Fontaine-Aupart, M.-P., 2008. Fluorescence correlation spectroscopy to study diffusion
and reaction of bacteriophages inside biofilms. Applied and environmental microbiology 74 (7), 2135
2143.
Bridier, A., Dubois-Brissonnet, F., Boubetra, A., Thomas, V., Briandet, R., 2010. The biofilm architecture of
sixty opportunistic pathogens deciphered using a high throughput CLSM method. Journal of
microbiological methods 82 (1), 6470.
Bridier, A., Tischenko, E., Dubois-Brissonnet, F., Herry, J.-M., Thomas, V., Daddi-Oubekka, S., Waharte, F.,
Steenkeste, K., Fontaine-Aupart, M.-P., Briandet, R., 2011. Deciphering biofilm structure and reactivity
by multiscale time-resolved fluorescence analysis. Advances in experimental medicine and biology 715,
333349.
Bryers, J.D., Drummond, F., 1998. Local Macromolecule Diffusion Coefficients in Structurally Non-Uniform
Bacterial Biofilms Using Fluorescence Recovery After Photobleaching (FRAP). Biotechnology and
bioengineering 60 (4), 462473.

ACCEPTED MANUSCRIPT

AC

CE
P

TE

MA

NU

SC
R

IP

Bttner, H., Mack, D., Rohde, H., 2015. Structural basis of Staphylococcus epidermidis biofilm formation:
mechanisms and molecular interactions. Frontiers in cellular and infection microbiology 5, 14.
Christen, M., Kulasekara, H.D., Christen, B., Kulasekara, B.R., Hoffman, L.R., Miller, S.I., 2010. Asymmetrical
distribution of the second messenger c-di-GMP upon bacterial cell division. Science (New York, N.Y.)
328 (5983), 12951297.
Christner, M., Franke, G.C., Schommer, N.N., Wendt, U., Wegert, K., Pehle, P., Kroll, G., Schulze, C., Buck, F.,
Mack, D., Aepfelbacher, M., Rohde, H., 2010. The giant extracellular matrix-binding protein of
Staphylococcus epidermidis mediates biofilm accumulation and attachment to fibronectin. Molecular
microbiology 75 (1), 187207.
Conover, M.S., Mishra, M., Deora, R., 2011. Extracellular DNA is essential for maintaining Bordetella biofilm
integrity on abiotic surfaces and in the upper respiratory tract of mice. PloS one 6 (2), e16861.
Daddi Oubekka, S., Briandet, R., Fontaine-Aupart, M.-P., Steenkeste, K., 2012. Correlative time-resolved
fluorescence microscopy to assess antibiotic diffusion-reaction in biofilms. Antimicrobial agents and
chemotherapy 56 (6), 33493358.
Daims, H., Lcker, S., Wagner, M., 2006. daime, a novel image analysis program for microbial ecology and
biofilm research. Environmental microbiology 8 (2), 200213.
Das, T., Kutty, S.K., Kumar, N., Manefield, M., 2013. Pyocyanin facilitates extracellular DNA binding to
Pseudomonas aeruginosa influencing cell surface properties and aggregation. PloS one 8 (3), e58299.
de los Rios, Asuncion, Wierzchos, J., Sancho, L.G., Ascaso, C., 2003. Acid microenvironments in microbial
biofilms of antarctic endolithic microecosystems. Environmental microbiology 5 (4), 231237.
Diaz, G., Melis, M., Batetta, B., Angius, F., Falchi, A.M., 2008. Hydrophobic characterization of intracellular
lipids in situ by Nile Red red/yellow emission ratio. Micron (Oxford, England : 1993) 39 (7), 819824.
Dige, I., Schlafer, S., Nyvad, B., 2012. Difference in initial dental biofilm accumulation between night and
day. Acta odontologica Scandinavica 70 (6), 441447.
Epstein, A.K., Pokroy, B., Seminara, A., Aizenberg, J., 2011. Bacterial biofilm shows persistent resistance to
liquid wetting and gas penetration. Proceedings of the National Academy of Sciences of the United
States of America 108 (3), 9951000.
Falsetta, M.L., Klein, M.I., Lemos, J.A., Silva, B.B., Agidi, S., Scott-Anne, K.K., Koo, H., 2012. Novel antibiofilm
chemotherapy targets exopolysaccharide synthesis and stress tolerance in Streptococcus mutans to
modulate virulence expression in vivo. Antimicrobial agents and chemotherapy 56 (12), 62016211.
Fish, K.E., Collins, R., Green, N.H., Sharpe, R.L., Douterelo, I., Osborn, A.M., Boxall, J.B., 2015.
Characterisation of the physical composition and microbial community structure of biofilms within a
model full-scale drinking water distribution system. PloS one 10 (2), e0115824.
Flemming, H.-C., Wingender, J., 2010. The biofilm matrix. Nature reviews. Microbiology 8 (9), 623633.
Forier, K., Messiaen, A.-S., Raemdonck, K., Deschout, H., Rejman, J., Baets, F. de, Nelis, H., De Smedt,
Stefaan C, Demeester, J., Coenye, T., Braeckmans, K., 2013. Transport of nanoparticles in cystic fibrosis
sputum and bacterial biofilms by single-particle tracking microscopy. Nanomedicine (London, England)
8 (6), 935949.
Foster, T.J., Hk, M., 1998. Surface protein adhesins of Staphylococcus aureus. Trends in microbiology 6
(12), 484488.
Frank, K.L., Patel, R., 2007. Poly-N-Acetylglucosamine Is Not a Major Component of the Extracellular Matrix
in Biofilms Formed by icaADBC-Positive Staphylococcus lugdunensis Isolates. Infection and immunity 75
(10), 47284742.

ACCEPTED MANUSCRIPT

AC

CE
P

TE

MA

NU

SC
R

IP

Franks, A.E., Nevin, K.P., Jia, H., Izallalen, M., Woodard, T.L., Lovley, D.R., 2009. Novel strategy for threedimensional real-time imaging of microbial fuel cell communities: monitoring the inhibitory effects of
proton accumulation within the anode biofilm. Energy Environ. Sci. 2 (1), 113119.
Galy, O., Latour-Lambert, P., Zrelli, K., Ghigo, J.-M., Beloin, C., Henry, N., 2012. Mapping of bacterial biofilm
local mechanics by magnetic microparticle actuation. Biophysical journal 103 (6), 14001408.
Geoghegan, J.A., Corrigan, R.M., Gruszka, D.T., Speziale, P., O'Gara, J.P., Potts, J.R., Foster, T.J., 2010. Role
of surface protein SasG in biofilm formation by Staphylococcus aureus. Journal of bacteriology 192 (21),
56635673.
Greiner, L.L., Edwards, J.L., Shao, J., Rabinak, C., Entz, D., Apicella, M.A., 2005. Biofilm Formation by
Neisseria gonorrhoeae. Infection and immunity 73 (4), 19641970.
Guiot, E., Georges, P., Brun, A., Fontaine-Aupart, M.P., Bellon-Fontaine, M.N., Briandet, R., 2002.
Heterogeneity of Diffusion Inside Microbial Biofilms Determined by Fluorescence Correlation
Spectroscopy Under Two-photon Excitation. Photochemistry and Photobiology 75 (6), 570578.
Guo, K., Freguia, S., Dennis, P.G., Chen, X., Donose, B.C., Keller, J., Gooding, J.J., Rabaey, K., 2013a. Effects of
surface charge and hydrophobicity on anodic biofilm formation, community composition, and current
generation in bioelectrochemical systems. Environmental science & technology 47 (13), 75637570.
Guo, L., Hu, W., He, X., Lux, R., McLean, J., Shi, W., 2013b. investigating acid production by Streptococcus
mutans with a surface-displayed pH-sensitive green fluorescent protein. PloS one 8 (2), e57182.
Hassan, A.N., Frank, J.F., Farmer, M.A., Schmidt, K.A., Shalabi, S.I., 1995a. Formation of Yogurt
Microstructure and Three-Dimensional Visualization as Determined by Confocal Scanning Laser
Microscopy. Journal of Dairy Science 78 (12), 26292636.
Hassan, A.N., Frank, J.F., Farmer, M.A., Schmidt, K.A., Shalabi, S.I., 1995b. Observation of Encapsulated
Lactic Acid Bacteria Using Confocal Scanning Laser Microscopy. Journal of Dairy Science 78 (12), 2624
2628.
Hidalgo, G., Burns, A., Herz, E., Hay, A.G., Houston, P.L., Wiesner, U., Lion, L.W., 2009. Functional
tomographic fluorescence imaging of pH microenvironments in microbial biofilms by use of silica
nanoparticle sensors. Applied and environmental microbiology 75 (23), 74267435.
Houari, A., Seyer, D., Kecili, K., Heim, V., Di Martino, P., 2013. Kinetic development of biofilm on NF
membranes at the Mry-sur-Oise plant, France. Biofouling 29 (2), 109118.
Hu, W., Li, L., Sharma, S., Wang, J., McHardy, I., Lux, R., Yang, Z., He, X., Gimzewski, J.K., Li, Y., Shi, W., 2012.
DNA builds and strengthens the extracellular matrix in Myxococcus xanthus biofilms by interacting with
exopolysaccharides. PloS one 7 (12), e51905.
Hunter, R.C., Beveridge, T.J., 2005. Application of a pH-sensitive fluoroprobe (C-SNARF-4) for pH
microenvironment analysis in Pseudomonas aeruginosa biofilms. Applied and environmental
microbiology 71 (5), 25012510.
Huseby, M.J., Kruse, A.C., Digre, J., Kohler, P.L., Vocke, J.A., Mann, E.E., Bayles, K.W., Bohach, G.A.,
Schlievert, P.M., Ohlendorf, D.H., Earhart, C.A., 2010. Beta toxin catalyzes formation of nucleoprotein
matrix in staphylococcal biofilms. Proceedings of the National Academy of Sciences of the United States
of America 107 (32), 1440714412.
Ilanchelian, M., Ramaraj, R., 2004. Emission of thioflavin T and its control in the presence of DNA. Journal of
Photochemistry and Photobiology A: Chemistry 162 (1), 129137.

ACCEPTED MANUSCRIPT

AC

CE
P

TE

MA

NU

SC
R

IP

Ju, H., Ryu, B.H., Doohun Kim, T., 2015. Identification, characterization, immobilization of a novel type
hydrolase (LmH) from Listeria monocytogenes. International journal of biological macromolecules 72,
6370.
Kara, D., Luppens, Suzanne B I, van Marle, J., Ozok, R., ten Cate, Jacob M, 2007. Microstructural differences
between single-species and dual-species biofilms of Streptococcus mutans and Veillonella parvula,
before and after exposure to chlorhexidine. FEMS microbiology letters 271 (1), 9097.
Kiedrowski, M.R., Crosby, H.A., Hernandez, F.J., Malone, C.L., McNamara, J.O., Horswill, A.R., 2014.
Staphylococcus aureus Nuc2 is a functional, surface-attached extracellular nuclease. PloS one 9 (4),
e95574.
Kim, J.-Y., Sahu, S., Yau, Y.-H., Wang, X., Shochat, S.G., Nielsen, P.H., Dueholm, M.S., Otzen, D.E., Lee, J.,
Delos Santos, May Margarette Salido, Yam, J.K.H., Kang, N.-Y., Park, S.-J., Kwon, H., Seviour, T., Yang, L.,
Givskov, M., Chang, Y.-T., 2016. Detection of Pathogenic Biofilms with Bacterial Amyloid Targeting
Fluorescent Probe, CDy11. J. Am. Chem. Soc. 138 (1), 402407.
Klein, M.I., Duarte, S., Xiao, J., Mitra, S., Foster, T.H., Koo, H., 2009. Structural and molecular basis of the
role of starch and sucrose in Streptococcus mutans biofilm development. Applied and environmental
microbiology 75 (3), 837841.
Klein, M.I., Xiao, J., Heydorn, A., Koo, H., 2011. An analytical tool-box for comprehensive biochemical,
structural and transcriptome evaluation of oral biofilms mediated by mutans streptococci. Journal of
visualized experiments : JoVE (47).
Ko, K.-C., Lee, B., Cheong, D.-E., Han, Y., Choi, J.H., Song, J.J., 2015. Bacterial cell surface display of a
multifunctional cellulolytic enzyme screened from a bovine rumen metagenomic resource. Journal of
microbiology and biotechnology.
Kuehn, M., Mehl, M., Hausner, M., Bungartz, H.J., Wuertz, S., 2001. Time-resolved study of biofilm
architecture and transport processes using experimental and simulation techniques: the role of EPS.
Water Science and Technology 43 (6), 143151.
Lacroix-Gueu, P., Briandet, R., Lvque-Fort, S., Bellon-Fontaine, M.-N., Fontaine-Aupart, M.-P., 2005. In
situ measurements of viral particles diffusion inside mucoid biofilms. Comptes rendus biologies 328
(12), 10651072.
Larsen, P., Nielsen, J.L., Dueholm, M.S., Wetzel, R., Otzen, D., Nielsen, P.H., 2007. Amyloid adhesins are
abundant in natural biofilms. Environmental microbiology 9 (12), 30773090.
Larsson, D., Larsson, B., Lundgren, T., Sundell, K., 1999. The effect of pH and temperature on the
dissociation constant for fura-2 and their effects on [Ca(2+)](i) in enterocytes from a poikilothermic
animal, Atlantic cod (Gadus morhua). Analytical Biochemistry 273 (1), 6065.
Lawrence, J., Neu, T., Swerhone, G., 1998. Application of multiple parameter imaging for the quantification
of algal, bacterial and exopolymer components of microbial biofilms. Journal of microbiological
methods 32 (3), 253261.
Lawrence, J.R., Swerhone, G. D. W., Leppard, G.G., Araki, T., Zhang, X., West, M.M., Hitchcock, A.P., 2003.
Scanning Transmission X-Ray, Laser Scanning, and Transmission Electron Microscopy Mapping of the
Exopolymeric Matrix of Microbial Biofilms. Applied and environmental microbiology 69 (9), 55435554.
Lawrence, J.R., Wolfaardt, G.M., Korber, 1994. Determination of Diffusion Coefficients in Biofilms by
Confocal Laser Microscopy. Applied and environmental microbiology 60 (4), 11661173.

ACCEPTED MANUSCRIPT

AC

CE
P

TE

MA

NU

SC
R

IP

Lupini, G., Proia, L., Di Maio, M., Amalfitano, S., Fazi, S., 2011. CARD-FISH and confocal laser scanner
microscopy to assess successional changes of the bacterial community in freshwater biofilms. Journal of
microbiological methods 86 (2), 248251.
Maixner, F., Noguera, D.R., Anneser, B., Stoecker, K., Wegl, G., Wagner, M., Daims, H., 2006. Nitrite
concentration influences the population structure of Nitrospira-like bacteria. Environmental
microbiology 8 (8), 14871495.
Martinez, K.A., Kitko, R.D., Mershon, J.P., Adcox, H.E., Malek, K.A., Berkmen, M.B., Slonczewski, J.L., 2012.
Cytoplasmic pH response to acid stress in individual cells of Escherichia coli and Bacillus subtilis
observed by fluorescence ratio imaging microscopy. Applied and environmental microbiology 78 (10),
37063714.
Mathias, J.D., Stoodley, P., 2009. Applying the digital image correlation method to estimate the mechanical
properties of bacterial biofilms subjected to a wall shear stress. Biofouling 25 (8), 695703.
Messner, P., Schffer, C., Kosma, P., 2013. Bacterial cell-envelope glycoconjugates. Advances in
carbohydrate chemistry and biochemistry 69, 209272.
Mills, E., Petersen, E., Kulasekara, B.R., Miller, S.I., 2015. A direct screen for c-di-GMP modulators reveals a
Salmonella Typhimurium periplasmic -arginine-sensing pathway. Science signaling 8 (380), ra57.
Mohsin, M., Ahmad, A., Iqbal, M., 2015. FRET-based genetically-encoded sensors for quantitative
monitoring of metabolites. Biotechnology letters 37 (10), 19191928.
Neu, T., Swerhone, G.D., Lawrence, J.R., 2001. Assessment of lectin-binding analysis for in situ detection of
glycoconjugates in biofilm systems. Microbiology (Reading, England) 147 (Pt 2), 299313.
Nguyen, M.H., Ojima, Y., Sakka, M., Sakka, K., Taya, M., 2014. Probing of exopolysaccharides with green
fluorescence protein-labeled carbohydrate-binding module in Escherichia coli biofilms and flocs induced
by bcsB overexpression. Journal of bioscience and bioengineering 118 (4), 400405.
Ojima, Y., Nguyen, M.H., Yajima, R., Taya, M., 2015a. Flocculation of Escherichia coli Cells in Association
with Enhanced Production of Outer Membrane Vesicles. Applied and environmental microbiology 81
(17), 59005906.
Ojima, Y., Suparman, A., Nguyen, M.H., Sakka, M., Sakka, K., Taya, M., 2015b. Exopolysaccharide assay in
Escherichia coli microcolonies using a cleavable fusion protein of GFP-labeled carbohydrate-binding
module. Journal of microbiological methods 114, 7577.
Okshevsky, M., Meyer, R.L., 2014. Evaluation of fluorescent stains for visualizing extracellular DNA in
biofilms. Journal of microbiological methods 105, 102104.
Oliver, A.E., Baker, G.A., Fugate, R.D., Tablin, F., Crowe, J.H., 2000. Effects of Temperature on CalciumSensitive Fluorescent Probes. Biophysical journal 78 (4), 21162126.
Rani, S.A., Pitts, B., Stewart, P.S., 2005. Rapid diffusion of fluorescent tracers into Staphylococcus
epidermidis biofilms visualized by time lapse microscopy. Antimicrobial agents and chemotherapy 49
(2), 728732.
Rasconi, S., Jobard, M., Jouve, L., Sime-Ngando, T., 2009. Use of calcofluor white for detection,
identification, and quantification of phytoplanktonic fungal parasites. Applied and environmental
microbiology 75 (8), 25452553.
Reed, Howard, 1999. Stereological estimation of covariance using linear dipole probes. Journal of
Microscopy 195 (2), 96103.

ACCEPTED MANUSCRIPT

AC

CE
P

TE

MA

NU

SC
R

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Rodenacker, K., Brhl, A., Hausner, M., Khn, M., Liebscher, V., Wagner, M., Wuertz, S., 2000.
Quantification of biofilms in multi-spectral digital volumes from confocal laser scanning microscopes.
Image Anal. Stereol. 19, 151156.
Rogers, S.S., van der Walle, C, Waigh, T.A., 2008. Microrheology of bacterial biofilms in vitro:
Staphylococcus aureus and Pseudomonas aeruginosa. Langmuir : the ACS journal of surfaces and
colloids 24 (23), 1354913555.
Romero, D., Aguilar, C., Losick, R., Kolter, R., 2010. Amyloid fibers provide structural integrity to Bacillus
subtilis biofilms. Proceedings of the National Academy of Sciences of the United States of America 107
(5), 22302234.
Rumin, J., Bonnefond, H., Saint-Jean, B., Rouxel, C., Sciandra, A., Bernard, O., Cadoret, J.-P., Bougaran, G.,
2015. The use of fluorescent Nile red and BODIPY for lipid measurement in microalgae. Biotechnology
for biofuels 8, 42.
Raaijmakers, J.M., Bruijn, I. de, Nybroe, O., Ongena, M., 2010. Natural functions of lipopeptides from
Bacillus and Pseudomonas: more than surfactants and antibiotics. FEMS microbiology reviews 34 (6),
10371062.
Sadovskaya, I., Vinogradov, E., Flahaut, S., Kogan, G., Jabbouri, S., 2005. Extracellular carbohydratecontaining polymers of a model biofilm-producing strain, Staphylococcus epidermidis RP62A. Infection
and immunity 73 (5), 30073017.
Sahu, P.K., Iyer, P.S., Oak, A.M., Pardesi, K.R., Chopade, B.A., 2012. Characterization of eDNA from the
clinical strain Acinetobacter baumannii AIIMS 7 and its role in biofilm formation.
TheScientificWorldJournal 2012, 973436.
Schaeffer, C.R., Woods, K.M., Longo, G.M., Kiedrowski, M.R., Paharik, A.E., Bttner, H., Christner, M.,
Boissy, R.J., Horswill, A.R., Rohde, H., Fey, P.D., 2015. Accumulation-associated protein enhances
Staphylococcus epidermidis biofilm formation under dynamic conditions and is required for infection in
a rat catheter model. Infection and immunity 83 (1), 214226.
Schillinger, C., Petrich, A., Lux, R., Riep, B., Kikhney, J., Friedmann, A., Wolinsky, L.E., Gbel, U.B., Daims, H.,
Moter, A., 2012. Co-localized or randomly distributed? Pair cross correlation of in vivo grown
subgingival biofilm bacteria quantified by digital image analysis. PloS one 7 (5), e37583.
Schlafer, S., Birkedal, H., Olsen, J., Skovgaard, J., Sutherland, D.S., Wejse, P.L., Nyvad, B., Meyer, R.L., 2016.
Calcium-Phosphate-Osteopontin Particles for Caries Control. Biofouling (In press).
Schlafer, S., Dige, I., 2015. Ratiometric imaging of extracellular pH in dental biofilms. Journal of visualized
experiments : JoVE, Accepted.
Schlafer, S., Garcia, J.E., Greve, M., Raarup, M.K., Nyvad, B., Dige, I., 2015. Ratiometric imaging of
extracellular pH in bacterial biofilms with C-SNARF-4. Applied and environmental microbiology 81 (4),
12671273.
Schlafer, S., Raarup, M.K., Meyer, R.L., Sutherland, D.S., Dige, I., Nyengaard, J.R., Nyvad, B., 2011. pH
landscapes in a novel five-species model of early dental biofilm. PloS one 6 (9), e25299.
Schneider, C.A., Rasband, W.S., Eliceiri, K.W., 2012. NIH Image to ImageJ: 25 years of image analysis. Nat
Meth 9 (7), 671675.
Schooling, S.R., Beveridge, T.J., 2006. Membrane vesicles: an overlooked component of the matrices of
biofilms. Journal of bacteriology 188 (16), 59455957.
Schooling, S.R., Hubley, A., Beveridge, T.J., 2009. Interactions of DNA with biofilm-derived membrane
vesicles. Journal of bacteriology 191 (13), 40974102.

ACCEPTED MANUSCRIPT

AC

CE
P

TE

MA

NU

SC
R

IP

Schwartz, K., Syed, A.K., Stephenson, R.E., Rickard, A.H., Boles, B.R., 2012. Functional amyloids composed of
phenol soluble modulins stabilize Staphylococcus aureus biofilms. PLoS pathogens 8 (6), e1002744.
Sekar, R., Griebe, T., Flemming, H.-C., 2010. Influence of Image Acquisition Parameters on Quantitative
Measurements of Biofilms using Confocal Laser Scanning Microscopy. Biofouling 18 (1), 4756.
Shabala, L., McMeekin, T., Budde, B.B., Siegumfeldt, H., 2006. Listeria innocua and Lactobacillus delbrueckii
subsp. bulgaricus employ different strategies to cope with acid stress. International journal of food
microbiology 110 (1), 17.
Shrestha, D., Jenei, A., Nagy, P., Vereb, G., Szllsi, J., 2015. Understanding FRET as a research tool for
cellular studies. International journal of molecular sciences 16 (4), 67186756.
Stewart, P.S., Davison, W.M., Steenbergen, J.N., 2009. Daptomycin rapidly penetrates a Staphylococcus
epidermidis biofilm. Antimicrobial agents and chemotherapy 53 (8), 35053507.
Stocks, S.M., 2004. Mechanism and use of the commercially available viability stain, BacLight. Cytometry.
Part A : the journal of the International Society for Analytical Cytology 61 (2), 189195.
Stone, G., Wood, P., Dixon, L., Keyhan, M., Matin, A., 2002. Tetracycline Rapidly Reaches All the Constituent
Cells of Uropathogenic Escherichia coli Biofilms. Antimicrobial agents and chemotherapy 46 (8), 2458
2461.
Stoodley, P., Beer, D. de, Lewandowski, Z., 1994. Liquid flow in biofilm systems. Applied and environmental
microbiology 60 (8), 27112716.
Sweity, A., Ying, W., Ali-Shtayeh, M.S., Yang, F., Bick, A., Oron, G., Herzberg, M., 2011. Relation between EPS
adherence, viscoelastic properties, and MBR operation: Biofouling study with QCM-D. Water research
45 (19), 64306440.
Takenaka, S., Pitts, B., Trivedi, H.M., Stewart, P.S., 2009. Diffusion of macromolecules in model oral
biofilms. Applied and environmental microbiology 75 (6), 17501753.
Tang, L., Schramm, A., Neu, T.R., Revsbech, N.P., Meyer, R.L., 2013. Extracellular DNA in adhesion and
biofilm formation of four environmental isolates: a quantitative study. FEMS microbiology ecology 86
(3), 394403.
Van Ommen Kloeke, F., Geesey, G.G., 1999. Localization and Identification of Populations of PhosphataseActive Bacterial Cells Associated with Activated Sludge Flocs. Microbial Ecology 38 (3), 201214.
Vroom, J.M., Grauw, K.J. de, Gerritsen, H.C., Bradshaw, D.J., Marsh, P.D., Watson, G.K., Birmingham, J.J.,
Allison, C., 1999. Depth Penetration and Detection of pH Gradients in Biofilms by Two-Photon Excitation
Microscopy. Applied and environmental microbiology 65, 35023511.
Waharte, F., Steenkeste, K., Briandet, R., Fontaine-Aupart, M.-P., 2010. Diffusion measurements inside
biofilms by image-based fluorescence recovery after photobleaching (FRAP) analysis with a commercial
confocal laser scanning microscope. Applied and environmental microbiology 76 (17), 58605869.
Walther, E., Richter, M., Xu, Z., Kramer, C., Grafenstein, S. von, Kirchmair, J., Grienke, U., Rollinger, J.M.,
Liedl, K.R., Slevogt, H., Sauerbrei, A., Saluz, H.P., Pfister, W., Schmidtke, M., 2015. Antipneumococcal
activity of neuraminidase inhibiting artocarpin. International journal of medical microbiology : IJMM
305 (3), 289297.
Webster, P., Wu, S., Gomez, G., Apicella, M., Plaut, A.G., St Geme, Joseph W, 2006. Distribution of bacterial
proteins in biofilms formed by non-typeable Haemophilus influenzae. The journal of histochemistry and
cytochemistry : official journal of the Histochemistry Society 54 (7), 829842.
Whitchurch, C.B., Tolker-Nielsen, T., Ragas, P.C., Mattick, J.S., 2002. Extracellular DNA required for bacterial
biofilm formation. Science (New York, N.Y.) 295 (5559), 1487.

ACCEPTED MANUSCRIPT

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Xiao, J., Klein, M.I., Falsetta, M.L., Lu, B., Delahunty, C.M., Yates, J.R., Heydorn, A., Koo, H., 2012. The
exopolysaccharide matrix modulates the interaction between 3D architecture and virulence of a mixedspecies oral biofilm. PLoS pathogens 8 (4), e1002623.
Xiao, J., Koo, H., 2010. Structural organization and dynamics of exopolysaccharide matrix and microcolonies
formation by Streptococcus mutans in biofilms. Journal of applied microbiology 108 (6), 21032113.
Zeng, G., Vad, B.S., Dueholm, M.S., Christiansen, G., Nilsson, M., Tolker-Nielsen, T., Nielsen, P.H., Meyer,
R.L., Otzen, D.E., 2015. Functional bacterial amyloid increases Pseudomonas biofilm hydrophobicity and
stiffness. Frontiers in microbiology 6, 1099.
Zuriani, R., Vigneswari, S., Azizan, M. N. M., Majid, M. I. A., Amirul, A.A., 2013. A high throughput Nile red
fluorescence method for rapid quantification of intracellular bacterial polyhydroxyalkanoates.
Biotechnol Bioproc E 18 (3), 472478.

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Figure 2

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Table 1. Overview of methods for visualizing the components of the biofilm matrix by confocal microscopy

References
Rasconi et al. 2009
Reviewed by Neu et al. 2001

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Staining principle
Calcofluor (for -1,3 and -1,4
glucans)
Fluorescently labeled lectins (for
various di- or tri-saccharides)
GFP fusion protein with
carbohydrate-binding modules
from polysaccharidedegrading enzymes

Nguyen et al. 2014

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Target molecule
Polysaccharide
adhesin

Cell impermeant DNA-binding


fluorescent stains: TOTO-1,
TO-PRO 3, PicoGreen,
DDAO, propidium iodide and
SYTOX stains

Specificity of stains evaluated by


Okshevsky and Meyer 2014

Extracellular proteins

Fluorescent stains that bind to all


proteins: FilmTracerTM SyPro
Fluorescently labeled antibodies
with specificity for individual
proteins
Dye with specificity for amyloid
proteins

Lawrence et al. 2003, Frank et al.


2007
Berk et al. 2012

Thioflavin T staining
Fluorescently labeled antibodies
with specificity for amyloid
proteins

Ilanchelian and Ramaraj 2004


Larsen et al. 2007, Kim et al.
2016

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Extracellular amyloid

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Extracellular DNA

Kim et al. 2016

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Table 2. Overview of methods for quantitative analyses of the biofilm matrix by confocal microscopy

Immobilization of pH/O2sensitive fluorescent dyes


in particles
Fluorescence lifetime
imaging (FLIM) of pHsensitive fluorescent dyes
pH ratiometry

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References
Lawrence et al. 1998, Xiao et al.
2010

Hu et al. 2012

Hidalgo et al. 2009, Acosta et al.


2012

Time-lapse imaging
Single particle tracking (SPT)

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Measurements of diffusion
properties

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Measurements of solute
concentrations

Employed technique
Area/volume quantification
of fluorescently stained
matrix components using
digital image analysis
Colocalization analyses of
bacteria and matrix
components using digital
image analysis

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Geometric measurements

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Fluorescence recovery after


photobleaching (FRAP)
Fluorescence correlation
spectroscopy (FCS)

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Rheology measurements

Magnetic microparticle
actuation

Vroom et al. 1999

Xiao et al. 2012, Schlafer et al.


2015
Takenaka et al. 2009
Stoodley et al. 1994, Birjiniuk et
al. 2014
Waharte et al. 2010, Daddi
Oubekka et al. 2012
Briandet et al. 2008; Daddi
Oubekka et al. 2012
Galy et al. 2012

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Highlights

Confocal laser scanning microscopy is a valuable tool to study the biofilm matrix

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Various extracellular compounds can be labelled fluorescently and visualized in 3D


Concentrations of diffusing molecules in the matrix can be monitored in real-time

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Diffusion of solutes through the matrix can be quantified

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