1. INTRODUCTION
In 2002, the Nobel prize in Physiology or Medicine was awarded to
Sydney Brenner, USA, H. Robert Horvitz, UK, and John E. Sulston, USA
for their seminal discoveries concerning genetic regulation of organ
development and programed cell death. They used the nematode
Caenorhabditis elegans as an experimental model to follow cell
division and differentiation from the fertilized egg to an adult. While
identifying the key genes, they have shown that corresponding genes
exist in higher species, including man, and the discovery has shed new
light on the pathogenesis of many diseases (Nobel
Foundation, 2002). The importance of C. elegans as a
model initiated the present work.
Soft X-ray contact microscopy (SXCM), this paper is devoted to the
study of C. elegans by a technique devised to produce images
of biological specimens using short wavelength radiation and provides a
resolution higher than optical microscopy. With respect to other
imaging techniques, such as electron microscopy (EM), it avoids the
drawbacks of sample preparation. In EM, the specimens must be fixed,
dehydrated, embedded in resin, cut in thin sections, and stained with
heavy metals. Potentially any of these processes may introduce
artifacts (Ford et al., 1992) and
thus the possibility of studying living samples is precluded. SXCM
allows living organisms to be imaged in a liquid environment (Ford et al., 1991; Fletcher et al., 1992), also allowing an
easier way of handling the samples. SXCM experiments exploit the
existence of a particular region of the electromagnetic spectrum,
called the Water Window (WW), which lies between the absorption edges
of carbon (280 eV) and of oxygen (530 eV). In the WW region, water is
almost transparent to X-rays, whereas carbon, which is the main
constituent of the organic structures, is highly opaque. Therefore it
is possible to produce high contrast images in organic samples.
Laser plasmas seems to be an ideal X-rays source for SXCM (Stead et al., 1995a). The hot and
dense plasma produced in the interaction of a laser pulse with a solid
target (Kondo & Tomie, 1994) can yield
high fluxes of radiation whose spectrum can be chosen by changing the
target material and the beam focusing. The short irradiation time of
the order of the laser pulse is far below the time needed (of the order
of milliseconds) to damage the specimens. Therefore this technique
produces images of the live specimens (Foster et
al., 1992; Stead et al.,
1992) and it is also not influenced by the movement of the
specimen. The images are obtained by exposing the specimens as a 1:1
radiograph on a photo resist which is then chemically developed and
finally analyzed using an atomic force microscope (AFM). In this way no
optics are required. This is significant because in the soft X-ray
region, optics are still expensive and not very efficient. X rays in
the water window range can be produced using low and high Z
materials as targets. There are certain advantages of using a high
Z target for producing X rays as X-ray conversion efficiency
is high and copious soft X rays are generated due to Bremsstrahlung and
recombination processes. However, there is a large spillover of
radiation around the water window in the case of high Z
materials.
In this article, we report the analysis of SXCM images of living
C. elegans. The X-ray spectra that irradiated the specimens
were also recorded using X-ray spectrometers. These spectra were used
to obtain the corresponding plasma parameters like plasma density and
temperature by spectroscopic analysis. Images obtained on the
polymethilmetacrylate (PMMA) photo resists are analyzed using an AFM
operating in contact mode. SXCM has already been used to image
biological specimens like yeast cells, unicellular green algae, human
red cells (Stead et al.,
1995b; Masini et al.,
1997; Batani et al., 1998;
Bortolotto, 2000). The experiments reported in
the present article allowed us to explore the potentialities of this
technique to study complex, multicellular organisms. To this aim, the
biologically well characterized nematode C. elegans has been
chosen. It is the first time that small (semimicroscopic) but complex
(but not too complex, like mammals), multicellular samples have been
studied by means of SXCM.
2. EXPERIMENTAL
X-ray microscopy experiments were performed using the Prague Asterix
Iodine Laser System (PALS; Jungwirth et
al., 2001), which emits 1.314-μm radiation. Laser pulse
duration was ∼400 ps. The maximum optical energy in a single beam
was ∼600 J and the beam diameter at the entrance of the plasma
chamber was ∼290 mm. Laser radiation was focused on the target
surface using a f = 600 mm focusing lens and the intensity was
varied in the range ∼1014–1016
W/cm2 by either changing the laser spot area (focal spot
was varied from 150 μm to 600 μm) or changing the incident
laser energy. The interaction chamber was evacuated to better than
10−4 mbar.
2.1. X-ray spectroscopy
Biological specimens were irradiated with X rays produced from three
types of planar solid targets, Teflon (CF2), molybdenum
(Mo), and gold (Au). Gold and molybdenum have similar flux in water
window regime (Batani et al., 2000).
A flat crystal Bragg's spectrometer was used to record the
spectrum emitted by the plasma. X-ray spectra can provide information
on the properties of the plasma, in particular, plasma density and
temperature during the process of X-ray emission, by the analysis of
the spectral shape, spectral width, and position of different lines and
their relative intensities (Tallents, 1987).
Teflon targets have a particularly simple K-shell centered at
0.9 KeV and are particularly useful for such a diagnostic purpose.
The spectrometer used in the present experiment had a RbAp crystal
(2d = 2.6121 nm). Time- and space-integrated X-ray spectra
were recorded on Kodak DEF films. In the present experiment, the
spectrometer was placed at a distance of 6.5 cm from the X-ray source
at an angle of 75° to the laser axis. Spectrometer placing was
restricted due to the presence of biological sample holders. Spectra
were recorded for every exposure of the biological sample. A 100-μm
slit was placed on the spectrometer. An aluminum foil of 5 μm acted
as a filter for the Teflon target whereas a 14-μm aluminum foil was
used for gold and molybdenum targets.
X rays are produced from the dense and coronal plasma of a
laser-irradiated target. The process lasts over a time span
(τx) comparable to laser pulse duration (τ).
Actually, X-ray emission duration depends on the plasma dynamics. Rapid
plasma expansion leads to faster cooling, thereby terminating the
process of X-ray emission. For picosecond pulses as in our case, the
duration of the soft X-ray component is comparable to the laser pulse
length. X-ray emission consists of continuous and characteristic line
emission determined by the target material. Continuum is produced due
to free–free Bremsstrahlung and free–bound transitions in a
partially ionized plasma from dense (ne
> nc) and coronal plasma
(ne ≤ nc),
and the line emission from highly excited target ions. This emission is
mostly in the soft X-ray region (below a few kiloelectron volts) and
has been largely investigated as a tool for plasma diagnostics. In the
case of a molybdenum target, soft X-rays are from the N shell
whereas for gold targets they are from N and O
shells.
2.2. Biological specimens
Encouraged by our earlier results on SXCM experiments on unicellular
organisms like yeast (Batani et al.,
2000), our interest in the present investigation was to
establish the suitability of the SXCM technique to study multicellular
living organisms. From this point of view, the C. elegans
nematodes seem to be a suitable choice for the following reasons:
- they are quite well studied and represented in the literature;
- they can be easily grown at 20°C on agar plates or in liquid
culture in the laboratory;
- they can be conserved in liquid culture;
- their life cycle is approximately 3 days;
- they are quite resistant to environmental changes;
- it is easy to study them at various developing phases;
C. elegans is a small, up to approximately 1 mm in length
and about 30 μm in diameter, free-living nematode with a remarkable
anatomic simplicity. An adult nematode contains 959 somatic cells. Its
natural environment is the soil where this organism feeds mainly on
bacteria.
In the sample holders, a droplet of water containing some biological
specimens was sandwiched between a silicon nitride window and a photo
resist (PMMA) with the minimum level of fluid (water), a little more
than the specimen body diameter. The assembly of the specimen holder
was similar to that of earlier works (Batani et al., 2000). X rays were incident
on the silicon nitride window, which is transparent to visible and X
radiation. Usually 4–5 specimens were simultaneously exposed to
every laser shot. The sample holders were placed at two different
distances (5–10 cm from the X-ray source) at an angle of 45°
to the laser axis. X rays are absorbed from various parts of specimen
in different levels. Therefore different parts of the photo resist
receive different X-ray doses, and the damage of the structure of the
PMMA is affected by the characteristics of the sample. The chemical
development of the photo resist gives rise to a profile of the sample,
the height of which is proportional to the absorbed X-ray intensity.
The height of the nematode profile is approximately 400 nm and the
traces of the internal organs are of the order of 10–12
nanometers superimposed on this layer. Such a profile has been analyzed
using AFM.
2.3. AFM analysis
Photo resists were analyzed using an Auto Probe CP Research AFM
(ThermoMicroscopes, Sunnyvale, CA). All the AFM measurements discussed
in the following were made in air operating in constant force mode.
Microfabricated V-shaped silicon cantilevers with a silicon conical tip
of resonant frequency of approximately 45 kHz and a force constant of
0.4 N/m were used. AFM images were processed using the Image
Processing Data Analysis 2.0 software provided by ThermoMicroscopes.
In constant force mode, while the scanner gently traces the tip
across the sample surface, the forces between tip and sample cause the
cantilever to bend to accommodate changes from the sample topography.
The spatial variation of the cantilever deflection can be used as input
to a feedback circuit that moves the scanner up and down in the
Z direction responding to the topography by keeping the
cantilever deflection constant, and the topography images are generated
using the signal applied to the scanner. In this operating mode, the
speed of scanning is limited by the response time of the feedback
circuit, but the total force exerted on the sample by the tip is well
controlled so that this mode is generally preferred for most
applications.
3. RESULTS AND DISCUSSION
3.1. Analysis of X-ray emission spectra
The X-ray spectra discussed in the following have been recorded at a
laser intensity I ∼ 1014 W/cm2
on target. X-ray intensities as a function of X-ray wavelength from
Teflon, gold, and molybdenum plasmas are shown in Figure 1a, b, and c, respectively. X-ray
intensities were corrected by taking into account the X-ray film
response (Rockett et al., 1985),
crystal reflectivity (Henke et al., 1985),
and protective foil transmission (Henke et al.,
1986). Prominent and separated satellite lines were observed at lower
photon energy for Teflon plasma as seen in Figure 1a
(λ > 15 Å).
X-ray intensity versus X-ray wavelength for (a) Teflon, (b) gold, and
(c) molybdenum. Laser intensity on the target surface was 5 ×
1014, 2.6 × 1014, 2.75 ×
1014 W/cm2, respectively. Al filter thickness
was 5 μm for Teflon and 14 μm for Au and Mo.
Spectra were analyzed by assuming purely free–free and
free–bound transitions, which is a rather crude assumption in the
present case. Results show that the X-ray intensity corresponds to an
exponential decrease with increasing photon energy. The electron
temperature for gold from the slope of the continuum (Fig. 1b) Te = 90
± 10 eV. The analogous evaluation of the continuum emitted by
the molybdenum plasma (Fig. 1c) shows a
temperature Te = 120 ± 10 eV. The
dominant bound–bound transitions are marked for Teflon plasma in
Figure 2. Spectra clearly show H-like and
He-like groups of lines and strong satellite lines. From these lines we
can estimate the plasma density and temperature that characterize the
plasma during the process of X-ray emission.
Normalized X-ray intensity versus X-ray wavelength for Teflon target:
(a) experimental results, (b) calculated by RATION.
Laser-produced plasma is inhomogeneous. X rays are generated from
different parts of the plasma that corresponds to different densities
and temperatures as it expands from the target surface. Therefore a
single temperature and density should not be appropriate to
characterize such plasmas. However, we tried to interpolate
experimental spectra using the minimum number of parameters, that is, a
single density and a single temperature that must then be considered as
a space and time average of the evolving plasma parameters during the
process of X-ray emission. Line radiation and continuum are emitted due
to different mechanism in the plasma. Line radiation is emitted due to
the electron transition between bound states of an ion. The spectrum of
the emitted radiation therefore consists of lines at distinct
wavelengths, whereas the free–free transition and
free–bound electron transitions give rise to continuum. In the
present case, carbon K-shell emission was not recorded, as the
lines were below the detectable range of the RbAP crystal. However, the
presence of carbon (33%) in Teflon plasma implies the existence of
carbon lines and recombination radiation due to carbon plasma. This can
be seen as a continuum superimposed on fluorine ion lines. The
continuum appearing at higher energy is mainly due to fluorine ions.
The exponential interpolation of the continuum at high photon energy
from Teflon spectra shows an average plasma temperature
Te = 125 ± 5 eV.
In multiply charged ions, satellite lines appear when two electrons
are in excited quantum states. One of the electrons undergoes a
transition resulting in the emission of a photon, while the other,
known as a spectator electron, remains in an excited quantum state,
thereby affecting the energy levels of the excited quantum state. These
satellite lines appear at a wavelength longer than the resonance lines.
A shift in the wavelength depends on the excited state of the spectator
electrons, and several satellites can be emitted depending on the
excited state of the spectator electrons.
We also used the code RATION to synthesize X-ray spectra. This code
was developed by R. Lee at the Lawrence Livermore National Laboratory
(Lee, 1990) and used to generate
K-shell spectra from carbon (Z = 6) to iron
(Z = 26). Experimentally recorded Teflon spectra correspond to
the X-ray wavelength in the range 10–16 Å. The calculations
assumed fluorine ions (Z = 9) with 33% impurity from carbon,
spot size of the order of laser focal spot on the target surface (150
μm), and instrumental width (FWHM) of 2 eV. Plasma was considered
to be in nonlocal thermodynamic equilibrium (NLTE). Figure 2 shows (a) experimental and (b) synthetic
spectra. The synthetic spectra show a quite good agreement with the
experimental spectrum. Ly-β, He series, and Ly-α can be
reproduced at Te = 145 eV and plasma
density Ne = 4.8 ×
1020/cm−3. However, the structure of
the satellite lines is not well reproduced.
3.2. Biological images
Two AFM topography images of SXCM imprints on PMMA photo resists of
C. elegans nematodes are shown in Figure
3. In particular, Figure 3a shows
regularly repeating bands (ranging from 0.9 to 1.2 μm) giving rise
to a structural periodicity along all the nematode profile. Both the
well-defined pattern and the good agreement between our measurements
and data reported in literature (Cox et
al., 1981) suggest that annuli of the nematode cuticle have
been imaged. During the 3 days development of the specimen, the cuticle
is replaced at each of four postembryonic molts. Literature data report
that the cuticles of these developmental stages of C. elegans
differ substantially from one another in ultrastructure and protein
composition (Cox et al., 1981). As
the organism grows, the cuticle formed at each successive stage becomes
thicker. The increase in cuticle thickness is proportional to the
increase in body diameter during these stages. In the Juvenile J1, J4,
and adult, the ratio of cuticle thickness to body diameter averages
approximately 1:88.
AFM topography images of SXCM imprints on a PMMA photo resist of a
Caenorhabditis elegans. (a) The nematode cuticle is
circumferentially indented at regular approximately 1.1 μm
intervals, creating pleated-appearing annuli. (b) Profile of the
anterior body end.
The anterior body end of a C. elegans is shown in Figure 3b. The small protrusions, visible in the
AFM image, are very similar in shape to the lips that symmetrically
surround the buccal cavity of C. elegans.
The features of the image shown in Figure 4
have been obtained in various samples. AFM images collected in the
central region of the nematode show a well-defined relief separating
two regions with different roughnesses.
A 10 × 10 μm2 AFM topography image of a SXCM
imprint on a PMMA photo resist of a Caenorhabditis elegans.
This image has been collected at the central region of the
body.
Several AFM images collected in the central region of the SXCM
imprint of the C. elegans nematode showed structures
morphologically well characterized presented in Figure 5. The comparison with data reported in
literature (Bargmann & Avery, 1995)
allowed us to regard them as nuclei of three different cell types. In
particular, Figure 5a shows round hypodermal
and gut nuclei with a large and prominent nucleolus, and Figure 5b shows neuronal nuclei that are round,
small, and lacking in prominent nucleoli. The structures in Figure 5c can again be considered as cell nuclei
(muscle nuclei?).
Some 6 × 6 μm2 AFM topography images of a SXCM
imprint on a PMMA photo resist of a nematode Caenorhabditis
elegans. The images have been collected in the central region of
the nematode. Nuclei of three different cell types have been
individuated: (a) hypodermal and gut nuclei, round with a large,
prominent nucleolus; (b) neuronal nuclei, smaller, round, and without
prominent nucleoli; (c) nuclei of a not individuated cell
type.
The present experimental data indicate our preliminary results.
C. elegans of different sizes and in different development
phases (length ranging from 250 to 1000 μm and corresponding
diameters ranging from 10 to 30 μm) were present. As a consequence,
the thickness of water around the various specimens was not constant.
This may have influenced the quality of several images. Nevertheless
good results are obtained from C. elegans of different
dimensions, confirming that the technique can be used as a research
tool top imaging non unicellular organism.
In spite of the large number of specimens (60 approximately) exposed
to the X rays, about 20% have been recorded on the PMMA photo resist.
Apart from the breakage of several extremely delicate silicon nitride
windows during the various phases of the experiment, the probability of
imaging a single C. elegans on the PMMA windows was low.
Moreover, only a few photo resists showed identifiable structures. In
spite of the above difficulties, the recorded images provide important
quantitative information on C. elegans. Other images of the
C. elegans will be published in detail elsewhere.
4. CONCLUSIONS
It is the first time that SXCM has been applied to the study of a
multicellular living organism such as C. elegans. Internal
structures and cuticle features of C. elegans have been
distinguished. In spite of several problems encountered during the
experiments, our preliminary results prove that the technique can be a
useful tool in biological research. The knowledge and control of
experimental parameters such as the water layer thickness, the X
radiation intensity, the device geometry, the photo resist chemical
development, and the sex and embryonic development of the biological
specimens will allow us to obtain better results in the future.
From the experimental spectra, we observe many X rays at higher
energy (hν ≥ 1 KeV) that can penetrate the sample. The
fact that we used infrared laser radiation and high Z target
materials could indeed justify a strong X emission outside the water
window. This can explain the low contrast found in many AFM images.
