Live imaging of alterations in cellular morphology and organelles during cornification using an epidermal equivalent model

The stratum corneum plays a crucial role in epidermal barrier function. Various changes occur in granular cells at the uppermost stratum granulosum during cornification. To understand the temporal details of this process, we visualized the cell shape and organelles of cornifying keratinocytes in a living human epidermal equivalent model. Three-dimensional time-lapse imaging with a two-photon microscope revealed that the granular cells did not simply flatten but first temporarily expanded in thickness just before flattening during cornification. Moreover, before expansion, intracellular vesicles abruptly stopped moving, and mitochondria were depolarized. When mitochondrial morphology and quantity were assessed, granular cells with fewer, mostly punctate mitochondria tended to transition to corneocytes. Several minutes after flattening, DNA leakage from the nucleus was visualized. We also observed extension of the cell-flattening time induced by the suppression of filaggrin expression. Overall, we successfully visualized the time-course of cornification, which describes temporal relationships between alterations in the transition from granular cells to corneocytes

A summarized schematic of the proliferation and migration of the epidermal basal cells, based on our results.

<p>(<b>A</b>) Schematic of parallel and oblique division. Parallel division generates two basal cells. Because the basal cell density is maintained, the number of parallel divisions is similar to the number of cells migrating to the suprabasal layer. Thus, after a parallel division occurs, a basal cell might gradually migrate into the suprabasal layer over at least 4 hours. On the other hand, oblique division generates one basal cell and another cell that is translocated into a suprabasal layer, without slow migration. Thus, oblique divisions might play an important role in maintaining the rapid upward stream of keratinocytes in thick epidermis. (<b>B</b>) A simple model of the maintenance of the thin and thick epidermis. The epidermis can be roughly considered as having three layers: a proliferative basal layer (layer a), a differentiated cell layer (layer b), and a cornified layer (layer c). Assuming that the increase and the decrease in cell numbers in each layer is approximately equal to the number of cell divisions (as described in the text), then the basal cell density, the number of cell divisions, and oblique division rate are increased in thick epidermis compared with thin epidermis. Thus, the upward stream of the keratinocytes in thick epidermis is more rapid than in thin epidermis.</p

Images of dorsal skin in living R26H2BEGFP hairless mice.

<p>(<b>A</b>) Low-magnification image of dorsal skin around the field-of-view of the images obtained by confocal microscopy at a 488 nm excitation wavelength. Scale bar = 1 mm. (<b>B</b>) A series of optically sectioned images using a two-photon microscope at a 900 nm excitation wavelength at the position indicated by the yellow rectangle in <b>A</b>. The depth from the surface of the skin is shown in the upper left of each image. The white asterisks indicate the hair follicles. Scale bar = 50 μm.</p

Transmissive liquid-crystal device for correcting primary coma aberration and astigmatism in biospecimen in two-photon excitation laser scanning microscopy

All aberrations produced inside a biospecimen can degrade the quality of a three-dimensional image in two-photon excitation laser scanning microscopy. Previously, we developed a transmissive liquid-crystal device to correct spherical aberrations that improved the image quality of a fixed-mouse-brain slice treated with an optical clearing reagent. In this study, we developed a transmissive device that corrects primary coma aberration and astigmatism. The motivation for this study is that asymmetric aberration can be induced by the shape of a biospecimen and/or by a complicated refractive-index distribution in a sample; this can considerably degrade optical performance even near the sample surface. The device's performance was evaluated by observing fluorescence beads. The device was inserted between the objective lens and microscope revolver and succeeded in improving the spatial resolution and fluorescence signal of a bead image that was originally degraded by asymmetric aberration. Finally, we implemented the device for observing a fixed whole mouse brain with a sloping surface shape and complicated internal refractive-index distribution. The correction with the device improved the spatial resolution and increased the fluorescence signal by similar to 2.4x. The device can provide a simple approach to acquiring higher-quality images of biospecimens

Our intravital imaging method using the newly established R26H2BEGFP hairless mice.

<p>(<b>A</b>) Photograph of a R26H2BEGFP hairless mouse and a WT littermate. The upper and lower panels show each mouse under ordinary white light and blue light. (<b>B</b>) A schematic showing the intravital imaging of the dorsal skin with an upright two-photon microscope. The inset in the red rectangle shows the region indicated by the red dashed rectangle in the upper image viewed from the side. The detailed procedure is provided in the Materials and Methods and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163199#pone.0163199.s002" target="_blank">S2 Fig</a>.</p

Three-Dimensional Analysis of Cell Division Orientation in Epidermal Basal Layer Using Intravital Two-Photon Microscopy

<div><p>Epidermal structures are different among body sites, and proliferative keratinocytes in the epidermis play an important role in the maintenance of the epidermal structures. In recent years, intravital skin imaging has been used in mammalian skin research for the investigation of cell behaviors, but most of these experiments were performed with rodent ears. Here, we established a non-invasive intravital imaging approach for dorsal, ear, hind paw, or tail skin using R26H2BEGFP hairless mice. Using four-dimensional (x, y, z, and time) imaging, we successfully visualized mitotic cell division in epidermal basal cells. A comparison of cell division orientation relative to the basement membrane in each body site revealed that most divisions in dorsal and ear epidermis occurred in parallel, whereas the cell divisions in hind paw and tail epidermis occurred both in parallel and oblique orientations. Based on the quantitative analysis of the four-dimensional images, we showed that the epidermal thickness correlated with the basal cell density and the rate of the oblique divisions.</p></div

Correcting spherical aberrations in a biospecimen using a transmissive liquid crystal device in two-photon excitation laser scanning microscopy

Two-photon excitation laser scanning microscopy has enabled the visualization of deep regions in a biospecimen. However, refractive-index mismatches in the optical path cause spherical aberrations that degrade spatial resolution and the fluorescence signal, especially during observation at deeper regions. Recently, we developed transmissive liquid-crystal devices for correcting spherical aberration without changing the basic design of the optical path in a conventional laser scanning microscope. In this study, the device was inserted in front of the objective lens and supplied with the appropriate voltage according to the observation depth. First, we evaluated the device by observing fluorescent beads in single-and two-photon excitation laser scanning microscopes. Using a 25x water-immersion objective lens with a numerical aperture of 1.1 and a sample with a refractive index of 1.38, the device recovered the spatial resolution and the fluorescence signal degraded within a depth of +/- 0.6 mm. Finally, we implemented the device for observation of a mouse brain slice in a two-photon excitation laser scanning microscope. An optical clearing reagent with a refractive index of 1.42 rendered the fixed mouse brain transparent. The device improved the spatial resolution and the yellow fluorescent protein signal within a depth of 0-0.54 mm