Hard x-ray phase contrast imaging of black lipid membranes

Abstract

We report hard x-ray phase contrast imaging of black lipid membranes, freely suspended over a micromachined aperture in an aqueous solution. Biomolecular and organic substances can thus be probed in hydrated environments by parallel beam propagation imaging, using coherent multi-kilo-electronvolt x-ray radiation. The width of the thinning film can be resolved from analysis of the intensity fringes in the Fresnel diffraction regime down to about 200 nm. The thinning process, in which solvent is expelled from the space in between two opposing monolayers, is monitored, and the domain walls between coexisting domains of swollen and thinned membrane patches are characterized.

X-Ray propagation imaging of a lipid bilayer in solution

“…The values of d, s and c 2 are given in the legend. The extracted values from profile 1 show an increase in the resolution below the minimum membrane thickness of 200 nm detectable in the previously reported parallel imaging geometry, 18 which now reaches down to values below 100 nm. In this case, membrane motion may dominate the profile broadening and yield larger s values than predicted by the GSM.…”

“…Since it is composed of symmetric monolayers, we can model the intensity profile in the simplest approach by a symmetric phase slit. 18 From the corresponding fit, a thickness of d ¼ 383.5 nm is obtained, showing that the monolayers are still separated by oil and have not fused yet. The fit result of 4 ¼ 0.593 reflects the stoichiometric composition (index of refraction) of the oil phase, and the path length of the X-rays through the membrane.…”

“…Note that compared to the parallel beam imaging, much smaller effective detector pixel sizes can be achieved, as well as smaller defocus values, along with much higher photon flux densities. 18 which now reaches down to values below 100 nm. In this case, membrane motion may dominate the profile broadening and yield larger s values than predicted by the GSM.…”

“…The image recording procedure is analogous to the previously reported parallel beam imaging experiments. 18 After flat (empty beam) and dark field correction of the raw images, the measured I(x,z) is then considered as the squared output of the 1D Fresnel operator acting on the one-dimensional optical transmission function, which for a pure phase object can be written as T(x) ¼ e i4(x) . T(x) reflects the structure of the membrane patch along its local normal axis in a plane directly behind the sample, see Fig.…”

“…), allowing for studies of functional transport across the bilayer at controlled compositional and environmental parameters, such as protein concentration, ionic strength, pH, and electrical field. In previous studies we have adapted this system to in situ X-ray structural studies, 17,18 and have shown that BLMs swollen with organic solvent can be imaged by means of hard X-ray Fresnel diffraction (propagation imaging), with phase contrast arising from free space propagation of a partially coherent, parallel X-ray beam traversing the sample. 19,20 To quantitatively monitor the BLM thickness and its changes during the thinning process of BLMs, the intensity fringes in the Fresnel diffraction image were recorded and analyzed down to about 200 nm thickness, where contrast and resolution were lost.…”