Blackwell Science, LtdOxford, UKMMIMolecular Microbiology 1365-2958Blackwell Publishing Ltd, 20035016168Original ArticleBiofilm mushrooms with a twitchM. Klausen, A. Aaes-Jørgensen, S. Molin and T. Tolker-Nielsen
Molecular Microbiology (2003) 50(1), 61–68
doi:10.1046/j.1365-2958.2003.03677.x
Involvement of bacterial migration in the development of
complex multicellular structures in Pseudomonas
aeruginosa biofilms
Mikkel Klausen, Anders Aaes-Jørgensen, Søren Molin
and Tim Tolker-Nielsen*
Molecular Microbial Ecology Group, BioCentrum-DTU,
Technical University of Denmark, DK-2800 Lyngby,
Denmark.
Summary
Detailed knowledge of the developmental process
from single cells scattered on a surface to complex
multicellular biofilm structures is essential in order to
create strategies to control biofilm development. In
order to study bacterial migration patterns during
Pseudomonas aeruginosa biofilm development, we
have performed an investigation with time-lapse confocal laser scanning microscopy of biofilms formed
by various combinations of colour-coded P. aeruginosa wild type and motility mutants. We show that
mushroom-shaped multicellular structures in P.
aeruginosa biofilms can form in a sequential process
involving a non-motile bacterial subpopulation and a
migrating bacterial subpopulation. The non-motile
bacteria form the mushroom stalks by growth in certain foci of the biofilm. The migrating bacteria form
the mushroom caps by climbing the stalks and aggregating on the tops in a process which is driven by
type-IV pili. These results lead to a new model for
biofilm formation by P. aeruginosa.
Introduction
Investigations of living bacterial biofilms by the use of
advanced microscopy have shown that these sessile communities often consist of mushroom-shaped complex multicellular structures separated by water-filled spaces
(Lawrence et al., 1991; DeBeer et al., 1994). Because of
their innate resistance to host immune systems, antibiotics and other biocides, biofilms in medical and industrial
settings are difficult, if not impossible, to eradicate
(Costerton et al., 1987, 1999). Biofilm formation therefore
leads to various persistent and sometimes lethal infecAccepted 13 June, 2003. *For correspondence. E-mail
[email protected]; Tel. (+45) 45 25 27 93; Fax (+45) 45 88 73 28.
© 2003 Blackwell Publishing Ltd
tions in humans and animals and to a variety of problems
in industry where solid–water interfaces occur. Detailed
knowledge of the developmental process from single cells
scattered on a surface to complex multicellular biofilm
structures is essential in order to create strategies to
control biofilm development.
The formation of complex multicellular structures in biofilms has been proposed to be the result of growth under
nutrient-transfer limited conditions (Wimpenny and Colasanti, 1997; Picioreanu et al., 1998) or alternatively to be
the result of bacterial differentiation and expression of
genes that directly control the spatial organization of the
organisms (Stoodley et al., 2002). The involvement of cellto-cell signals in the development of mushroom-shaped
structures in Pseudomonas aeruginosa biofilms suggested that the development of complex multicellular biofilm structures has similarities to fruiting body formation
by the multicellular myxobacteria (Davies et al., 1998).
Myxococcus fruiting body formation occurs through
aggregation of bacteria via social gliding motility and
requires type-IV pili, a set of chemotaxis related genes
and cell-to-cell signalling molecules (Kaiser, 2001).
Although cellular migration plays a key role in the formation of complex multicellular structures in Myxococcus
(Kaiser, 2001), as well as in eukaryotic morphogenesis
and organogenesis (Firtel and Meili, 2000; Locascio and
Nieto, 2001; Moscoso, 2002), the role of bacterial migration in the formation of complex multicellular structures in
the later phases of biofilm development has not been
investigated.
We have previously shown that structural development
of P. aeruginosa biofilms is dependent on the carbon
source (Klausen et al., 2003). When citrate was used as
a carbon source P. aeruginosa PAO1 formed a flat and
very dynamic biofilm, whereas when glucose was used as
a carbon source P. aeruginosa PAO1 formed a heterogeneous biofilm containing mushroom-shaped multicellular
structures separated by water-filled channels. In the previous study (Klausen et al., 2003), we characterized the
developmental steps in the formation of the flat citrategrown P. aeruginosa biofilm using colour-coded bacteria
and confocal laser scanning microscopy (CLSM). It
appeared that the most important factor for the formation
of the flat biofilm was extensive twitching motility exhibited
62 M. Klausen, A. Aaes-Jørgensen, S. Molin and T. Tolker-Nielsen
by the cells past the initial phase of biofilm formation. Here
we present a detailed characterization of the developmental steps occurring in the formation of the mushroomshaped multicellular structures in P. aeruginosa biofilms.
Our analysis with time-lapse CLSM of biofilms formed by
various combinations of colour-coded P. aeruginosa wildtype and P. aeruginosa pilA mutants shows that the mushroom stalks are formed by proliferation of a non-motile
subpopulation, and that the mushroom caps are formed
by a motile subpopulation which use type-IV pili to climb
the stalks and aggregate on the tops.
Results
In order to investigate bacterial migration occurring during
development of the heterogeneous P. aeruginosa biofilm,
we inoculated a flow-chamber with a 1:1 mixture of Yfptagged and Cfp-tagged P. aeruginosa PAO1, irrigated the
flow-chamber with glucose minimal medium and followed
the structural development in the biofilm by time-lapse
CLSM. After 1 day of development the biofilm consisted
of microcolonies which contained either cyan fluorescent
or yellow fluorescent cells and the surface between the
microcolonies was covered with a layer of yellow and cyan
fluorescent cells in a mixture (data not shown). This indicated that the initial microcolonies in P. aeruginosa biofilm
form by clonal growth of non-motile bacteria and that the
bacteria move in the layer between the microcolonies.
After 4 days of development, the surface was only partially
covered with bacteria and the biofilm consisted of mushroom-shaped structures with either cyan or yellow fluorescent stalks, and with caps composed of various mixtures
of cyan and yellow fluorescent bacteria (Fig. 1). These
locations of the colour-coded bacteria suggested that the
mushroom stalks were formed by clonal growth of nonmotile bacteria and that the mushroom caps were formed
subsequently by aggregation of motile bacteria on the top
of the existing microcolony stalks.
Because P. aeruginosa is able to move on surfaces by
means of type-IV pili driven, so-called twitching, motility
(Mattick, 2002), we speculated that mushroom formation
in P. aeruginosa biofilms might occur in a sequential process of stalk formation by a subpopulation of non-motile
bacteria and cap formation by a subpopulation of bacteria
which use type-IV-pili to migrate towards, and climb up on,
the stalks. In order to investigate a role of type-IV pili in
the formation of mushroom structures in P. aeruginosa
biofilms we used a mutant, pilA, which is deficient in the
synthesis of type-IV pili. A flow-chamber was inoculated
with a 1:1 mixture of Yfp-tagged and Cfp-tagged P. aeruginosa pilA bacteria and the structural development was
followed in the biofilm irrigated with glucose minimal
medium. The P. aeruginosa pilA biofilm did not undergo a
stage with complete surface coverage, but consisted of
irregularly shaped microcolonies which were either yellow
or cyan fluorescent and grew bigger at fixed locations
without the formation of caps (Fig. 2). This supported the
proposed hypothesis, that the formation of regular mushrooms with caps in wild-type biofilms requires type-IV pili
driven motility and that dispersal of the cells on the surface
between the microcolonies also occur by twitching motility.
In order to study a role of differential type-IV pili driven
motility in biofilm mushroom formation in more detail, we
investigated the development in biofilms which were initiated with 1:1 mixtures of Yfp-tagged P. aeruginosa PAO1
wild-type bacteria and Cfp-tagged P. aeruginosa pilA
mutants. According to the hypothesis of mushroom formation by non-motile and motile subpopulations, the wild-
Fig. 1. Mushroom-shaped multicellular biofilm structures with mixed colour caps and either yellow or cyan stalks. Confocal laser scanning
microscopy (CLSM) images were acquired in a 4-day-old biofilm which was initiated with a 1:1 mixture of cyan fluorescent and yellow fluorescent
Pseudomonas aeruginosa PAO1 and grown on glucose minimal medium. The central picture in (A) shows a horizontal CLSM section, while the
two flanking pictures in (A) show vertical CLSM sections. The thin white lines in the central picture indicate the positions of the vertical sections,
while the thin white lines in the flanking pictures indicate the position of the horizontal section. (B) shows the corresponding 3-D CLSM image.
Bars, 20 mm.
© 2003 Blackwell Publishing Ltd, Molecular Microbiology, 50, 61–68
Biofilm mushrooms with a twitch 63
Fig. 2. Irregularly shaped P. aeruginosa pilA microcolonies of either yellow or cyan colour. CLSM images were acquired in a 4-day-old biofilm
which was initiated with a 1:1 mixture of cyan fluorescent and yellow fluorescent P. aeruginosa pilA mutants and grown on glucose minimal
medium. Bars, 20 mm. See legend to Fig. 1 for details on the image presentation.
type population in glucose-grown biofilms should split in
a subpopulation of motile bacteria exhibiting twitching
motility and another subpopulation of non-motile stalkforming bacteria, while the pilA bacteria would constitute
a second subpopulation of non-motile stalk-forming bacteria. With the used colour coding we would then expect
a wild-type/pilA mixed biofilm developing into mushroom
structures with yellow fluorescent caps and with stalks
which were either cyan fluorescent (the majority) or yellow
fluorescent. A CLSM micrograph of a 4-day-old biofilm
which was irrigated with glucose minimal medium and had
been initiated with a 1:1 mixture of yellow fluorescent
PAO1 wild type and cyan fluorescent pilA mutant is shown
in Fig. 3. In accordance with the hypothesis of mushroom
stalk formation by non-motile bacteria and mushroom cap
formation by twitching bacteria, all the mushroom caps
were formed by the yellow fluorescent wild-type bacteria,
while most of the mushroom stalks were formed by the
cyan fluorescent pilA bacteria and a minor fraction of the
stalks were formed by the yellow fluorescent wild-type
bacteria.
We have previously shown that when citrate is used as
the carbon source P. aeruginosa wild-type bacteria exhibit
extensive twitching motility and form a flat biofilm without
mushroom structures (Klausen et al., 2003). It was of
interest to investigate if the presence of a stalk-forming P.
aeruginosa pilA population would lead to mushroom formation in a citrate-grown wild-type/pilA mixed biofilm. If
so, the expected result should be even clearer than in the
case with the glucose-grown wild-type/pilA mixed biofilm,
as with citrate as the carbon source the motile population
should be all the wild-type bacteria, while the non-motile
population should be the pilA mutants only. In a citrategrown biofilm consisting of Yfp-tagged P. aeruginosa wild
type and Cfp-tagged P. aeruginosa pilA mutants all the
mushrooms should therefore have cyan fluorescent stalks
Fig. 3. Mushroom-shaped multicellular biofilm structures with yellow caps and cyan or yellow stalks. CLSM images were acquired in a 4-day-old
biofilm which was initiated with a 1:1 mixture of yellow fluorescent P. aeruginosa PAO1 wild type and cyan fluorescent P. aeruginosa pilA derivative
and grown on glucose minimal medium. Bars, 20 mm. See legend to Fig. 1 for details on the image presentation.
© 2003 Blackwell Publishing Ltd, Molecular Microbiology, 50, 61–68
64 M. Klausen, A. Aaes-Jørgensen, S. Molin and T. Tolker-Nielsen
Fig. 4. Yellow P. aeruginosa PAO1 wild type carpet-like structure and
blue P. aeruginosa pilA irregularly shaped microcolonies. The 3-D
CLSM image was acquired in a 1-day-old biofilm which was initiated
with a 1:1 mixture of yellow fluorescent wild-type bacteria and cyan
fluorescent pilA mutants and grown on citrate minimal medium. Bar,
20 mm.
and yellow fluorescent caps. To test the hypothesis, we
followed the structural development in a biofilm which was
irrigated with citrate minimal medium and had been initiated with a 1:1 mixture of yellow fluorescent PAO1 wild
type and cyan fluorescent pilA mutants. The yellow fluorescent PAO1 wild type initially formed a flat and expanding bacterial lawn, while the cyan fluorescent pilA mutants
formed microcolonies at fixed positions (Fig. 4). In the
later stages of biofilm development, mushroom-shaped
structures formed, with the cyan fluorescent pilA mutant
constituting the stalks and the yellow fluorescent PAO1
wild type constituting the caps (Fig. 5). The experiments
thus showed that formation of mushroom-shaped structures may occur in citrate-grown P. aeruginosa biofilms if
a stalk-forming subpopulation is present and suggested
that the difference in structure between the citrate-grown
and the glucose-grown P. aeruginosa wild-type biofilms is
due to different capabilities to form the mushroom stalks.
Vertical sections of a time-lapse CLSM image series
captured in a biofilm consisting of a 1:1 mixture of Yfptagged P. aeruginosa wild-type bacteria and Cfp-tagged
P. aeruginosa pilA mutants showed directly that the yellow
fluorescent wild-type cells climbed the existing cyan fluorescent pilA microcolonies (Fig. 6). The increase in biomass in the mushroom caps may, in addition to type-IV pili
driven aggregation of the yellow fluorescent wild-type bacteria, to some extent be attributed to growth. It should be
noted, however, that the cyan fluorescent pilA mutants did
not grow significantly in the time period.
In order to show that the results obtained with the P.
aeruginosa pilA mutant were not due to a secondary
mutation in our strain, we repeated the experiments with
the wild-type/pilA mixed biofilms using the strain P. aeruginosa PAKDpilA (Kagami et al., 1998) and got the same
results (data not shown).
Discussion
The present work leads to the model for development of
the heterogeneous P. aeruginosa biofilm which is shown
in Fig. 7. The model suggests that the formation of
mushroom-shaped structures in P. aeruginosa wild-type
biofilms occurs through stalk formation by proliferation of
bacteria which have downregulated twitching motility and
cap formation by bacteria which climb the microcolony
stalks by the use of type-IV pili and aggregate on the top.
According to the model, type-IV pili driven bacterial migration plays a key role in structure formation in the late
phase of biofilm development. A role for type-IV pili in the
initial phase of P. aeruginosa biofilm development has
been proposed in previous studies. O’Toole and Kolter
(1998) studied the initial phase of P. aeruginosa biofilm
Fig. 5. Mushroom-shaped multicellular biofilm structures with yellow caps and cyan stalks. CLSM images were acquired in a 4-day-old biofilm
which was initiated with a 1:1 mixture of yellow fluorescent P. aeruginosa PAO1 wild type and cyan fluorescent P. aeruginosa pilA derivative and
grown on citrate minimal medium. Bars, 20 mm. See legend to Fig. 1 for details on the image presentation.
© 2003 Blackwell Publishing Ltd, Molecular Microbiology, 50, 61–68
Biofilm mushrooms with a twitch 65
Fig. 6. Development of caps of yellow P. aeruginosa wild-type bacteria upon stalks of cyan P. aeruginosa pilA mutants. A biofilm was
initiated with a 1:1 mixture of yellow fluorescent P. aeruginosa PAO1
wild type and cyan fluorescent P. aeruginosa pilA mutants and grown
on citrate minimal medium. The structural development in the biofilm
was followed by CLSM time-lapse microscopy. The shown CLSM
vertical sections were acquired after 42 (A), 64 (B), and 86 h (C) of
biofilm development. The complete CLSM time-lapse movie (Film2)
is accessible at the internet site http://www.im.dtu.dk/mol-mic-avi.
Bar, 20 mm.
formation in a static system with glucose medium and
based on the observations (i) a pil mutant formed only a
monolayer of scattered cells whereas the wild-type formed
small microcolonies and (ii) the wild-type bacteria moved
on the surface so that small cell clusters formed and
dispersed, it was proposed that the initial microcolonies in
P. aeruginosa biofilms form by aggregation of bacteria
mediated by type-IV pili. Because fruiting body formation
by Myxobacteria also occurs via type-IV pili driven aggregation, a parallel between P. aeruginosa microcolony formation and Myxococcus fruiting body formation was
proposed in a review by O’Toole et al. (2000). Recently,
however, it was shown that P. aeruginosa type-IV pili
mutants are able to form protruding microcolonies in cit-
rate-grown biofilms (Heydorn et al., 2002; Klausen et al.,
2003) and in Tryptic Soy Broth-grown biofilms (Singh
et al., 2002) and evidence was presented that the initial
microcolonies in citrate-grown and Tryptic Soy Brothgrown P. aeruginosa wild-type biofilms do not form by cell
aggregation but by clonal growth (Singh et al., 2002;
Klausen et al., 2003). The present study with time-lapse
CLSM of biofilm formation by colour-coded bacteria suggests that the initial microcolonies (mushroom stalks) in
glucose-grown wild-type P. aeruginosa biofilms form by
clonal growth and that P. aeruginosa pilA mutants with
glucose as a carbon source are able to form biofilms
consisting of irregularly shaped protruding microcolonies
without mushroom caps.
Stalk formation may initiate in certain foci of a P. aeruginosa biofilm as a consequence of twitching motility suppression in response to local environmental cues. In
agreement with a requirement of downregulation of twitching motility for initial microcolony formation, it was recently
reported that a component of innate immunity inhibits the
formation of large microcolonial structures in P. aeruginosa biofilms by preventing downregulation of twitching
motility (Singh et al., 2002). However, it is also possible
that stalk formation initiates because the cells in certain
foci of the biofilms adhere strongly to each other creating
an intercellular matrix, so that twitching motility becomes
arrested. Because the ability to sense local population
sizes by means of cell-to-cell signalling evidently has a
role in the formation of P. aeruginosa biofilm mushroom
structures (Davies et al., 1998), the possibility exists that
the expression of cell-to-cell adherence factors is regulated by cell-to-cell communication. Because stalk formation is prevented by type-IV pili driven migration in P.
aeruginosa wild-type biofilms grown on citrate minimal
medium (Klausen et al., 2003), the process appears to be
dependent on nutritional conditions. In this connection it
is interesting that Vfr, the P. aeruginosa homologue of the
Escherichia coli catabolite repressor protein (Crp), is
involved both in the regulation of quorum sensing (Albus
et al., 1997) and in the regulation of twitching motility
(Beatson et al., 2002) and it has moreover been shown
Fig. 7. Formation of the P. aeruginosa biofilm with mushroom-shaped multicellular structures. After attachment, a subpopulation of non-motile
bacteria forms the initial microcolonies (mushroom stalks) by clonal growth, while another subpopulation of motile bacteria spread out on the
substratum in a process which depends on type-IV pili. In the next stage of biofilm formation the migrating bacteria climb up on the stalks using
type-IV pili in a coordinated process which leads to formation of the complete biofilm mushrooms. An example of the coordinated colony-climbing
can be seen in the CLSM time-lapse movie ‘Film1’ at the internet site http://www.im.dtu.dk/mol-mic-avi.
© 2003 Blackwell Publishing Ltd, Molecular Microbiology, 50, 61–68
66 M. Klausen, A. Aaes-Jørgensen, S. Molin and T. Tolker-Nielsen
that downregulation of twitching motility is prevented by
iron limitation (Singh et al., 2002).
Cap formation through climbing of P. aeruginosa bacteria on top of microcolonial stalks by means of type-IV pili
is consistent with the current belief that these pili mediate
cellular motility by an extension-grip-retraction mechanism (Skerker and Berg, 2001), although type-IV pili in P.
aeruginosa were until now only known to be involved in
bacterial translocation over solid surfaces (Mattick, 2002).
Because chemotaxis-related genes are involved in twitching motility by P. aeruginosa (Darzins, 1994), and local
consumption creates nutrient gradients in biofilms
(DeBeer et al., 1994; Wimpenny and Colasanti, 1997;
Picioreanu et al., 1998), it is possible that the bacteria
climb on top of the existing microcolonies because more
nutrients are available on the top. Twitching motility is not
regulated by any of the known cell-to-cell signalling systems in P. aeruginosa (Beatson et al., 2002), indicating
that migration of the cells on top of the stalks is not
coordinated by a known cell-to-cell communication system. Because a role of quorum sensing in formation of the
heterogeneous P. aeruginosa biofilm structures has been
shown previously (Davies et al., 1998), there may, however, be parallel quorum sensing controlled pathways
required for mushroom cap formation. It was recently
shown that rhamnolipid surfactant production affects P.
aeruginosa biofilm architecture and that a rhlA mutant did
not maintain open channels in biofilms (Davey et al.,
2003). Because rhamnolipid synthesis is quorum sensing
regulated (Pearson et al., 1997), and surfactants may
facilitate twitching motility, it is likely that quorum sensing
plays a role in mushroom cap formation through these
processes. Quorum sensing could also be involved in a
process that makes the twitching bacteria settle on the top
of the mushrooms. Because the presence of the stalkforming P. aeruginosa pilA bacteria also resulted in mushroom formation in a citrate-grown wild-type/pilA mixed
biofilm, where the wild-type bacteria exhibit extensive
twitching motility and unaccompanied form a flat biofilm,
it is likely that formation of some kind of cell-to-cell glue
causes the twitching bacteria to settle on top of the mushrooms. A role of quorum sensing in the formation of mushroom structures is consistent with our previous finding that
a P. aeruginosa wild type and lasI mutant formed similar
flat biofilms in citrate medium (Heydorn et al. 2002), as
citrate medium, unlike glucose medium, does not support
mushroom formation by the P. aeruginosa wild type in the
flow-chamber biofilms (Klausen et al., 2003).
Mushroom formation by P. aeruginosa during biofilm
growth has similarities to fruiting body formation by Myxococcus which occur during the sporulation process
through aggregation of bacteria via social gliding motility
(Kaiser, 2001). Twitching motility and social gliding motility
are essentially the same process (Semmler et al., 1999)
and it appears that Myxococcus and P. aeruginosa by and
large have recruited the same genes for the formation of
fruiting bodies and biofilm mushrooms. Both examples
demonstrate how development of complex morphogenetic
structures may be directed by regulated cellular motility
and cellular responses to the nutrient conditions. P. aeruginosa mushroom development in biofilms is evidently
directed by two independent activities involving a first
stage of stalk development influenced by the nutritional
conditions and a second stage of cap formation dependent on twitching motility and possibly influenced by nutrient gradients and quorum sensing. In case of the true
multicellular Myxococcus, the two activities seem to be
under joint control resulting in a coordinate developmental
programme for fruiting body formation. Multicellular fruiting body formation and sporulation confer several selective advantages to myxobacteria, one being that the
fruiting body package may adhere to a small animal and
be transported to a new place where nutrients are not
scarce (Kaiser, 2001). The formation in biofilms of mushroom structures separated by water-filled spaces has
been proposed to be a multicellular process, which creates a circulation system that ensures efficient nutrient
supply to most cells and efficient removal of waste products (Costerton et al., 1995). It is intriguing that also the
highly regulated events involved in eukaryotic organogenesis and morphogenesis are based on the interplay
between cell proliferation, cell migration and the generation of an intercellular matrix (Firtel and Meili, 2000;
Locascio and Nieto, 2001; Moscoso, 2002). The involvement of these factors in the development of complex multicellular structures in biofilms as in organogenesis and
morphogenesis in multicellular organisms, suggests that
they are common denominators to the formation of complex multicellular structures in nature.
Experimental procedures
Bacterial strains and growth conditions
The P. aeruginosa PAO1 (Holloway and Morgan, 1986) strain
from John Mattick’s laboratory and a DpilA derivative constructed by allelic displacement (Klausen et al., 2003) were
used in the study. The P. aeruginosa PAK derivative PAKDpilA
(Kagami et al., 1998) was used in control experiments. The
strains were fluorescently tagged at an intergenic neutral
chromosomal locus with cfp or yfp in mini-Tn7 constructs
(Klausen et al., 2003). Modified FAB medium (Heydorn et al.,
2000) was used supplemented with 30 mM glucose or 10 mM
citrate for batch overnight cultures and with 0.3 mM glucose
or 0.1 mM citrate for biofilm cultivation. Biofilms and batch
cultures were grown at 30∞C.
Cultivation of biofilms
Biofilms were grown in flow chambers with individual channel
© 2003 Blackwell Publishing Ltd, Molecular Microbiology, 50, 61–68
Biofilm mushrooms with a twitch 67
dimensions of 1 ¥ 4 ¥ 40 mm. The flow system was assembled and prepared as described previously (Møller et al.,
1998). The flow-chambers were inoculated by injecting 350 ml
overnight culture diluted to an OD600 of 0.001 into each flowchannel with a small syringe. After inoculation flow-channels
were left without flow for 1 h, after which medium flow
(0.2 mm s-1) was started using a Watson Marlow 205S peristaltic pump.
Microscopy and image acquisition
All microscopic observations and image acquisitions were
done with a Zeiss LSM 510 CLSM (Carl Zeiss, Jena, Germany) equipped with detectors and filter sets for monitoring
of Cfp and Yfp fluorescence. Images were obtained using a
63¥/1.4 objective or a 40¥/1.3 objective. Simulated 3-D
images and sections were generated using the IMARIS software package (Bitplane AG).
Acknowledgements
This work was supported by the Danish Biotechnological
Research Program.
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