X-Ray Holography of Fast-Frozen Hydrated Biological Samples

Athough x-ray holography has a long history [11], it is only in recent times that short-wavelenth lasers and undulator x-ray sources have opened the possibility for competitive imaging performance. Experiments using the in-line (Gabor) geometry have been favored by most groups and have been carried out at Orsay [12], Brookhaven [9], Livermore [31], Tsukuba [34], Chilton [32], Osaka [27] and Grenoble [28] while the Fourier-transform geometry has been developed at Brookhaven [23]. Submicron resolution has been reported by the Orsay group [12] and by some of the present authors working at Brookhaven [10]. These holographic schemes all operated in or near the water window and were mostly conceived as technical developments toward imaging of natural biological material. Their motivations were mostly the practical simplicity of the hologram recording process, which is similar to contact imaging, and the fact that such recordings can be made to deliver both the amplitude and phase imgages. The chief disadvantages are that holography provides no excape from the large radiation dose that is required by all x-ray and electron imaging methods and that the in-line style of holography produces an inherent corruption to the desired image known as the twin image. We have discussed the question of how to exploit the potential of x-ray holography in light of these disadvantages in an earlier paper [18] and have concluded that part of the solution is to cool the sample to cryogenic temperatures. Under this condition, features as small as 5 nm remain intact at doses of up to 108 Gray which thus allows a tilt series containing scores of holographic images to be taken.