combination of microscopic techniques reveals a comprehensive visual impression of biofilm structure...

R E S EA RCH AR T I C L E

Combination of microscopic techniques reveals acomprehensive visual impression of biofilm structure and

composition

Morten Alhede1, Klaus Qvortrup2, Ramon Liebrechts2, Niels Høiby1,3, Michael Givskov1,4 &Thomas Bjarnsholt1,3

1Institute for International Health, Immunology and Microbiology, University of Copenhagen, Copenhagen, Denmark; 2Department of Biomedical

Sciences, University of Copenhagen, Copenhagen, Denmark; 3Department of Clinical Microbiology, Rigshospitalet, University Hospital of

Copenhagen, Copenhagen, Denmark; and 4Singapore Centre on Environmental Life Sciences Engineering, Nanyang Technological University,

Singapore, Singapore

Correspondence: Morten Alhede, Institute

for International Health, Immunology and

Microbiology, University of Copenhagen,

2200 Copenhagen, Denmark.

Tel.: +45 3532 6657; fax: +45 3532 7853;

e-mail: [email protected]

Received 25 October 2011; revised 11

January 2012; accepted 14 March 2012.

DOI: 10.1111/j.1574-695X.2012.00956.x

Editor: Gianfranco Donelli

Keywords

biofilm; microscopy; scanning electron

microscopy; ESEM; CRYO-SEM; FIB-SEM.

Abstract

Bacterial biofilms are imaged by various kinds of microscopy including confo-

cal laser scanning microscopy (CLSM) and scanning electron microscopy

(SEM). One limitation of CLSM is its restricted magnification, which is

resolved by the use of SEM that provides high-magnification spatial images of

how the single bacteria are located and interact within the biofilm. However,

conventional SEM is limited by the requirement of dehydration of the samples

during preparation. As biofilms consist mainly of water, the specimen dehydra-

tion might alter its morphology. High magnification yet authentic images are

important to understand the physiology of biofilms. We compared conven-

tional SEM, Focused Ion Beam (FIB)-SEM and CLSM with SEM techniques

[cryo-SEM and environmental-SEM (ESEM)] that do not require dehydration.

In the case of cryo-SEM, the biofilm is not dehydrated but kept frozen to

obtain high-magnification images closer to the native state of the sample.

Using the ESEM technique, no preparation is needed. Applying these methods

to biofilms of Pseudomonas aeruginosa showed us that the dehydration of bio-

films substantially influences its appearance and that a more authentic biofilm

image emerges when combining all methods.

Introduction

Bacteria are found in at least two distinct states – either as

planktonic or sessile cells. Planktonic cells are classically

defined ‘as free flowing bacteria in suspension’ as opposed to

the sessile biofilm state: ‘a structured community of bacterial

cells enclosed in a self-produced polymeric matrix and adher-

ent to an inert or living surface’ (Costerton et al., 1999) but

may also be suspended in host material as seen in many

chronic infections (Burmølle et al., 2010). Microbiologists

have up until the last few decades focused and emphasized

the planktonic state over the biofilm state. However, the

importance of the biofilm mode of growth is becoming

increasingly recognized as improved methods to study

sessile bacteria have become available, and hence the subse-

quent accumulation of evidence for its widespread pres-

ence. It has been suggested that bacteria are predominantly

growing as sessile communities rather than as single cells

(Costerton et al., 1987; Davey & O’Toole, 2000).

Sessile growing bacteria are defined as an assemblage of

cells embedded ‘in a self-produced polymeric matrix’. This

matrix is very important for the properties of the biofilm,

because it offers structural stability and increased tolerance

to antimicrobials and immune cells (Stoodley et al., 2002;

Anderson & O’Toole, 2008; Mulcahy et al., 2008; Ma et al.,

2009). To gain further information on this phenomenon,

one has to investigate how a biofilm is established and propa-

gated. The most common method is the continuous-culture

once-through flow system using the model organism

Pseudomonas aeruginosa. In this system, media are slowly

passed over the biofilm-growing bacteria, which have

attached to a cover slip on a flow cell. This in vitro process of

FEMS Immunol Med Microbiol && (2012) 1–8 ª 2012 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

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P. aeruginosa biofilm formation can be divided into at least

five stages: in the first stage, planktonic cells reversibly attach

to a vacant surface. Irreversible binding follows this attach-

ment and then multiplication into microcolonies. The

microcolonies produce an extracellular polymeric matrix,

which in turn envelopes the colonies. After a couple of days,

the microcolonies attain tower- or mushroom-like struc-

tures measuring up to 50 lm in the flow cell (Costerton

et al., 1995; Davey & O’Toole, 2000; Stoodley et al., 2002).

The extracellular matrix contains a mixture of polysac-

charides, proteins, and DNA (Wingender et al., 2001;

Whitchurch et al., 2002; Costerton et al., 2003). When the

biofilm grows to a size not beneficial for bacterial survival

and growth (e.g., owing to nutrient limitations), focal areas

of the biofilm are sloughed off. It is hypothesized this

enables the otherwise sessile biofilm bacteria to spread and

colonize new surfaces and biofilms to spread. Hence, it

seems that the biofilm lifecycle by P. aeruginosa is a dynamic

process capable of renewing itself (Costerton et al., 1995;

Davey & O’Toole, 2000; Stoodley et al., 2002).

The biofilm lifecycle and the matrix components have

preferably been investigated by means of confocal laser scan-

ning microscopy (CLSM). This method has provided valu-

able insight into the biofilm development; however, the

information on the detailed ultrastructure of the biofilm is

difficult to image by light microscopes. Electron microscopy

(EM) has the needed resolution and hence magnification to

offer a more detailed insight into the ultrastructure of the

biofilm as well as its environment. Several EM techniques

have been used to investigate biofilms, with scanning elec-

tron microscopy (SEM) as the predominant choice (Sutton

et al., 1994; Priester et al., 2007; Sangetha et al., 2009). Con-

ventional SEM methods are far from optimal for investiga-

tion of water-containing specimens such as biofilms,

because the technique requires dehydration of the sample.

In most cases, the choice of microscope is based on avail-

ability and not the suitability. We here present a micro-

graph survey of P. aeruginosa biofilm development with

four different SEM techniques: standard SEM, cryo-SEM

and environmental-SEM as well as focused ion beam (FIB)-

SEM.

Materials and methods

Growth of bacteria

All bacteria were grown in ABtrace minimal medium con-

taining 0.3 mM glucose for continuous cultures and 0.5%

glucose for batch cultures, as previously described (Bjarns-

holt et al., 2005). Planktonic cultures were grown in shake

flasks at 37 °C. Continuous biofilms were cultivated in

once-through flow chambers, perfused with sterile media, as

previously described (Bjarnsholt et al., 2005).

Conventional SEM

The biofilms were imaged by SEM as previously described

(Qvortrup et al., 1995). Briefly, bacteria were harvested

and fixed in 2% glutaraldehyde, postfixed in 1% OsO4,

critical point–dried using CO2 and sputter-coated with

gold according to standard procedures. Specimens for

SEM were investigated with a Philips XL Feg30 SEM

operated at 2–5 kV accelerating tension.

Cryo-SEM

Glass-pieces from the flow cell were broken and plunge-

frozen in slushed liquid nitrogen at �210 °C and

transferred in a special transfer container, which is under

continuous vacuum to the cryo-preparation chamber

attached to the Quanta 3D FEG (FEI). The sample temper-

ature was raised to �95 °C for approximately 3 min to

sublime any condensed ice from the surface gained during

transfer. The temperature of the sample was then reduced

to �125 °C. Essentially, to avoid charging problems while

searching for a suitable site, the sample was sputter-coated

with platinum for 160 s, giving a thickness of approxi-

mately 15 nm. The sample was then passed through the

transfer lock to the FIB-SEM cryo-stage, which was main-

tained at �125 °C. Imaging was performed using an accel-

erating voltage of 3–10 kV.

Environmental-SEM

Biofilm containing glass-pieces from the flow cell were

broken of and were mounted onto double-sided carbon

tape on a small, circular metal stub, and samples were

imaged with a Quanta 3D FEG SEM (FEI) operated in

ESEM mode. The biofilm samples were viewed with a

gaseous secondary electron detector in a humidified

environment. The system was operated under high accel-

erating voltages (5–15.0 kV), and the low chamber pres-

sures were gained with a special ESEM final lens insert,

so a maximum pressure of 2700 Pa could be obtained.

FIB–SEM

The biofilms were fixed with 2% glutaraldehyde in 0.05

phosphate buffer (pH = 7.2) and postfixed in 1% osmium

tetroxide with 1.5% potassium ferrocyanide (Knott

et al., 2008) and embedded in Epon according to stan-

dard protocols (Hayat, 2000). Specimens were sputter-

coated with gold and imaged with a Quanta 3D FEG

(FEI). Features within the FIB–SEM dataset were seg-

mented using Amira (Visage Imaging Inc.), and 3D

images were created.

ª 2012 Federation of European Microbiological Societies FEMS Immunol Med Microbiol && (2012) 1–8Published by Blackwell Publishing Ltd. All rights reserved

2 M. Alhede et al.

Results

To compare the different microscope techniques, we inves-

tigated the biofilm development (day 1 trough 4) of

P. aeruginosa PAO1 in once-through flow chambers, perfused

with media as described previously (Bjarnsholt et al., 2005).

Conventional SEM

SEMs are used to examine topographies of materials with

magnifications that range from that of optical microscopy

to the nanoscale. SEM scans the surface of the specimen

with a finely focused electron beam to produce an image.

SEM micrographs have a large depth of field yielding a

three-dimensional appearance, which is useful for under-

standing the surface structure of the sample. Accordingly,

SEM is a good option to visualize the bacteria residing in

the biofilms.

As shown in Fig. 1, it is possible to obtain high-resolu-

tion images of P. aeruginosa aggregating on the glass

substratum of a flow cell. As with CLSM, it is possible to

see the spatial distribution of bacteria including the

so-called mushrooms (for comparison se Fig. 2). It seems

that the bacteria are uncovered but interconnected by

fiber-like structures. Most biofilm literature agrees that an

alginate- and water-containing matrix, which protects the

bacteria against adverse conditions, surrounds the bacteria.

We were not able to show or find any evidence of a gel-like

matrix covering the bacteria using conventional SEM. This

is not surprising because an important step in conventional

SEM preparation is dehydration. It is hard to evaluate

whether the biofilm structures, including the fibers, that

are visualized with this method are influenced by the prep-

aration. We speculate that these structures are condensed

matrix components or are actual polymers found under-

neath the water-containing matrix.

Cryo-SEM

When investigating a biological structure in the electron

microscope, the problem of artifact formation because

of specimen preparation always needs to be considered

and analyzed carefully. It is generally considered that

vitrification by ultra fast freezing, for example high-

pressure freezing, is the gold standard for nonsolid

specimen fixation (Walther & Ziegler, 2002; Hohenberg

et al., 2003; Walther, 2003a). The clear advantage of

cryo-SEM is the lack of preoperational steps including

dehydration and the investigation of time-based speci-

mens ‘frozen in time’. The total preparation occurs

within a minute of time, which is significantly less than

with conventional SEM that takes days.

The sample in the current study was fixed by plung-

ing it into sub-cooled nitrogen (nitrogen slush) close to

Fig. 1. Conventional SEM examination of Pseudomonas aeruginosa biofilm development day 1–4.


Comparing SEM techniques in biofilm imaging 3

the freezing point of nitrogen at �210 °C. The sample

is then transferred in vacuo to the temperature con-

trolled cold stage of the SEM cryo-preparation chamber

where the sample is sputter-coated with platinum,

followed by sample transfer into the SEM chamber.

The sample remains frozen during imaging on the cold

stage, which is cooled by liquid nitrogen.

Even though the frozen state stabilizes the soft material

and liquids of the biofilm (which would otherwise be

impossible to examine at high magnification, because of

Fig. 2. Comparison of biofilm imaging by conventional CLSM and SEM.

Fig. 3. Cryo-SEM examination of Pseudomonas aeruginosa biofilm development day 1–4.


4 M. Alhede et al.

sample movement or beam damage), the cryo-SEM images

(Fig. 3) appear to be of a lower resolution compared to

conventional SEM. This is partly attributable to a lower

conductivity of the frozen surface compared to the dehydrated

gold-sputtered surface we employ in conventional SEM.

Another downside of cryo-SEM is that the frozen sur-

face melts and cracks at high magnifications because of

the heat generated by the focused electron beam. How-

ever, we were able to produce images of the biofilm that

clearly show that the bacteria are enveloped in a gel-like

matrix. We were not able to obtain high-magnification

images showing details of the matrix. Cryo-SEM also

allows for freeze-fracture, which exposes the internal

structure of the biofilm and may thus reveal how the bac-

teria are interconnected.

Environmental-SEM

The ESEM was developed in the late 1980s (Danilatos,

1988). The ESEM retains many of the advantages of a

conventional SEM, without the high vacuum requirement

by varying the sample environment through a range of

pressures, temperatures, and gas compositions. Wet and

nonconductive samples may be examined in their natural

state without modification or preparation. The ESEM

offers high-resolution secondary electron imaging in a

gaseous environment.

The obvious advantage of ESEM is the total lack of

preparation. The sample is placed directly on a stub and

placed in the SEM chamber. However, with ESEM, we

had problems obtaining high-resolution images of the

biofilm because of the lack of conductivity in the wet

sample. We experienced that close to magnifications of

10 0009 and more, where the beam current is locally

very high, the focused electron beam seemed to destroy

the 3D biofilm structure. We also observed that during

prepumping, the sample also slightly dehydrates, but not

to near the same extent as the dehydration used in con-

ventional SEM (Fig. 4).

FIB–SEM

A superior, yet more sophisticated alternative to the con-

ventional SEM and CLSM is the FIB–SEM. Similar to con-

Fig. 4. ESEM examination of Pseudomonas aeruginosa biofilm development day 1–4.



focal scanning microscopy, it is possible with FIB–SEM to

create 3D reconstructions. With a process termed ‘slice and

view’, the FIB can sequentially mill away down to 10-nm-

thick sections from the surface of a resin embedded speci-

men and subsequently record a SEM image (Fig. 5a) of the

exposed block surface using a back scattered electron

(a) (b)

(c) (d)

Fig. 5. FIB–SEM reconstruction of a 3-day-old Pseudomonas aeruginosa biofilm. The reconstruction is based on successive images similar to (a).

(b, c) 3D reconstruction of the biofilm. (d) Matrix components marked red by manual labor.

Fig. 6. Comparison of biofilm details obtained by conventional SEM, cryo-SEM, and ESEM.


6 M. Alhede et al.

detector (BSED). Following acquisition of the successive

image slices, the image data are processed to perform a 3D

volume reconstruction (Fig. 5b and c).

We were able to produce stunning 3D reconstructions

of the spatial interaction of bacteria down through the 3-

day-old biofilm (Supporting information, Movie S1). At

present, we have not been able to automate the 3D

reconstruction of matrix components. Instead, we have

to manually mark matrix components on each successive

image. Thus, we are able to reconstruct the interconnect-

ing fibers also seen in conventional SEM, but as it relies

on manual labor, it is not very precise (Fig. 5d).

We find this tool very useful for ex vivo imaging of

infected tissue. Further improvements in heavy metal

contrasting of the specimens could potentially yield better

BSED imaging of the matrix.

Discussion

We have tested four different techniques of SEM on

P. aeruginosa biofilms (Fig. 6). Each method has obvious

drawbacks but also distinct strengths, making it difficult

to determine which method is the most suitable for bio-

film visualization. The conventional SEM together with

FIB–SEM provides good information on spatial structure;

however, Fig. 5 shows that the dehydration preparative

step leaves the bacteria exposed. Therefore, the technique

is not suitable visualizing substances in the biofilm matrix.

Here, the Cryo-SEM and environmental-SEM techniques

are more suited, because they appear to leave the matrix

unaffected (Fig. 5). However, the problem with these tech-

niques is the poor resolution and hence limited magnifica-

tion when compared to conventional SEM. Obviously, no

single method for visualization exists at present time for

visualizing the true architecture of the biofilm matrix.

Therefore, it is important to first ask the scientific ques-

tions and subsequently chose the most appropriate

method. In this study, no single method revealed the true

nature of the biofilm, but if combined, the image data

from the different methods are better able to predict the

true architecture of the matrix. Probably, not many

research centers will have all the above methods in hand,

but caution should be taken when drawing conclusions

based on only one method. Figure 7 outlines the advanta-

geous contribution from each method to a more realistic

biofilm structure.

Acknowledgements

The authors would like to thank Grazyna Hahn Poulsen,

for the artistic presentation of the biofilm model, and the

Villum Foundation and Novo Nordic Foundation for

support to MG.

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Fig. 7. Contributions of different microscopes to biofilm

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environment. Last FIB–SEM is used for creating high-resolution 3D

images of the biofilm.



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Supporting Information

Additional Supporting Information may be found in the

online version of this article:

Movie S1. Illustration of 3D FIB-SEM reconstruction of a

3-day-old P. aeruginosa biofilm. Image dataset captured

with a Quanta 3D DualBeam FEG and subsequently edi-

ted in Amira(R). A threshold was set to mask the bacteria

(green) and DNA (red) was masked manually.

Please note: Wiley-Blackwell is not responsible for the

content or functionality of any supporting materials sup-

plied by the authors. Any queries (other than missing

material) should be directed to the corresponding author

for the article.


8 M. Alhede et al.

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