SummaryStructural investigation of tissue biopsies requires the coupling of optimal structural sample preservation with detailed information collected at the light and electron microscopic level. Unfortunately, although cryo-immobilization by highpressure freezing provides the best structural preservation, it is used routinely only for electron microscopic (EM) investigations, whereas for light microscopy chemical fixation protocols have been established. These chemically invasive fixation protocols have the drawback of introducing unpredictable fixation artefacts. Therefore, comparative histopathological (i.e. light microscopic) and ultrastructural (i.e. EM) results are usually obtained from parallel samples that have not been prepared identically and never by examining exactly the same features in exactly the same, optimally preserved sample. Finally, finding an area of interest for EM investigation within a complex tissue is like searching for a needle in a haystack. To overcome these handicaps, we modified the well-established freeze-substitution technique (FS) to allow us to investigate resin-embedded cryo-immobilized tissue by confocal laser scanning microscopy (CLSM) prior to EM examination. Thus (1) selected cells throughout the whole tissue block can be depicted by CLSM and subsequently (2) selectively prepared by targeted sectioning for follow-up investigation of the identical structure by transmission electron microscopy. This is facilitated by the addition of specific fluorescent dyes during the first FS exchange step. Selective binding properties of various dyes to different cellular structures allow a direct histological description of the tissue at the light microscope level. After embedding and preparation of a blockface, the specimen can first be examined by CLSM. For areas of interest, the depth in the resin block is determined followed by removal of the tissue lying above. Then, the cell layer can be cut into a series of ultrathin sections and examined by EM for determination of the subcellular and nanostructural organization.

Current transmission electron microscopy techniques do not permit simultaneous visualization of skin ultrastructure and stratum corneum extracellular lipids. We developed a new procedure, which entails application of high-pressure freezing followed by freeze-substitution with acetone containing uranyl acetate, followed by low temperature embedding in HM20. Electrospray ionization mass spectrometry showed that the amount of lipids lost during preparation was minimal. The ultrastructure of cryoprocessed skin was compared with that of conventionally prepared skin samples. Cryoprocessing, but not conventional processing, enabled visualization of lipid stacks within epidermal lamellar bodies, as well as the extracellular lipid domains of the stratum corneum and the ultrastructure within keratinocytes. Anti-filaggrin immunocytochemistry also showed, e.g., excellent preservation of filaggrin on cryoprocessed samples. Additionally, the cytosol of keratinocytes appeared to be organized in "microdomain"-like areas. Finally, the stratum corneum appeared more compact with smaller intercellular spaces and hence tighter cell-cell interactions, after cryoprocessing, than after conventional tissue preparation for transmission electron microscopy. We conclude here that only cryoprocessing preserves skin in a close to native state.

The stratum corneum (SC) requires ceramides, cholesterol, and fatty acids to provide the cutaneous permeability barrier. SC lipids can be analyzed by normal-phase high-performance thin-layer chromatography (HPTLC). However, without further analysis, some uncertainty remains about the molecular composition of lipids represented by every TLC band of an unknown sample. We therefore analyzed each ceramide band further by subjecting the isolated lipids to a direct coupling of reversed-phase high-performance liquid chromatography and electrospray ionization-mass spectrometry (HPLC/ESI-MS, or LC/MS). LC/MS analysis and ESI-MS/MS negative ion and collision-induced dissociation experiments revealed that ceramide band 4 contained not only N-(omega-OH-acyl)acyl-6-OH-sphingosine, Cer(EOH), but also N-(alpha-OH-acyl)-sphingosine. Band 5 exclusively contained N-acyl-6-OH-sphingosine. Our results demonstrate the benefit of LC/MS analysis for selective identification of human SC ceramides. Moreover, the combination of HPTLC for pre-separation and LC/MS for identification of lipids is an even more powerful tool for detailed ceramide analysis.

Topically applied water exerts mechanical stress on individual corneocytes as well as on the whole stratum corneum (SC), resulting in an alteration of barrier function. In this study we used complete skin biopsies and showed that the SC reacts to water stress as a highly optimized and well-regulated structure against osmotic changes. Following a relatively new cryo-processing protocol for cryo-SEM, it is possible to reliably maintain and investigate the hydrated state of the SC and individual corneocytes after treatment with solutions of different ionic strength. Treatment with distilled water results in swelling of SC cells together with formation of massive water inclusions between adjacent cell layers. Treatment with 5–20% NaCl reveals three different hydration zones within the SC: Corneocytes near the live-dead transition zone can swell to nearly double their thickness. The second zone is the most compact, as the corneocytes here show the smallest thickness variation with all treatments. Within the outermost zone, again a massive swelling and loosening of intracellular filament packing can be observed. We therefore conclude that the SC itself is subdivided into three functional zones with individual water penetration and binding potentials. Since the second zone remains nearly unaffected by water stress, we propose that this zone hosts the functional SC barrier.

Ceramides and glucosylceramides are pivotal molecules in multiple biologic processes such as apoptosis, signal transduction, and mitogenesis. In addition, ceramides are major structural components of the epidermal permeability barrier. The barrier ceramides derive mainly from the enzymatic hydrolysis of glucosylceramides. Recently, anti-ceramide and anti-glucosylceramide anti-sera have become available that react specifically with several epidermal ceramides and glucosylceramides, respectively. Here we demonstrate the detection of two epidermal covalently bound omega-hydroxy ceramides and one covalently bound omega-hydroxy glucosylceramide species by thin-layer chromatography immunostaining. Moreover, we show the ultrastructural distribution of ceramides and glucosylceramides in human epidermis by immunoelectron microscopy on cryoprocessed skin samples. In basal epidermal cells and dermal fibroblasts ceramide was found: (i) at the nuclear envelope; (ii) at the inner and outer mitochondrial membrane; (iii) at the Golgi apparatus and the endoplasmic reticulum; and (iv) at the plasma membrane. The labeling density was highest in mitochondria and at the inner nuclear membrane, suggesting an important role for ceramides at these sites. In the upper epidermis, ceramides were localized: (i) in lamellar bodies; (ii) in trans-Golgi network-like structures; (iii) at the cornified envelope; and (viii) within the intercellular space of the stratum corneum, which is in line with the known analytical data. Glucosylceramides were detected within lamellar bodies and in trans-Golgi network-like structures of the stratum granulosum. The localization of glucosylceramides at the cornified envelope of the first corneocyte layer provides further proof for the existence of covalently bound glucosylceramides in normal human epidermis.