A Comparative Study of the Ultrastructure of Living Cells of the Green Alga Chlamydomonas

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1 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. Ford 1, A.M. Page 1, W. Meyer-Ilse 2, J.T. Brown 2, J. Heck 2, A.D. Stead 1 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 X- ray microscopy. We have been imaging living Chlamydomonas cells using the X- ray 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

2 II T.W. Ford et al. 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 X- ray microscope XM-1 at the Advanced Light Source (ALS) at the Berkeley Laboratory

3 A Comparative Study of the Ultrastructure of Living Cells II [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 8.8x10 5 Gy) (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 8.8x10 5 Gy) (3) or 10 sec. (accumulated dose 7.53x10 6 Gy) (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 7.53x10 6 Gy 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.

4 II T.W. Ford et al. 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 8.6x10 5 Gy) (5), 4 sec. (accumulated dose 4.3x10 6 Gy) (6) or 0.5 sec. (accumulated dose 4.5x10 6 Gy) (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.

5 A Comparative Study of the Ultrastructure of Living Cells II Loss of myofibril contraction occurs at around 1-2x10 4 Gy [3],[4] whilst loss of membrane function occurs in hamster cells at Gy with morphological damage visible at 10 5 Gy [5]. Ultrastructural damage was visible in Chlorella cells at 1.5x x10 4 Gy [6]. Yeast cell death (LD 50 ) was reported after irradiation with 2x10 5 photons.µm -2 (approximately 2.5x10 4 Gy) [4]. The images of living Chlamydomonas cells reported here showed no detectable damage after exposure to 8.6x10 5 Gy whilst dosages in excess of 10 6 Gy 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. References 1 T.W. Ford, R.A. Cotton, A.M. Page, and A.D. Stead, in X-Ray Microscopy IV, 276 (Institute of Microelectronics Technology, Chernogolovka, Russia 1994). 2 W. Meyer-Ilse, H. Medecki, L. Jochum, E. Anderson, D.T. Attwood, C. Magowan, R. Balhorn, and M. Moronne, Synchrotron Rad. News 8, 29 (1995). 3 P.M. Bennett, G.F. Foster, C.J. Buckley and R.E. Burge, J. Microscop. 172, 109 (1993). 4 H. Fujisaki, S. Takahashi, H. Ohzeki, K. Sugisaki, H. Kondo, H. Nagata, H. Kato and S. Ishiwata, J.Microscop. 182, 79 (1996). 5 J. Kirz, C. Jacobsen and M. Howells, Quat. Rev. Biophys. 28, 33 (1995). 6 T.W. Ford, A.M. Page, G. Foster, and A.D. Stead, in Soft X-Ray Microscopy, Conference Volume 1741, 325 (1993). 7 G. Schneider, B. Niemann, P. Guttmann, D. Rudolph and G. Schmahl, Synchrotron Rad. News 8, 19 (1995).