Computed tomography of cryogenic biological specimens based on X-ray microscopic images

Soft X-ray microscopy employs the photoelectric absorption contrast between water and protein in the 2.34-4.38 nm wavelength region to visualize prote in structures down to 30 nm size without any staining methods. Due to the l arge depth of focus of the Fresnel zone plates used as X-ray objectives, co mputed tomography based on the X-ray microscopic images can be used to reco nstruct the local linear absorption coefficient inside the three-dimensiona l specimen volume. High-resolution X-ray images require a high specimen rad iation dose, and a series of images taken at different viewing angles is ne eded for computed tomography. Therefore, cryo microscopy is necessary to pr eserve the structural integrity of hydrated biological specimens during ima ge acquisition. The cryo transmission X-ray microscope at the electron stor age ring BESSY I (Berlin) was used to obtain a tilt series of images of the frozen-hydrated green alga Chlamydomonas reinhardtii. The living specimens were inserted into borosilicate glass capillaries and, in this first exper iment. rapidly cooled by plunging into liquid nitrogen. The capillary speci men holders allow image acquisition over the full angular range of 180 degr ees. The reconstruction shows for the first time details down to 60 nm size inside a frozen-hydrated biological specimen and conveys a clear impressio n of the internal structures. This technique is expected to be applicable t o a wide range of biological specimens, such as the cell nucleus. It offers the possibility of imaging the three-dimensional structure of hydrated bio logical specimens close to their natural living state. (C) 2000 Elsevier Sc ience B.V. All rights reserved.