Soft X-ray microscopes can be used to examine whole, hydrated cells up to 10 microm thick and produce images approaching 30 nm resolution. Since cells are imaged in the X-ray transmissive "water window", where organic material absorbs approximately an order of magnitude more strongly than water, chemical contrast enhancement agents are not required to view the distribution of cellular structures. Although living specimens cannot be examined, cells can be rapidly frozen at a precise moment in time and examined in a cryostage, revealing information that most closely approximates that in live cells. In this study, we used a transmission X-ray microscope at photon energies just below the oxygen edge (lambda = 2.4 nm) to examine rapidly frozen mouse 3T3 cells and obtained excellent cellular morphology at better than 50 nm lateral resolution. These specimens are extremely stable, enabling multiple exposures with virtually no detectable damage to cell structures. We also show that silver-enhanced, immunogold labelling can be used to localize both cytoplasmic and nuclear proteins in whole, hydrated mammary epithelial cells at better than 50 nm resolution. The future use of X-ray tomography, along with improved zone plate lenses, will enable collection of better resolution (approaching 30 nm), three-dimensional information on the distribution of proteins in cells.
SummaryThe soft X-ray microscope at the Lawrence Berkeley National Laboratory was developed for visualization of biological tissue. Soft X-ray microscopy provides high-resolution visualization of hydrated, non-embedded and non-sectioned cells and is thus potentially an alternative to transmission electron microscopy. Here we show for the first time soft X-ray micrographs of structures isolated from the guinea-pig inner ear. Sensory outer hair cells and supporting pillar cells are readily visualized. In the hair cells, individual stereocilia can easily be identified within the apical hair bundle. The underlying cuticular plate is, however, too densely composed or too thick to be clearly visualized, and thus appears very dark. The cytoplasmic structures protruding from the cuticular plates as well as the fibrillar material surrounding and projecting from the cell nuclei can be seen. In the pillar cells the images reveal individual microtubule bundles. Soft X-ray images of the acellular tectorial membrane and thin two-layered Reissner's membrane display a level of resolution comparable to low-power electron microscopy. IntroductionTo investigate the true structural and functional properties of cells, it is essential to study them under as intact and physiological conditions as possible. Each experimental method, however, reflects a combination of advantages and disadvantages. For example, light microscopy allows studying living cells but with a limited spatial resolution. In addition, for high spatial resolution imaging of thin objects, the contrast for biological samples is quite poor and contrast enhancement techniques, e.g. fluorophore staining and phase contrast microscopy, are frequently used. Transmission electron microscopy, by contrast, offers much greater spatial resolution but is primarily used for fixed, dehydrated, embedded and, finally, sectioned tissue. In addition, the processing efforts required prior to visualization are laborious and time consuming. Clearly, a microscope technique combining the advantages of light and electron microscopes would be very powerful.The soft X-ray microscope offers a highly interesting method for imaging intact hydrated cells, with high natural absorption contrast and with a spatial resolution beyond what is achievable with visible light microscopy (Schmahl et al ., 1993;Kirz et al ., 1995;Meyer-Ilse et al ., 1995; see also review by Yamamoto & Shinohara, 2002). The reason is that in the soft X-ray region the interaction between matter and electromagnetic radiation is particularly strong and highly wavelength dependent (Attwood, 1999). By a slight change in wavelength, the photon absorption can change by several orders of magnitude and, for a selected wavelength, one material can be almost transparent and another material highly absorbent. Its resolution falls between that of light microscopy and high-resolution electron microscopy, and is presently limited by the available diffractive X-ray optics to 20 nm. Exposure times in the soft X-ray microscope are usually a fe...
Construction of the XM-1 microscope, located on beamline 6.1.2 of the Advanced Light Source Facility at the E. O. L Berkeley National Laboratory, has enabled the application of soft X-ray microscopy for examination of biological specimens by providing a high thrughput of high-spatial resolution images from hydrated samples up to 10 µm thick. Previous biological applications of soft X-ray microscopy included examination of a variety of cells and unicellular organisms, including intracellular malaria parasites [1,2,3] and tracing intracellular distribution of proteins and nucleic acids [4]. To determine whether this technique could have wider application in life sciences and allow morphological studies of multicellular organisms and fragile protozoa, we have examined by soft X-ray microscopy glutaraldehyde-fixed newborn larvae of Trichinella spiralis, microfilariae of Dirofilaria immitis, trophozoites of Giardia lamblia and oocysts of Cryptosporidium parvum. Radiation damage to the specimens was minimal. In the vacuolated cytoplasm of G. lamblia trophozoite, the following internal structures are readily discernible: the two nuclei, each with its nucleolus, median bodies, located almost at right angle to the axostyle that originates in the central area anterior to the nuclei, and the anterior and posterior flagella (Fig. 1). Examination of multicellular organisms revealed aspects of their internal organization and arrangement of organ precursors that are extremely difficult to obtain by other microscopy techniques. New information obtained about the structure of T. spiralis larvae (Fig. 2) indicated that the larva has attained a remarkable degree of development and differentiation that was hitherto not suspected; the relationship between the cellular components, including the Excretory and the R1-Anal Vesicle complexes, was elucidated in the microfilaria; and the development of sporozoites within the oocysts of C. parvum (Figs. 3,4) could be discerned. These studies have demonstrated that the unique capabilities of soft X-ray microscopy can be successfully applied to examine the structure of protozoa and small, multicellular organisms. Soft X-ray microscopy can provide new information unattainable by other microscopy techniques, and it is also especially useful to correlate and integrate results obtained by scanning and transmission electron microscopy.
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