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MICROSCOPY RESEARCH AND TECHNIQUE 46:319–324 (1999)
Imaging Faces of Shadowed Magnetite (Fe3O4) Crystals From
Magnetotactic Bacteria With Energy-Filtering Transmission
Electron Microscopy
ULYSSES LINS,1 BECHARA KACHAR,2 AND MARCOS FARINA3*
1Setor
de Microscopia Eletrônica e Departamento de Microbiologia Geral, Instituto de Microbiologia Professor Paulo de
Góes,Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil
2Laboratory of Cellular Biology, National Institute on Deafness and Other Communication Disorders, National Institutes of Health,
Bethesda, Maryland 20892
3Departamento de Anatomia, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, CCS, Bl. F,
21941–590 Rio de Janeiro, Brasil
KEY WORDS
magnetotactic bacteria; bacterial magnetite; energy filtering transmission electron microscopy; biomineralization
ABSTRACT
We used energy-filtering transmission electron microscopy to image magnetite
crystals isolated from uncultured magnetotactic bacteria. These magnetite crystals were shadowed
in high vacuum with platinum at 45°. The shadowed crystals were observed in a Zeiss (Thornwood,
NY) CEM902 transmission electron microscope. Imaging shadowed crystals with inelastically
scattered electrons provided information of the decoration pattern of small platinum particles over
crystal surfaces, and thus information on surface characteristics of crystals. Results were comparable to those obtained from scanning electron microscopy using a field emitter gun. Electron energy
loss spectra of the crystals as well as of the supporting film were recorded to evaluate variations of
image contrast with energy losses. Results indicated that the contrast is attenuated with inelastic
imaging and that the effect of contrast tuning caused a contrast inversion at a given point between
100 and 150 eV. We believe this approach can be useful for studying multilayered materials by
transmission electron microscopy. Microsc. Res. Tech. 46:319–324, 1999. r 1999 Wiley-Liss, Inc.
INTRODUCTION
Magnetotactic bacteria navigate along magnetic field
lines because of the presence of magnetosomes (Lins de
Barros et al., 1990). Magnetosomes contain a magnetic
crystal enveloped by a membrane (Gorby et al., 1988),
and usually form chains. Magnetite (Fe3O4) or iron
sulfides form the magnetic crystals of magnetosomes
(Stolz, 1993; Pósfai et al., 1998), which are usually 50 to
100 nm in length. The membrane that envelops the
crystals may influence this size restriction and morphology. Because of the presence of a magnetosome membrane, it is thought that bacteria control the deposition
of crystals within magnetosomes and, as a consequence,
singular morphologies, usually different from inorganic
magnetite, are formed. It has been considered that
magnetosomes are species-specific, which reflects a
genetic control on their precipitation (Vali and Kirschvink, 1990). Thus, the study of the morphology and
structure of magnetosomes may contribute to understanding the process involved in magnetosome formation.
High-resolution transmission electron microscopy
(Matsuda et al., 1983; Mann et al., 1990), electron
diffraction, X-ray microanalysis, and recently scanned
probe microscopy (Farina et al., 1994; Proksch et al.,
1995) have been applied in the study of bacterial
magnetite. With these approaches, several morphologies of bacterial magnetite crystals were reported:
octahedral with truncated faces, pseudo-hexagonal prismatic, and bullet-shaped (Matsuda et al., 1983; Thornr 1999 WILEY-LISS, INC.
hill et al., 1994). In samples of uncultured magnetotactic bacteria, it is desirable to directly visualize the faces
of the magnetic crystals or to distinguish crystals of
different morphologies when mixed. In this work, we
used an energy-filtering transmission electron microscope to image bacterial magnetite crystal faces with
low-loss inelastic electrons. The objective was to directly observe faces of magnetite crystals from bacterial
origin. The results obtained using this approach are
comparable to those obtained by high-resolution scanning electron microscopy (this work) and atomic force
microscopy (Farina et al., 1994).
MATERIALS AND METHODS
Sampling and Magnetic Enrichment of Bacteria
Sediments were collected in Itaipu lagoon (43° 04’
W ⫻ 22° 57’ S), near Rio de Janeiro city, placed in
bottles, and stored under dim light for several weeks.
Periodically, a drop with sediment was exposed to a
properly aligned magnetic field and observed under a
light microscope. If the presence of a relatively large
number of magnetotactic bacteria was confirmed, the
bottle was considered suitable for enrichment. In this
Contract grant sponsor: PRONEX; Contract grant sponsor: FINEP; Contract
grant sponsor: TWAS; Contract grant sponsor: NIDCD-NIH.
Correspondence to: Dr. Marcos Farina, Laboratório de Biomineralização,
Departamento de Anatomia, Instituto de Ciências Biomédicas, Universidade
Federal do Rio de Janeiro, CCS, Bl. F, 21941–590, Rio de Janeiro, RJ Brasil.
E-mail: mfarina@anato.ufrj.br
Received 4 December 1998; accepted in revised form 6 April 1999
320
U. LINS ET AL.
Fig. 1. a: Isolated magnetite crystals deposited over a formvar-coated grid observed by TEM in bright
field mode. The crystals appear dark in most of the cases. b: Image obtained by inelastically scattered
electrons (⌬E ⫽ 50 eV). Crystals present different optical densities though faces are not well defined.
Bar ⫽ 400 nm.
case, a specially designed glass chamber was filled with
sediment and water in a proportion of approximately
1:3, and exposed to the magnetic field for 15 minutes.
Afterwards, drops with enriched bacteria were withdrawn with a capillary tube and placed in an eppendorf
tube for isolation.
Isolation of Magnetic Crystals
Triton X-100 or SDS detergents were added to an
Eppendorf tube containing enriched magnetotactic bacteria and agitated vigorously using a Pasteur pipette.
The tube was then washed several times with distilled
water to remove the detergent. Afterwards, the crystals
were treated with a solution of 40% NaOH in water at
60°C for 1 hour to remove the organic matter, particularly nucleic acids. To avoid losing crystals during the
isolation procedures, a strong magnet was placed at the
lower end of the Eppendorf tube during the whole
procedure and after each step the tube was left still for
30 minutes. The isolated crystals were then washed
extensively with distilled water and spread over formvar-coated copper grids. Some samples were treated
with NaOCl in place of the detergent, however, all other
procedures are identical.
Scanning Electron Microscopy
For scanning electron microscopy observation, isolated crystals were deposited on silicon chips specimen
supports (Pelco International, Redding, CA, USA) and
air-dried. Samples were mounted on aluminum stubs
and observed uncoated at 5 kV in a Hitachi S-4500
scanning electron microscope equipped with a cold
cathode field emission gun.
Shadowing of Isolated Crystals and Observation
Grids with isolated crystals were shadowed unidirectionally with platinum at 45° in a Balzers freezefracture apparatus and observed in a Zeiss CEM902
transmission electron microscope operating at an accelerating voltage of 80 kV. This microscope has a spectrometer that permits the selection and imaging of inelastically scattered electrons of defined energy losses
(Reimer, 1991). The diameter of the objective aperture
was 60 µm (collection angle of approximately12 mrad)
and the energy-selecting slit aperture was approximately 20 eV. All negatives from shadowed crystals
were photographically reversed before printing. This is
a common procedure in quick-freeze, deep-etch preparation of biological samples for 3D electron microscopy
(Heuser, 1989). For energy loss spectroscopy, the microscope was operated at 80 kV and an energy-selecting
slit aperture of approximately 2 eV was used. Energy
loss intensities from 0 to 200 eV were registered at 2-eV
intervals by a photomultiplier attached to the microscope and connected to a digital multimeter that loaded
the output voltage into the computer memory of a
Kontron Zeiss image analysis system.
RESULTS
There was a mixture of morphologically different
crystals in all samples observed. Most of the crystals
presented the already described pseudo-hexagonal morphology (Farina et al., 1994), showing long faces 51016
and small oblique faces of the type 51006 and 51106 and
51116 end faces. These crystals were probably isolated
from some of the two most abundant morphotypes
present in the samples (Spring et al., 1998). Observation of isolated crystals was carried out in different
imaging modes of TEM. Crystals without platinum
shadowing were imaged with zero-loss electrons (Fig.
1a) and inelastic imaging mode (Fig. 1b). Little surface
information could be obtained from these images no
matter which diffraction and photographic exposure
conditions were employed during imaging of the crystals. Magnetosomes shadowed with platinum were
imaged in both in zero-loss mode (Fig. 2a; see also Fig.
IMAGING FACES OF Fe3O4 CRYSTALS
321
Fig. 2. a: Zero loss filtered transmission electron microscopy image
of shadowed magnetosomes. Very little surface information about
the crystals can be assessed particularly in the region of large crystals. b: Inelastically filtered image (⌬E ⫽ 77 eV) of the same shadowed
crystals. Due to contrast tuning, the surface of the crystals can
now be observed. Arrow indicates a 51016 crystal face. Bar ⫽ 120 nm.
Fig. 3. Inelastically filtered image of shadowed crystal. The effect
of the decoration pattern of the platinum microcrystals can be
observed and the faces of crystals in different orientations can be
observed. ⌬E ⫽ 68 eV; Bar ⫽ 100 nm. Inset: Idealized faces for some of
the shadowed crystals. (䊏) Faces of the type 51006 or 51106; (ⴱ) Faces of
the type 51016.
4a) and with inelastically scattered electrons (Figs. 2b,
3, and 4b). In zero-loss, the contrast between magnetosome and support film was very high. However, because
of the superposition of the platinum layer on the whole
magnetite crystal, very little surface information could
be obtained from images formed primarily by the
322
U. LINS ET AL.
Fig. 4. Shadowed isolated octahedral crystals. a: Zero loss filtered image. Note that no information on
crystal faces can be observed. b: Inelastically filtered image of the same area in the previous figure (⌬E ⫽
104 eV). Now the characteristic 51116 crystal faces can easily be evaluated. Bar ⫽ 90 nm.
Fig. 5. Electron energy loss spectra of the supporting film (I) and of a shadowed crystal (II). The
intensities of the spectra obtained from the supporting film alone and over the crystal are closer in the
inelastic part of the spectra. This means that the contrast is much reduced in the inelastic images. At a
certain point (⬇110 eV) there is an inversion of the intensities.
scattering contrast of the elastically scattered electrons, especially in the case of the larger magnetosomes
(Fig. 2a). A much better imaging condition was obtained
when shadowed crystals were imaged with inelastically
scattered electrons (Figs. 2b, 3, and 4b). There was a
contrast attenuation in the images, associated to the
shadowing of the platinum layer covering the crystals,
and the surface information could be retrieved from the
crystals. It was possible to visualize the decoration
pattern of the platinum crystals on the surface of the
underlying magnetite crystals. Faces of the crystals
could be directly observed and the relative disposition
of one face to another could be established in some
cases. In the inset of Figure 3, elongated faces 51016, as
well as smaller faces of the crystals of the type 51006 or
51106, can be seen. Besides pseudo-hexagonal prismatic
crystals, we also observed a few octahedral crystals
presenting the representative well-defined 51116 faces
(Fig. 4a and b).
Electron energy loss spectra from regions of the
crystal and the supporting film were obtained (Fig. 5).
In these spectra, it can be noted that the differences in
intensities between the crystal and the supporting film
were much closer in the inelastic part of the spectra
(note the vertical log scale which means that the ratio
between the elastically scattered electron signal of both
curves is very high). The effect of contrast tuning
observed for energy losses caused an inversion of the
contrast in the images in a given point between 100 and
150 eV (Fig. 5).
Scanning electron microscopy of similar preparations
imaged using a field emission microscope produced
IMAGING FACES OF Fe3O4 CRYSTALS
323
Fig. 6. Scanning electron microscopy image of isolated magnetite crystals from magnetotactic
bacteria. Crystals are disposed in chains and arranged in different orientations. Several faces can be
directly visualized. Bar ⫽ 150 nm.
results of comparable image quality (Fig. 6). Magnetic
crystals appeared in linear chains and were disposed in
different orientations within a chain. The faces could be
directly visualized.
DISCUSSION
Magnetosomes have been studied with a structural
approach particularly in aspects of biomineralization
and crystallography (Stolz, 1993; Bazylinski, 1995).
With atomic force microscopy (Farina et al., 1994) and
the methodology described here, it is possible to obtain
data from different faces of previously known crystals,
and to discriminate different morphologies when a
mixed population of crystals is studied. We used an ‘‘in
column’’ magnetic spectrometer to directly observe surfaces of magnetic crystals decorated with metal aggregates of platinum. The replication at 45° produced a
decoration pattern of platinum particles of probably 2.5
to 3.0 nm. The use of vertical deposition of a thin (1 nm)
platinum-carbon metal layer (Aksiyote-Benbasat et al.,
1990) would have produced smaller platinum particles
with a better resolution at normal (global) imaging
conditions. In these normal imaging conditions, quantification of particles sizes is possible by calibrating
shadow widths with gold ball standards (Ruben, 1995).
However, when using inelastic electrons to image crystals in an energy-filtering electron microscope, the
resolution is limited not only by the size of the platinum
crystals but also by the delocalized nature of the
inelastic collisions (Reimer, 1991). This could reduce
the possibility of improvement of final image resolution
in inelastic mode even with a thinner metal layer of
smaller platinum particles. This improvement will be
checked in the future. Indexation of planes is not
possible with contrast tuning of metal-shadowed crystals, which is different from the results obtained by
high-resolution transmission electron microscopy and
electron diffraction. On the other hand, our approach is
less time-consuming, and does not require tilting of
crystals to determine correct faces, being a useful tool
for qualitative work.
Inelastic scattering results from the interaction of
incident electron with atomic electrons. The total kinetic energy of the incident electron is reduced by a
certain amount of energy necessary to excite an electron of the specimen atom (Reimer, 1991). The effect of
the scattering events can be observed in the electron
energy loss spectrum and many of its features are
dependent on the nature of the elements present in the
samples. Differences observed between the electron
energy loss spectra of the crystal region and the supporting film can be explained by differences in mass thickness between these two regions. The electrons diffracted by
the whole magnetite crystal, which is a crystallographic
single domain, do not generate contrast necessary for
the visualization of surfaces because the whole crystal
is in the same Bragg condition. However, different
regions of the crystal present different mass thickness
of the decoration pattern of platinum particles, which
result in differential scattering of the inelastic electrons. Therefore, shadowed faces of the underlying crystal
can be selectively visualized in one micrograph due to
differences in scattering properties of inelastically scattered electrons from the bulk and from the film on the
surfaces of the magnetite crystals. This process of image
formation is the same regardless of the size of the
platinum particles. As no interference fringes were observed on carbon holey films by defocusing (not shown)
in the energy losses used in the present work, it was
concluded that the contrast observed for the platinum
film has no contribution from the phase contrast effect.
We recently reported unusually large magnetosomes,
twice as large as usual magnetosomes, in Southseeking magnetotactic bacteria from a lagoon near Rio
de Janeiro city (Farina et al., 1994). These magnetosomes were considered unusual because they could fall
outside the narrow size range for single domains of
magnetite according to theoretical calculations raising
questions about the phylogenetic relationship among
bacteria that produced them (Spring et al., 1998). In
this work, we observed the elongated faces and also the
smaller oblique faces of the large crystals. The pseudohexagonal crystals are capped by 51116 faces that are
larger than similar crystals already described. Probably, the crystals remain in chains because these faces
are quite large as a consequence of the exceptional
width of the crystals. The precise characterization of
the faces, however, requires high-resolution transmis-
324
U. LINS ET AL.
sion electron microscopy studies. The oblique faces 51006
and 51106 present in these larger crystals have a relatively smaller area if compared to other pseudohexagonal crystals already described for magnetotactic
bacteria. Sometimes these faces are even absent. This
probably means that the velocity of growth in the
lateral faces of the crystals with respect to their 51116
capping faces is higher in these larger crystals than in
the smaller ones usually described in the literature.
Control by the organisms over the biominerals formed
is exerted by a series of possible mechanisms involving
controlled ionic environments and chemical processes
(Mann, 1986).
We conjecture that the present technique, despite its
relatively low resolution compared to other shadowing
techniques (Ruben, 1998), represents an easy method
for the discrimination of morphologies of previously
known small particles when mixed and may contribute
as complementary data for studying multilayered objects under transmission electron microscopy.
ACKNOWLEDGMENTS
We thank Carolina N. Keim and Dr. Angela Hampshire for discussion and Marianne Parakkal for technical help.
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