High Spatial Resolution Soft X-ray Microscopy

INTRODUCTION

A new soft x-ray microscope (XM-1) with high spatial resolution has been constructed by the Center for X-ray Optics (Fig. 1). It uses bending magnet radiation from beamline 6.1 at the Advanced Light Source, and is used in a variety of projects and applications in the life and physical sciences [1]. Most of these projects are ongoing.

Figure 1: Schematic of the high resolution soft x-ray microscope XM-1, built and operated by the Center for X-ray Optics. Bending magnet radiation from the ALS is monochromatized and focused onto the sample with a condenser zone-plate lens [2]. X-rays transmitted through the sample are enlarged with an objective (micro-) zone plate lens and detected with an x-ray CCD camera system. Except for an air gap containing the sample of about 100 microns, the x-ray optics are in vacuum. A mutual indexing system is used to position and focus the sample with an external visible light microscope.

The instrument uses zone plate lenses and achieves a resolution of 43 nm, measured over 10% to 90% intensity with a knife edge test sample [3]. X-ray microscopy permits the imaging of relatively thick samples, up to 10 µm thick, in water. XM-1 has an easy to use interface, that utilizes visible light microscopy to precisely position and focus the specimen.

Figure 2: Malaria Parasite in a human red blood cell imaged with XM-1

BIOLOGICAL APPLICATIONS

In our most extensive study with C. Magowan (Life Sciences Division, LBNL) [4,5], the life cycle of malaria parasites (Plasmodium falciparum) in intact normal human red blood cells (Fig. 2) has been observed, using hundreds of images. Building on the knowledge about the parasites' normal development, abnormalities which occur in the parasite after protease inhibitor treatments or in erythrocytes deficient in membrane protein 4.1, have been investigated.

In a collaboration of T. Ford and A. Stead (Royal Holloway University of London, UK) structures in green alga (Chlamydomonas), uniquely visible with soft x-rays, have been analyzed in unfixed samples [6]. By using x-ray wavelengths near the L-absorption edges, we were able to detect trace amounts of Fe, Mg, and Co in this alga. Bacterial spores (Bacillus subtilis) were studied in collaboration with J. Judge (Unilever plc., UK). Structural differences between hydrated and dehydrated spores were observed and reported [7].

Figure 3: Transgenic sperm cell from mouse.

The uniformity of chromatin organization within the heads of sperm from several mammals was analyzed in collaboration with R. Balhorn (LLNL). Sperm chromatin is particularly well suited for imaging with x-rays. Since the DNA is packaged in a highly compacted state, x-ray images of the sperm heads show structural details that cannot be observed using other techniques. These images are providing new insight into the importance of the timely synthesis of protamine 1, one of the two nuclear proteins that package DNA in spermatids and sperm (Fig. 3).

Figure 4: Cryptosporidium. (a) sporozoite emerging from oocyst, (b) empty oocyst with residium.

Cryptosporidium is a parasite commonly found in lakes and rivers contaminated with animal waste and sewage. Occasionally municipal drinking water supplies may become contaminated. The parasite oocyst is about 4 to 6 microns in size and resistant to chlorination treatment used in public water systems. Recent outbreaks of Cryptosporidium infections in Las Vegas (1994) and Milwaukee (1993) caused about 140 deaths and approximately 400,000 cases of severe diarrhea and vomiting. Cryptosporidium is a public health concern because of its debilitating effects, and it can be fatal to immuno-compromised individuals. We recorded a first series of x-ray microscope images of Cryptosporidium prepared in the laboratory of C. Petersen (UCSF, San Francisco General Hospital). These images were made from formalin fixed, wet samples. The images show a sporozoite emerging from the oocyst (Fig. 4 a) and an empty oocyst with the associated residuum (Fig. 4 b).

Other biological projects include investigations of human chromosomes (D. Arndt-Jovin, T. Jovin, Max-Planck-Institute Göttingen, Germany); mammalian cells (J. Hearst, M. Albertie, Structural Biology Division, LBNL, and S. Lelievre, Life Sciences Division, LBNL); human fibroblast cells (S. Krauss, Life Sciences Division, LBNL); human lung tissue (J. Bastacky, Life Sciences Division, LBNL); two projects with rat neurons (S. Brubaker, Univ. Oregon, Eugene and G. DeStasio, Univ. Rome, Italy); and development of lanthanide based luminescent labels
(M. Moronne, Life Sciences Division, LBNL).

PHYSICAL SCIENCES APPLICATIONS

Figure 5: Silicate (SiO_x) spheres of 0.2 µm diameter in toluene. Images taken at 539 eV (left) and at 533 eV (right).

Soft x-ray microscopy is advantageous wherever high spatial resolution transmission images from samples several microns thick are required. If the sample is in a liquid environment, x-rays might be the only possible method. In addition to that, it may be possible to gain elemental or chemical information through spectromicroscopy, making x-ray transmission the method of choice in a variety of applications in materials sciences and environmental research.

Fig. 5 shows images of silicate (SiO_x) micro-spheres suspended in toluene (C₆H₅CH₃; liquid at room temperature) imaged at different wavelengths near the absorption edge of the oxygen K-shell. At a photon energy of 539 eV (2.30 nm) the absorption of oxygen is at a maximum, whereas at the slightly lower photon energy of 533 eV (2.33 nm) the absorption of oxygen is negligible. As only the silicate micro-spheres contain oxygen, they are clearly visible at 2.30 nm and almost invisible (actually slightly brighter than the medium) at 2.33 nm.

In a collaboration with G. Mitchell and E. Rightor (Dow Chemical) we started to use XM-1 for a number of industrial applications. The samples investigated included different types of structured polymers with sizes that take full advantage of our spatial resolution, and latex spheres, which were imaged wet.

CONCLUSION AND OUTLOOK

The high resolution soft x-ray microscope XM-1 has gained new insights in a large variety of applications.

We are currently developing a cryogenic sample holder to be used in our soft x-ray microscope. The holder will allow us to use longer exposure times and perform multiple view imaging without increasing visible sample damage. For multiple view imaging, which provides tomographic information, a rotation stage is planned. A new electron beam writing tool (Nanowriter) is being commissioned; it will provide us with higher resolution condenser and objective zone plate lenses.

REFERENCES

1. W. Meyer-Ilse, H. Medecki, J. T. Brown, J. Heck, E. Anderson, C. Magowan, A. Stead, T. Ford, R. Balhorn, C. Petersen, D. T. Attwood, in X-ray Microscopy and Spectromicroscopy, J. Thieme, G. Schmahl, E. Umbach, D. Rudolph, eds. (Springer, Heidelberg 1997, in press)
2. M. Hettwer and D. Rudolph, ibid.
3. J. M. Heck, W. Meyer-Ilse, D. T. Attwood, this volume.
4. C. Magowan, J.T. Brown, N. Mohandas, W. Meyer-Ilse, this volume.
5. C. Magowan, J. T. Brown, J. Heck, M. Narla, W. Meyer-Ilse, "Developmental Anomalies of Plasmodium falciparum Malaria Parasites in Abnormal Erythrocytes Imaged by Soft X-ray Microscopy," submitted
6. T. W. Ford, A. M. Page, W. Meyer-Ilse, A. D. Stead, in ref. 1.
7. A. D. Stead, J. T. Brown, J. Judge, W. Meyer-Ilse, D. Neely, A. M. Page, E. Wolfrum, T. W. Ford, ibid.

This work was supported by the U.S. Department of Energy, Office of Basic Energy Sciences, and the Office of Health and Environmental Research and the Laboratory Directed Research and Development Program of the E. O. Lawrence Berkeley National Laboratory.

Principal investigator: Werner Meyer-Ilse, Center for X-ray Optics, E. O. Lawrence Berkeley National Laboratory,
e-mail: W_MEYER-ILSE@LBL.GOV, Phone: (510) 486-6892.