Laboratory Soft X-Ray Microscopy
An Application in Life Science
- Laboratory Soft X-Ray Microscopy - An Application in Life Science
- Fig. 1: Schematic representation of the experimental arrangement for the LTXM.
- Fig. 2: LTXM-measurements of 500nm resin embedded human skin slices. a) stratum corneum with intercellular lipids (L) and corneocytes (CC); b) overview of the epidermis‘ layered structure (with Richardson‘s staining) imaged with an optical microscope (courtesy of F. Rancan et al.); c) keratinocyte in the epidermis.
- Fig. 3: X-ray microscopy image of a plunge frozen cultivated primary keratinocyte.
Soft X-ray microscopy in the water window has the advantage of a high spatial resolution as well as a good contrast and high penetration depth within biological samples. This makes it an ideal tool for the structural analysis of living cells without extensive sample preparation. The laboratory transmission X-ray microscope (LTXM) at the Berlin Laboratory for innovative X-ray Technologies (BLiX) has various fields of applications, one of which is the investigation of the penetration of nanoparticles in human skin.
The Benefits of Soft X-Rays
X-ray microscopy (XRM) is a useful method to obtain a structural analysis of (biological) samples that are smaller than the wavelength of visible light. Whereas super-resolution microscopy methods, such as stimulated depletion microscopy (STED) [1, 2], only provide a functional analysis, the small wavelength of X-rays can be used to obtain structural information. Resolutions in the region of 10 nm can be achieved , filling the resolution gap between transmission electron microscopy (resolutions of approximately 1Å) and conventional confocal microscopy (diffraction limited to about 200 nm) .
The so-called water window between 284 eV (absorption edge of oxygen) and 543 eV (carbon) presents an additional advantage for XRM applications in biology, medicine, material and environmental sciences. In this spectral region, the absorption coefficient of oxygen, and thus water, is particularly low whereas the one of carbon is more than one order of magnitude higher. As a consequence, this causes a high natural contrast between water and carbon-based structures such as proteins or a cell nucleus. The low absorption of water also provides a high penetration depth of X-rays in aqueous samples rendering 3D-analysis of biological samples possible without invasive sample preparation, e.g. embedding, slicing and milling, as known from electron microscopy .
Soft X-ray sources with a sufficient brightness for imaging are mostly provided by synchrotrons. However, beamtimes at synchrotron facilities are limited and need to be applied for in advance.
The transfer of XRM to a laboratory therefore increases the user‘s flexibility, which can be crucial for the quality of sample preparation.
In this article we report on a laboratory transmission soft X-ray microscope (LTXM), which is operated at BLiX (Berlin Laboratory for innovative X-ray Technologies). Advantages of this method will be demonstrated by visualizing the pe-netration of nanoparticles in human skin.
The LTXM employs a highly brilliant laser-produced plasma source. Figure 1 shows a schematic illustration of the microscope‘s components. A high average power (up to 140 W) pulsed laser (1.3 kHz repetition rate, 0.5 ns pulse duration) is focused onto a liquid nitrogen jet (2) creating a plasma which emits clusters, ions and electrons as well as photons in the soft X-ray regime [6, 7]. A multilayer condenser mirror (1), which only reflects photons with an energy of 500 eV, focuses the X-rays onto the sample (5). A central stop (3) avoids direct illumination of the sample by plasma radiation and an aluminum filter (4) protects the sample from stray light and debris. The photons that cross the sample are finally focused by a zone plate objective (6) into the image plane, where an X-ray-sensitive CCD-camera is placed (7). Because of the small attenuation length of X-ray photons, the experiment needs to be conducted in vacuum.
Application - Do Nanoparticles Traverse Human Skin?
Nanoparticles are compounds of a few thousand atoms or molecules with sizes between 1 and 100 nm. Their increased use in everyday products like deodorants, sunscreens or textiles raises questions about the effect of nanoparticles on living organisms. The outermost layer of the skin (epidermis) represents the first protection of the human body against exterior influences. However, due to their small size, it is uncertain how well nanoparticles are retained by this natural shield. In cooperation with the Center of Experimental and Applied Cutaneous Physiology (Department of Dermatology, Charité-Universitätsmedizin Berlin), measurements were carried out in order to explore two distinct questions. Firstly, embedded skin slices were investigated to elucidate how far nanoparticles can traverse the skin through its different layers. In a second series of experiments, we investigated whether or not the skin cells (primary keratinocytes) and in particular the cell‘s nucleus are permeable by nanoparticles, once they have attained the lower epidermal layers.
For the first sample system, commercially available gold colloids with a diameter of (70 ± 4) nm were topically applied on human skin samples. The sectioning procedure was carried out with an ultramicrotome, for which the samples were dehydrated and embedded in epoxy resin. The resulting sections of 350 nm and 500 nm were put onto nickel grids with a diameter of 3 mm . Figure 2 shows two LTXM micrographs and their respective positions within the skin. The imaged areas are part of the epidermis‘ stratum corneum (SC, top), which represents the outer skin layer, and the stratum spinosum (SSp, bottom), a lower epidermal layer. Although the contrast seems to be deteriorated due to the previous dehydration and embedding of the sample, the measurements can resolve characteristic details of the respective layers, such as corneocytes (CC) embedded in the hydrophobic lipid (L) matrix in the SC. In the bottom image from figure 2, roundly shaped keratinocytes (KC) are recognizable. Unfortunately, nanoparticles were detected neither in the skin layers, nor on the skin surface for this set of samples. A possible explanation for their absence is that they might have fallen off the skin section during the preparation process and during the slicing in particular. Nevertheless, the investigation can be seen as a successful proof of principle experiment, since the different layers as well as characteristic elements of these layers were clearly resolved. The results confirm previous investigations at the Swiss Light Sources (SLS) . For the second set of samples, 70 nm silver nanoparticles were directly applied onto cultivated primary keratinocytes (PK) that were grown on carbon film coated gold grids. In order to prevent radiation damage, the grids containing the samples were plunge frozen and then transferred to the LTXM. Allegedly, figure 3 shows a nucleus of a PK surrounded by accumulations of 70 nm nanoparticles. The results of these measurements show that the nanoparticles as well as the cell structures can be resolved by the LTXM. Despite of the crack in the ice film (wavelike structure that covers the whole image), it can be considered that the plunge freezing procedure has been successfully performed. Unfortunately, no cell boundaries (possibly outside the small field of view of the LTXM; 20 µm), which might have indicated whether or not the nanoparticles have penetrated into the cell, are visible on this image.
Another limiting aspect for determining the nanoparticles‘ position, is that one 2D projection is not sufficient to localize any element in space. Therefore, the ambiguity if the nanoparticles are inside, below or above a specific structure (in the case of figure 3 the structure would be the cell‘s outer membrane or nuclear membrane) could be solved by imaging the sample from different angles.
Conclusion and Outlook
The investigation of the penetration and the localization of nanoparticles in human skin have shown the benefits of soft X-ray microscopy for the structural analysis of biological samples. Three-dimensionally resolved measurements (tomography) at resolutions in the sub 50-nanometer regime, allowing a more precise localization of nanoparticles in cell compartments are in progress. In addition to that, the recent integration of a visible light microscope (VLM) into the setup will facilitate the work flow and ease sample positioning. The larger field of view of the VLM compared to the LTXM‘s provides an overview of the sample and allows to quickly locate the area of interest, while simultaneously paving the way for correlative methods such as fluorescence microscopy, which would add functional information to the structural data gained from the LTXM's micrographs.
This article was written in closed collaboration with M. Meinke, F. Rancan and S. Ahlberg from the Department of Dermatology, Charité Medical University Berlin, H. Stiel (BLiX, Max-Born-Institute) and B. Kanngießer (BLiX, Technical University Berlin). The realization of this project was supported by Bruker ASC GmbH.
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Dr. Christian Seim
Berlin Laboratory for innovative X-ray Technologies (BLiX)
Technische Universität Berlin / Max-Born-Institut