SummarySoft X-ray contact microscopy (SXCM) is, at present, a useful tool for the examination at submicrometre resolution of biological systems maintained in their natural hydrated conditions. Among current X-ray-generating devices, laser-plasma sources are now easily available and, owing to their pulse nature, offer the opportunity to observe living biological samples before radiation damage occurs, even if the resolution achievable is not as high as with synchrotron-produced Xrays. To assess the potential of laser-plasma source SXCM in the study of cellular organelles, we applied it for the analysis of chloroplasts extracted from spinach leaves and mitochondria isolated from bovine heart and liver. X-ray radiation was generated by a nanosecond laser-plasma source, produced by a single shot excimer XeCl laser focused onto an yttrium target. The images obtained with SXCM were then compared with those produced by transmission electron microscopy observation of the same samples prepared with negative staining, a technique requiring no chemical fixation, in order to facilitate their interpretation and test the applicability of SXCM imaging.
IntroductionThe possibility of examining the structure of cells in their normal living state by an X-ray microscope has been pursued by biologists and physicists for many years. Soft X-ray contact microscopy (SXCM), which utilizes a radiation corresponding to the wavelengths between 2.3 and 4.4 nm, known as the water window region, allows untreated living biological specimens to be examined at optimal contrast. The water window lies between the oxygen and carbon k absorption edges, where the absorption of carbon is 10 times that of oxygen (and hence water).Several groups have developed microscopes working in the soft X-ray spectral region, using synchrotron or table-top (primarily laser-plasmas) X-ray sources. Current high-brightness synchrotron radiation sources, together with Fresnel Zone Plates microfocusing optics, allow high-resolution imaging of untreated biological samples (Kirz et al ., 1995;Attwood, 1999). However, synchrotron machines are not always readily accessible to investigators. Laser-plasma sources, by contrast, are more easily available and suited for the construction of more compact microscopes. Moreover, they have the advantage of generating very short X-ray pulses, thus reducing blurring resulting from brownian motion and the possibility of damage caused by radiation to biological structures. The main drawbacks of laser-plasma-based X-ray microscopy are poorer resolution and longer time needed to obtain images, as the contact technique usually employed requires chemical development and subsequent AFM imaging of the X-ray exposed resists.Whatever the X-ray source, soft X-ray microscopy can be considered a useful complementary technique to light and electron microscopy. The theoretical resolution of light microscopy is limited by the wavelength of light to about 200 nm, whereas transmission electron microscopy (TEM) offers subnanometre resolution; however, th...
