Use of Soft X-Rays
to Image Hydrated and Dehydrated Bacterial Spores
Using Either Soft X-Ray Contact Microscopy
or Soft X-Ray Transmission Microscopy
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A.D. Stead , J.T. Brown , J. Judge , W. Meyer-Ilse , D. Neely , A.M. Page ,
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E. Wolfrum , T.W. Ford
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Biological Sciences, Royal Holloway (Univ. of London), Egham, UK
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Center for X-ray Optics, Lawrence Berkeley National Laboratory,
Berkeley, CA94720, USA
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Unilever plc, Colworth House, Sharnbrook, Bedforshire, MK44 1LQ, UK
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Rutherford Appleton Laboratory, Didcot, Oxfordshire, OX11 0QX, UK
Abstract. The appearance of dehydrated and hydrated bacterial spores has
been studied by soft X-ray contact microscopy and X-ray transmission
microscopy. Structural differences between dehydrated and hydrated spores,
and the effects of heat activation and incubation in germination media, are
reported. The potential benefits of soft X-ray microscopy for imaging spores
are discussed.
1 Introduction
Bacterial spores are very resistant to environmental extremes, including desiccation,
but the physiological and structural aspects that confer tolerance is not understood.
The preparative techniques used for conventional transmission electron microscopy
allow the spores to hydrate and so dehydrated spore structure cannot be studied. Soft
X-ray microscopy avoids specimen preparation and can therefore provide novel
structural information [1].
By transmission electron microscopy, spores have been shown to have an electron
dense wall and central core separated by an electron-translucent cortex [2, 3].
However, the fixation procedures prevent the study of dehydrated spores. Light
microscopy has shown that prior to germinating the spores change from being phase
bright to phase dark [4], but these changes in the phase properties of spores cannot be
related to ultrastructural changes as it is not possible to determine whether individual
spores embedded for electron microscopy were phase bright or dark.
Biochemical analyses have shown that dormant spores lose large quantities of
dipicolinic acid and calcium [5] prior to germination. Such studies require the use of
large quantities of spores and it is not possible to differentiate between the loss of all
the dipicolinic acid from the few spores which germinate rapidly or the loss of a small
proportion of the dipicolinic acid from all of the spores. By measuring the X-ray
transmission it has been possible to determine whether the mass loss from all spores is
equal or if the behaviour of spores differs within the population.
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2 Material and Methods
Bacterial spores (Bacillus subtilis) were prepared [4] and transported frozen. After
thawing the spores were aliquoted and refrozen; each experiment used a fresh aliquot.
Initial experiments compared the structure of hydrated spores to dehydrated spores
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and in subsequent experiments spores were activated (70 C for 30min; phosphatecitrate buffer (50mM, pH 7.0)) followed by resuspension in nutrient broth (Oxoid)
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with 10mM L-alanine added and incubated at 30 C. Before, or at various times after,
addition of germination media a 2 l sample was removed and imaged.
2.1 Soft X-Ray Contact Microscopy
Spores were mounted on a photoresist (0.9 m thick polymethyl-methacrylate) in an
environmental cell [6]. The specimen was protected from the vacuum by a 120nm
thick Si3N4 window (aperture 250x250µm; FaSTec, UK) [7]. Using a rapid feedthrough interlock, specimens could be imaged within 3-5mins. Soft X-rays were
generated from a yttrium target using up to 15J laser energy (1.053µm; 1-3nsec pulse)
generated from a Nd:glass laser (Rutherford Appleton Lab., UK). The exposed
photoresist was developed with mixtures of methyl isobutyl ketone (MIBK) and
isopropyl alcohol (IPA) to reveal a topographical map of the soft X-ray absorbance by
the original specimen [6]. Development depth was monitored by interference light
microscopy (Leitz Dialux) and then the developed photoresist was viewed by atomic
force microscopy (Topometrix or Park Scientific).
2.2 Transmission X-Ray Microscopy
2µl of the spore suspension was placed between two 120nm thick Si3N4 windows. The
preparation was examined by phase contrast light microscopy and fields of view
selected which included phase bright, phase dark and, if possible, vegetative cells.
Each field of view was photographed for comparison with the X-ray images. The field
co-ordinates were recorded and imaged spores with soft X-rays using XM-1 on
beamline 6.1 at the ALS, Berkeley, USA [8] using monochromatic soft X-rays
(2.4 nm).
3 Results and Discussion
3.1 Soft X-Ray Contact Microscopy
Very little soft X-ray flux penetrates the dehydrated spores and when the photoresist is
developed the background develops away leaving a very strong image of the dry
spore. The images are very smooth, indicating that the soft X-ray absorbing properties
of the spore are very uniform across the spore (Fig. 1a). This ability to absorb the soft
X-rays is due to the carbon content of the spore and therefore shows that the carbon
density of the spore is high, furthermore it is distributed very homogeneously within
the dehydrated spore. In contrast, the carbon density of the wet (but not activated)
spore is very heterogenous as the images, whilst still very strong (ie the spores are still
very carbon-dense) are very uneven (Fig. 1b). This implies that the ability to absorb
Use of Soft X-Rays to Image Hydrated and Dehydrated Bacterial Spores
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soft X-rays is not uniform and this reflects the non-uniform carbon distribution within
the spore. In some spores the cental core is a prominent feature (Fig. 1c) indicating
that this region has greater X-ray density. The cell wall of the spores could not be
differentiated from the rest of the spore.
Fig. 1. Atomic force image of the developed photoresist produced by soft X-ray contact
microscopy. a) dehydrated; b-c) hydrated (but not activated).
3.2 Transmission X-Ray Microscopy
The hydrated spores have an X-ray dense core and an X-ray dense wall but the region
between is relatively X-ray transparent. This area corresponds to the cortex, as seen by
electron microscopy, where it is electron translucent [2,3]. Since soft X-rays (2.4nm)
and electrons are transmitted through this region of the spore it is clear that the
carbon-containing density of this region must be very different to that of the core. The
difference in X-ray absorption is exaggerated by repeated exposure of the spores,
indicating that the spores are radiation-sensitive (Fig. 2a-b), great care is therefore
needed in the interpretation of the images and in particular the existence of the X-ray
dense wall needed to be confirmed. To achieve this the thickness of the wall was
measured and then the pixels combined such that the resolution was not reduced below
that of the wall thickness, by doing this the exposures times could be reduced to a time
in which radiation damage was considered unlikely. In a exposure of 1sec with 6x6
binning it was still possible to distinguish the X-ray dense wall and core and the X-ray
lucent area between these (Fig. 2a). In this image the number of transmitted photons
per pixel element was 12908 ± 495 in the central X-ray dense core but 14625 ± 793 in
the cortex area. There was therefore a reduction of photons of about 12% in the core
area as compared to the cortex.
Dehydrated spores were exposed for shorter times to ensure that the radiation
doses were similar to those given to hydrated spores, thus an exposure of 0.8sec was
equivalent to 5sec and 5sec was equivalent to 30sec. In both cases the spores appeared
uniformly X-ray dense with no indication of an X-ray lucent cortex (Fig. 2c).
Extremely long exposures also show no indication of radiation damage (Fig. 2d). The
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radiation dose received by the specimen during such a long exposure was 1.9 x 10 Gy
which is considerably higher than that reported to cause functional or structural
damage in other biological tissue [9-12].
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A. D. Stead et al.
Fig. 2. Soft X-ray transmission images of hydrated spores (a-b) and dehydrated spores (c-d). a)
1 sec exposure with 6x6 pixel binning; b) 10sec exposure (1sec previous exposure) with 6x6
pixel binning. c) 5sec exposure, no binning; d) 150sec exposure, no binning.
These images are therefore similar to those obtained by contact microscopy and
show that spores have an X-ray translucent cortex with an X-ray dense core,
furthermore the wall was distinguishable due its greater X-ray absorbance. As with
contact microscopy the dehydrated spore was found to be uniformly X-ray dense.
Whilst the hydrated spores appear to be radiation-sensitive the dehydrated spores
appear to be structurally unaffected by exposure to high doses of soft X-rays.
Spore structure after activation and during germination was studied by monitoring
which spores were phase bright or phase dark prior to soft X-ray imaging. Short
exposures, ie with a minimum radiation dose, showed no suggestion of an X-ray
translucent cortex in activated spores. In subsequent exposures the X-ray dense core
and wall were clearly separated by a less X-ray dense cortex (Fig. 3a-c). This is in
contrast to the images of hydrated, but not activated spores, in which the X-ray lucent
cortex was seen in even the shortest of exposures and suggests that there are
fundamental structural differences between hydrated and activated spores. Radiation
damage, caused by prolonged exposure to X-rays was demonstrated by quantifying
soft X-ray transmission or measuring the linear dimensions of the spores (Fig. 4a,b).
Phase bright and phase dark spores, as well as vegetative cells, were subject to
radiation damage (Fig. 5) when imaged sequentially. Such mass loss during exposure
to soft X-rays is similar to that reported for isolated chromosomes [9] and once again
demonstrates the need for caution when interpreting soft X-ray images of hydrated
biological material which have required prolonged exposure times.
The most striking difference between phase bright and phase dark spores is their
X-ray transmission characteristics; phase bright spores are X-ray dense but phase dark
spores are more X-ray translucent (Fig. 3, 5). Quantitative analysis shows a c.40% loss
of material from the phase dark spores relative to the phase bright spores with no
change in the spore size. Vegetative cells absorb an even lower proportion of the
incident radiation and, on a per area basis, there is a c.80% loss of material relative to
phase bright spores, however the vegetative cells are considerably larger than the
spores and the loss of material must, in part, reflect a dilution of the X-ray absorbing
materials. It is not known if the reduced X-ray absorbance is due to the loss of calcium
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dipicolinate but biochemical analyses shows that over 600nM of dipicolinate is lost
per mg of spores within 15min of adding germination media [5]. Previous attempts at
soft X-ray contact microscopy at the N or CaIII edge showed that the spores were rich
in calcium but failed to detect the distribution of calcium [3] within the spore.
However, because of the long exposure times needed, the material was dry and as the
present study has shown X-ry absorbing properties of dehydrated spores is very
uniform. With the benefit of third generation high brightness synchrotron sources and
modern CCD cameras it should be possible to gain further data on the distribution of
calcium within the spore and the loss of calcium as the spore is activated and
eventually germinates.
Fig. 3. Sequential images of phase bright (B) and phase dark spores (a-c)
Fig. 4. a) Spore (open symbols) or vegetative cell (closed symbols) size reduction caused by
successive exposures. b) Mass loss from spores after sequential imaging with soft X-rays.
One of the advantages of studies using XM-1 at the ALS for soft X-ray
transmission microscopy is the ability to determine which spores are phase bright or
phase dark immediately prior to imaging with soft X-rays. In previous studies using
either electron microscopy or biochemical techniques populations of spores are
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A. D. Stead et al.
studied and the physiological state of individual spores is unknown. Studies have
shown that the behaviour of spores within a population can be very variable [4] or
even biphasic [13] and this complicates data interpretation. By determining which
spores are phase bright and phase dark prior to imaging, soft X-ray microscopy can
contribute more information about the timing of the loss of material relative to the
shift in phase properties and the time of germination.
Fig. 5. Quantification of the soft X-ray absorbance by phase bright, phase dark and vegetative
cells and the effect of subsequent re-exposure. In each case the thickness of carbon giving the
same aborsbance has been calculated. The variation in radiation dose (due to the changing beam
conditions was minimal between samples and exposures).
4 Conclusion
Spores structure, as seen by soft X-ray transmission microscopy, changes as the spores
hydrate, become activated and finally germinate. Long exposures affect the
appearance of all but dehydrated spores. The changes can be summarised as follows:
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Dehydrated spores - are uniformly X-ray dense without any indication of an Xray lucent cortex and are extremely resistant to damage by radiation
Hydrated spores - have an X-ray dense core and wall separated by an X-ray lucent
cortex, radiation exaggerates these differences
Activated spores (phase bright) - initially show no indication of an X-ray lucent
cortex but exposure to radiation very quickly causes condensation of the core to
leave an X-ray lucent cortex
Activated spores (phase dark) - overall are much more X-ray lucent (equivalent to
a loss of about 40% of the X-ray absorbing properties) but otherwise behave the
same as the phase bright spores (ie subject to radiation damage)
Use of Soft X-Rays to Image Hydrated and Dehydrated Bacterial Spores
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Vegetative cells - even more X-ray lucent than the phase dark spores (equivalent
to a loss of about 80% of the X-ray absorbing properties of phase bright spores)
and still very radiation sensitive although the affect of radiation is somewhat
different.
Acknowledgments
The direct imaging work has been carried out with the "High Resolution X-ray
Microscope" (XM-1) at the Advanced Light Source, build and operated by Berkeley
Labs Center for X-ray Optics (CXRO) and supported by the United States Department
of Energy under contract DE-AC 03-76SF00098 and the Laboratory Directed
Research and Development Program (LDRD). Financial support for this work was
received from Unilever plc and EPSRC (Grant refs GR/J76248 and GR/K23522).
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