A Comparative Study of the Ultrastructure of Living Cells
of the Green Alga Chlamydomonas Using Both
Soft X-Ray Contact and Direct Imaging Systems
and an Evaluation of Possible Radiation Damage
T.W. Ford1, A.M. Page1, W. Meyer-Ilse2, J.T. Brown2, J. Heck2, A.D. Stead1
1
Division of Biology, School of Biological Sciences, Royal Holloway, University of London,
Egham Hill, Egham, Surrey TW20 0EX, UK
2
Center for X-ray Optics, Lawrence Berkeley National Laboratory,
Berkeley, CA 94720, USA
Abstract. The extremely short exposure times of soft X-ray contact microscopy
(SXCM), have allowed the imaging of living specimens without the problems of
radiation damage. However, the technique has a number of practical and
logistical drawbacks which could be largely overcome by using direct imaging Xray microscopy. We have been imaging living Chlamydomonas cells using the Xray microscopy beamline 6.1 on the Advanced Light Source for comparison with
SXCM images. We have also been investigating the radiation sensitivity of these
cells and possible dosage limits for imaging living cells.
1 Introduction
One of the main attractions of X-ray microscopy for biologists interested in cell
ultrastructure is the potential for examining the organization of living cells at a resolution
superior to that of light microscopy. However the image obtained must be an accurate
representation of the original specimen and protocols used must be rigorously examined
for the possibility of artefacts being introduced. This is rarely a problem with light
microscopy but preparation procedures required for electron microscopy may introduce
such structural artefacts. With X-ray microscopy the main hazard is radiation damage to
the specimen before the image is collected and this is a major concern for the future
development and usefulness of this technique.
There are two possible ways by which radiation damage can be avoided. Firstly, by
using very short exposure times (of the order of nanoseconds) so that the image is
captured before damage can occur. Secondly by ensuring that the specimen radiation
dose is below the threshold at which damage is known to occur.
2 Soft X-Ray Contact Microscopy
By using the very short pulse (1-3 ns) from a high energy laser to generate a soft X-rayrich plasma it is possible to capture the image of a living specimen in a photoresist before
the sample is damaged by radiation. This technique has produced good images of the
fine structure of living cells. Comparison of SXCM images of the unicellular green alga
Chlamydomonas with images produced by conventional transmission electron
microscopy (TEM) shows both similarities and differences. Atomic Force Microscope
(AFM) readout of an SXCM image shows a ovoid cell with smooth outline and thin
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carbon-dilute cell covering which is thicker at the anterior end where the two flagella
emerge (Fig. 1). The main features visible within the cell body are several X-ray-dense
spherical inclusions approximately 1µm in diameter. The TEM image shows
considerably more detail but the outline and cell covering are rather convoluted (Fig. 2).
In addition, there are no obvious spherical electron-dense inclusions which could be
equated with those seen by SXCM. It is possible that SXCM, by using living cells, is
revealing features which are damaged or destroyed during processing for TEM. The only
other method available for studying living cells is light microscopy (LM). This again
shows a smooth ovoid Chlamydomonas cell with small refractile bodies in the anterior
cytoplasm when viewed by Nomarski interference LM [1].
Fig. 1. & 2. Chlamydomonas cells examined by SXCM/AFM (1), scale bars in µm and TEM (2),
scale bar=2µm. F-Flagellum; C-Chloroplast; N-Nucleus; V-Vacuole; P-Pyrenoid;
*-Spherical Inclusion.
Although SXCM produces good images it does have drawbacks. Firstly it is timeconsuming since, after imaging, the resist must be chemically developed then scanned by
AFM to produce a high resolution readout. Secondly, over-development of the resist can
increase surface roughness which reduces the potential resolution of the image. Thirdly,
since development is often non-linear, it is difficult to obtain quantitative information on
relative carbon density of structures. Finally, national laboratories housing high energy
lasers are usually multi-user facilities which can be problematical for biologists imaging
delicate living specimens.
3 Direct Imaging X-Ray Microscopy
Direct imaging of living cells overcomes the problems listed above for SXCM. However,
since the soft X-ray fluence is monochromatic and of relatively low energy, long
exposures times are usually needed. There is, therefore, the very real danger that the cell
will suffer radiation damage before the image is collected and so it is necessary to
determine radiation dosages which are sufficient to produce a good contrast image but
not introduce radiation-induced artefacts. Our experiments were carried out using the Xray microscope XM-1 at the Advanced Light Source (ALS) at the Berkeley Laboratory
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[2]. Living specimens were enclosed in an environmental holder and irradiated with
2.4nm soft X-rays. The main aim of the preliminary experiments was to determine if this
microscope could produce images of living cells at dosages which did not result in
radiation damage. From previous experience with SXCM, we used the unicellular green
alga Chlamydomonas as a test organism.
3.1 Imaging of Living Chlamydomonas Cells
Images of living Chlamydomonas cells could be obtained following a 1 second exposure
(equivalent to a total dose of 8.8x105Gy) (Fig. 3). The cell can be clearly seen with
several spherical, X-ray-absorbing inclusions at the anterior end of the cell. Towards the
basal end of the cell is a dense structure surrounded by a less carbon-dense halo. This has
the appearance of the pyrenoid as seen in these cells by TEM (Fig. 2) which is located
within the chloroplast. Between this structure and the edge of the cell are several lines
suggesting chloroplast membranes (thylakoids).
Fig. 3. & 4. X-ray images of living Chlamydomonas cells following 2.4nm irradiation for 1 sec.
(accumulated dose 8.8x105Gy) (3) or 10 sec. (accumulated dose 7.53x106Gy) (4).
By irradiating the same cell for a further 10 seconds the image (Fig. 4) has a sharper
outline with improved detail in the chloroplast, particularly in the pyrenoid, starch sheath
and thylakoids. However, the spherical inclusions show some distortion and loss of
contents. This second image has an accumulated dose of 7.53x106Gy and it would
appear that such doses can cause radiation-induced damage.
3.2 Enhanced Contrast v. Radiation Damage
The critical question concerning the feasibility of direct imaging of living biological
specimens is whether it is possible to provide a dosage of soft X-rays which will produce
an image of sufficient contrast but which causes no alteration to sub-cellular components.
It is therefore essential to know if features visible in X-ray images are radiation-induced
artefacts. A living cell of Chlamydomonas imaged for 2 seconds shows a chloroplast and
possibly some thylakoid membranes (Fig. 5). The position of the pyrenoid can also be
identified. Spherical inclusions are visible and appear intact suggesting that there has
been no radiation damage and therefore the structures are not radiation-induced artefacts.
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A subsequent exposure of the same cell for 4 seconds appears to show greater clarity of
the pyrenoid and its surrounding membranes but the distortion and collapse of the
spheres indicates that the cell has suffered some radiation damage (Fig. 6).
Figures 5–7. X-ray images of living
Chlamydomonas cells following irradiation
at 2.4nm for 2 sec. (accumulated dose
8.6x105Gy) (5), 4 sec. (accumulated dose
4.3x106Gy) (6) or 0.5 sec. (accumulated
dose 4.5x106Gy) (7).
One way to test whether the features are radiation-induced artefacts is to subject the
same cell to a third, much shorter exposure. If the features are genuine then the low
contrast of this image would produce less clarity than the first exposure. If radiation
damage has caused condensation of cell material, then these would still be visible with
good clarity even when only a few photons are collected. A third exposure of this cell for
only 0.5 second reveals the chloroplast thylakoids at a greater clarity than the first
exposure but not as good as in the second (Fig. 7). This suggests that these features are
probably genuine but what appears to be enhanced clarity following longer exposures
results from radiation damage.
4 Conclusions
Direct imaging systems can overcome many of the drawbacks of SXCM but it must be
certain that images of living cells obtained in this way do not show radiation damage.
Several measurements of dosages of soft X-rays causing cell damage have been reported.
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Loss of myofibril contraction occurs at around 1-2x104Gy [3],[4] whilst loss of
membrane function occurs in hamster cells at 104-105Gy with morphological damage
visible at 105Gy [5]. Ultrastructural damage was visible in Chlorella cells at 1.5x1031.5x104Gy [6]. Yeast cell death (LD50) was reported after irradiation with
2x105photons.µm-2 (approximately 2.5x104Gy) [4]. The images of living
Chlamydomonas cells reported here showed no detectable damage after exposure to
8.6x105Gy whilst dosages in excess of 106Gy showed clear damage, at least to the
spherical inclusions. Imaging of living specimens using this system is therefore possible
but with limitations to the length of exposure if radiation damage is to be avoided. One
possible solution to this dilemma is to use frozen samples where it is claimed that much
higher radiation dosages can be used without observable cellular damage [7]. However,
freezing itself can cause cell damage if the process results in ice crystal formation which
can damage cell membranes.
Acknowledgements
Funding from the EPSRC (UK) for purchase of the AFM (Grant GR/K23522) is
gratefully acknowledged as is access to the High Resolution X-ray Microscope XM-1 at
the ALS, built and operated by Berkeley Laboratory Center for X-Ray Optics and
supported by the US Department of Energy under contract DE-AC 03-76SF00098 and
the Laboratory Directed Research and Development Program. We are also grateful to
Stephen Janes for assistance with the production of the figures.
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