Table-top combined scanning X-ray small angle scattering and transmission microscopies of lipid vesicles dispersed in free-standing gel

Abstract

A mm thick free-standing gel containing lipid vesicles made of 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC) was studied by scanning Small Angle X-ray Scattering (SAXS) and X-ray Transmission (XT) microscopies. Raster scanning relatively large volumes, besides reducing the risk of radiation damage, allows signal integration, improving the signal-to-noise ratio (SNR), as well as high statistical significance of the dataset. The persistence of lipid vesicles in gel was demonstrated, while mapping their spatial distribution and concentration gradients. Information about lipid aggregation and packing, as well as about gel density gradients, was obtained. A posteriori confirmation of lipid presence in well-defined sample areas was obtained by studying the dried sample, featuring clear Bragg peaks from stacked bilayers. The comparison between wet and dry samples allowed it to be proved that lipids do not significantly migrate within the gel even upon drying, whereas bilayer curvature is lost by removing water, resulting in lipids packed in ordered lamellae. Suitable algorithms were successfully employed for enhancing transmission microscopy sensitivity to low absorbing objects, and allowing full SAXS intensity normalization as a general approach. In particular, data reduction includes normalization of the SAXS intensity against the local sample thickness derived from absorption contrast maps. The proposed study was demonstrated by a room-sized instrumentation, although equipped with a high brilliance X-ray micro-source, and is expected to be applicable to a wide variety of organic, inorganic, and multicomponent systems, including biomaterials. The employed routines for data reduction and microscopy, including Gaussian filter for contrast enhancement of low absorbing objects and a region growing segmentation algorithm to exclude no-sample regions, have been implemented and made freely available through the updated in-house developed software SUNBIM.

Fig. 1. Transmission (a and e), relative thickness (b and f), raw SAXS (c and g) and normalized SAXS (d and h) intensity maps of the wet GEL and POPC samples, respectively. SAXS intensity of each pixel is integrated over the full accessible Q-range. In the case of raw SAXS maps (c and g), the intensity values in the colour bar are normalized to the mean value. In the case of the normalized SAXS maps (d and h), the absolute intensity values are reported and directly indicate the relative abundance of lipids. The dotted lines delimit the ROIs from which integrated SAXS profiles (in Fig. S1 and S2†) were extracted, whereas the (×) symbols indicate the points from which single SAXS profiles were extracted: the colour codes for GEL and POPC samples correspond to those in Fig. S1 and S2. Scale bar: 1 mm.

Fig. 2. Transmission (a and e), relative thickness (b and f), raw SAXS (c and g) and normalized SAXS (d and h) intensity maps of the dried GEL and POPC samples, respectively; t (e) and n (f) maps were obtained after row by row compensation (see ESI: Section D†) of background irregularities and Gaussian filtering application. SAXS intensity in each pixel is integrated over the full accessible Q-range. In the case of raw SAXS maps (c and g), the intensity values in the colour bar are normalized to the mean value. In the case of the normalized SAXS maps (d and h), the absolute intensity values are reported and directly indicate the relative abundance of lipids. The dotted lines delimit the ROIs from which integrated SAXS profiles (in Fig. S7†) were extracted. The colour codes for gel and POPC samples correspond to those in Fig. S7a. In the case of gel samples, ROIs were drawn on the as-collected SAXS microscopy, as features related to density inhomogeneities were levelled upon absorption/thickness correction. Scale bar: 1 mm.

Fig. 3. SAXS maps in different q-ranges for the wet POPC sample. From left to right (in both rows): 0.15 ÷ 0.20, 0.36 ÷ 0.90, 0.92 ÷ 1.09 nm⁻¹. Upper row: as collected data; lower row: reduced data; in the area delimited by the dotted line, corresponding to low POPC concentration, the contrast is visibly changing as a function of the chosen Q-range, compared to the surrounding regions. Scale bar: 1 mm.

Fig. 4. SAXS maps in different Q-ranges for the dried POPC sample. From left to right (in both rows): 0.15 ÷ 0.45, 0.45 ÷ 0.85, 1.02 ÷ 1.20 nm⁻¹. Upper row: as collected data; lower row: reduced data; in the areas delimited by dotted lines, the contrast is visibly changing as a function of the chosen Q-range. Scale bar: 1 mm.