Subcellular Structure of Bacterial Cell
Observed by Flash Contact X-Ray Microscopy
T. Majima, H. Shimizu, T. Tomie, E. Miura, T. Kanayama, M. Yamada
Electrotechnical Laboratory, Umezono, Tsukuba, Ibaraki 305, Japan
E-mail: majima@etl.go.jp
Abstract. X-ray images of living bacteria in water were obtained using soft
X-ray flash contact microscopy. In X-ray images of bacteria, cells are clearly
not of uniform carbon density. In many cases the image has a dent of 500 to
1,000 nm in diameter which corresponds to low carbon density structure in the
cell. Size of the dent suggested that it was image of nucleoid in bacterium.
Estimated density of bacterial nucleoid from reported size and molecular
weight supported the results of X-ray microscopy. Nucleoid of bacterium was
expressed in the X-ray image as lower density structure in the cell. Soft X-ray
flash contact microscopy is useful to clarify subcellular structure of live
specimens.
1 Introduction
X-ray microscope has been expected as a tool for observations of subcellular structure
in living organisms. X-ray images of subcellular structure of living organisms are
given as a result of X-ray absorption by carbon and nitrogen compounds in cells.
Absorption by oxygen in specimen and surrounding water gives background noise on
the X-ray images of specimens. One should suppress the background noise to obtain
fine images.
Developments in soft X-ray technology enable us to make a X-ray microscope
which works in wavelength range of 2.3 to 4.3 nm, so called "water window". In this
wavelength range, absorption of carbon is dominant. In this wavelength range, the
penetration length of X-rays to water is 2,000 to 10,000 nm. It is much longer than the
penetration length of electron beams to material which is about 200 to 600 nm. This
means that dehydration and thin sectioning processes which are necessary for
observations by electron microscope is not required in cases of X-ray microscope
observations if one uses X-rays in water window range.
Various types of X-ray microscopes have been developed. An advantage of a flash
contact-type X-ray microscope is its simple compositions in X-ray optics. It is,
however, a defective instrument as a microscope, because it gives images of the same
size of specimen. The expanding procedures of X-ray images are required. In many
cases one should make replica of X-ray resists to observe the X-ray image by using
the electron microscope. It is inevitable to introduce easier ways of expansion of the
X-ray images for improvement of the X-ray microscope as a popular instrumentation
in biological field.
In case of a contact-type X-ray microscope, the X-ray images are given as relieves
on X-ray resists. The relieves contain information on distribution of carbon containing
components in the specimens. Application of an atomic force microscope (AFM) as a
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reading instrumentation of the images resulted us free from making replica of the
images and enables us to obtain precise information on height of the images.
2 Materials and Methods
Salmonella typhumurium cells were grown under shaking culture in LB culture
medium containing 10 g of peptone, 5 g of yeast extract and 5 g of NaCl per 1,000 ml
of water at 37° C.
A contact type X-ray microscope system is composed of a KrF laser, a vacuum
chamber containing a yttrium target and a sample holder with a X-ray resist
membrane, and an AFM as a X-ray image reader. The cross section of sample holder
is schematically shown in Fig. 1. In the holder, specimens are maintained under
atmospheric conditions. A silicon nitride membrane of 100 nm thickness supported on
a silicone wafer works both as a X-ray window and a pressure partition wall.
Fig. 1. Cross section of sample holder. The holder is set in vacuum chamber and inside of the
holder is maintained under atomospheric conditions at room temperature. By the exposure
of X-rays, atomospheric conditions are broken.
Cells in the stationary state of growth were used as specimens. A drop of culture
medium containing specimens was put on a X-ray resist membrane of
polymethylmethacrylate (PMMA) (500nm thickness spun on another silicon wafer)
and covered with the X-ray window as reported previously [1].
Structure of Bacterial Cell Observed by Flash Contact X-Ray Microscopy
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The laser plasma was produced by a KrF laser pulse (10 nsec). The specimens
were placed 7 mm far from the X-ray source. The X-ray energy density on the resist
was a few tens mJ/cm2.
The specimens on the exposed resist were removed by a sodium hypochlorite
solution before development. The exposed resist was developed with 1 : 1 mixture of
methylisobutylketone and isopropanol. The image of X-ray window of 0.2 mm square
appeared within a few seconds. After the development, X-ray images of specimens
are expanded and observed by using AFM instead of electron microscope.
3 Results and Discussions
3 Results and Discussions
The X-ray images of living Salmonella typhumurium cells in culture medium are
obtained. In Fig. 2 and Fig. 3, the X-ray image of a cell and a cross section are shown.
The images of cells are clearly not of uniform carbon density. Within each AFM
image, there is a depression of the chemically developed PMMA resists which
suggests a domain of lower carbon density. The depressions have a diameter of
between 500 to 1,000 nm. The dents represent low density structures in bacterial cell.
In many cases we observed another small dent at the center of the large dent.as shown
in Fig.4.
Fig. 2. X-ray image of Salmonella typhumurium cell with a dent.
The line in the inserted picture of Fig. 3 shows a sectioning position. Corresponding positions of the image and the cross section are shown in figures. In this
image the profile of cross section shows a single dent. At the base of the profile,
roughness of the background is estimated. It has high frequency components
comparing with that of the X-ray image of specimen. The background level itself
shows drift. It is caused by a change in thickness of water layer between PMMA and
X-ray window due to bending of the X-ray window by pressure difference.
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Fig. 3. Cross section of X-ray image of bacterial cell. The line in the insertion shows
a sectioning position.
Fig. 4. X-ray image of bacterial cell with large and small dents.
Structure of Bacterial Cell Observed by Flash Contact X-Ray Microscopy
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The nucleoid of bacterial cell is a DNA-containing structure and it has a globular
structure with clefts [2]. It is composed of a large circular DNA molecule [3] of about
4,400 Kilobase pairs [3,4]. The diameter of the nucleoid is between 500 to 1,000 nm
[5]. We can estimate the weight of DNA molecule in a bacterial chromosome as
1.9x10-15 g assuming an average molecular weight of 260 Dalton per base pair. The
density of nucleoid, estimated by the weight of DNA and the diameter of 550 nm, is
0.03 g/ml. If the nucleoid is larger, the density will be reduced still further. This
estimated value is lower than that of cell plasma of the same volume. It is likely that
the depression in the X-ray image of the bacterial cell is corresponding to the image
of nucleoid in the cell.
Cryoelectron microscope study of thin sections of E. coli. indicated spherical
structure of the nucleoid with central core [5]. Electron density of the core is lower
than that of surroundings of the core. This suggests that the small dent at the center of
the large dents corresponds to the image of the core in the spherical nucleoid.
The X-ray image of bacterial cells shows that the nucleoid has a low density of
carbon and that the central core of the spherical nucleoid is a empty space. These
results suggest that the empty space in the core of the nucleoid would be important for
growing daughter chromosomes into diffuse sub regions of the nucleoid.
Soft X-ray microscopy is a potential tool to observe subcellular structure of living
cells in water. It visualizes distribution pattern of carbon and nitrogen compounds in
the cell, if X-rays in wavelength range of 2.32 to 4.37 nm (so-called "water window"
wavelength) are used. At this range we can obtain X-ray images of specimens without
disturbance due to X-ray absorption caused by oxygen of water.
Soft X-ray flash contact microscopy still has defects. Deformation of specimens
due to X-ray exposure is a quite considerable problem. Denaturation of specimens by
thermal expansion results disordering of the fine X-ray images. Flash exposure of Xrays is one of countermeasure to avoid the disordering of the image due to damage of
specimens by X-ray exposure.
Soft X-ray flash contact microscopy enables us to observe subcellular structures of
living cells in water, which are not obtained by electron microscopy after dehydration
of specimens.
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