Soft X-ray contact microscopy of biological materials

Light microscopy (LM) enables biological specimens to be examined without fixation or dehydration but the resolution is insufficient for studies of cell ultrastructure. Electron microscopy (EM) improves the resolution, but requires the specimen to be fixed or frozen, which may cause alterations in cell structure. Using soft X-rays to image specimens improves the resolution, relative to LM, and avoids tissue pretreatment. Staining is not required since within the ‘water window’ (2.3–4.4nm), carbon absorbs more strongly than oxygen. The lower attenuation of soft X-rays, relative to electrons, by biological material allows specimens several microns thick to be examined.

Several sources for generating water-window X-rays are briefly described and examples of images obtained with each are presented. The specimens imaged include both plant and animal material either in the fixed or natural state. Of the different systems currently used to collect images only contact imaging is considered in detail. By placing the specimen against photosensitive resist, which acts as the image recording medium, an absorption map of the specimen is produced. This latent image is then chemically developed and, after coating, the resist is examined by scanning EM, or, if a replica is produced, by transmission EM. Using laser-produced plasmas such images are produced within a very short exposure time, typically 1–10 nsec, thus avoiding any radiation-induced damage to the specimen which other X-ray imaging techniques may cause.