Accurate and fast mitotic detection using an anchor-free method based on full-scale connection with recurrent deep layer aggregation in 4D microscopy images.

BACKGROUND: To effectively detect and investigate various cell-related diseases, it is essential to understand cell behaviour. The ability to detection mitotic cells is a fundamental step in diagnosing cell-related diseases. Convolutional neural networks (CNNs) have been successfully applied to object detection tasks, however, when applied to mitotic cell detection, most existing methods generate high false-positive rates due to the complex characteristics that differentiate normal cells from mitotic cells. Cell size and orientation variations in each stage make detecting mitotic cells difficult in 2D approaches. Therefore, effective extraction of the spatial and temporal features from mitotic data is an important and challenging task. The computational time required for detection is another major concern for mitotic detection in 4D microscopic images. RESULTS: In this paper, we propose a backbone feature extraction network named full scale connected recurrent deep layer aggregation (RDLA++) for anchor-free mitotic detection. We utilize a 2.5D method that includes 3D spatial information extracted from several 2D images from neighbouring slices that form a multi-stream input. CONCLUSIONS: Our proposed technique addresses the scale variation problem and can efficiently extract spatial and temporal features from 4D microscopic images, resulting in improved detection accuracy and reduced computation time compared with those of other state-of-the-art methods.

Polyoxyethylene/polyoxypropylene dimethyl ether (EPDME) random copolymer improves lipid structural ordering in stratum corneum of an epidermal-equivalent model as seen by two-photon microscopy.

BACKGROUND/PURPOSE: Topical application of polyoxyethylene/polyoxypropylene dimethyl ether (EPDME) random copolymer improves the barrier function of skin, whereas polyethylene glycol (PEG) and polypropylene glycol (PPG) are ineffective. The aim of this work was to examine the interaction between these polymers and lipid molecules in the stratum corneum in order to establish whether EPDME-specific changes in the structural ordering of lipids might account for the improvement of barrier function. METHODS: We used two-photon microscopy to evaluate the effects of EPDME, PEG, and PPG on the structural ordering of lipids in an epidermal-equivalent model in terms of the fluorescence changes of Laurdan, a fluorescent dye that responds to changes of membrane fluidity. The generalized polarization (GP) value, a parameter that reflects lipid ordering, was measured at various depths from the surface of the stratum corneum. RESULTS: EPDME increased the GP value to a depth of about 3 µm from the surface, indicating that lipid ordering was increased in this region, while PEG and PPG of the same molecular weight had no effect. Diffusion of Lucifer yellow into the epidermis was reduced after application of EPDME, indicating that the barrier function was improved. CONCLUSION: These results support the view that EPDME improves barrier function by increasing the ordering of lipid structures in the stratum corneum. The methodology described here could be useful for screening new compounds that would improve the structural ordering of lipids.

A Cascade of 2.5D CNN and Bidirectional CLSTM Network for Mitotic Cell Detection in 4D Microscopy Image.

Mitosis detection is one of the challenging steps in biomedical imaging research, which can be used to observe the cell behavior. Most of the already existing methods that are applied in detecting mitosis usually contain many nonmitotic events (normal cell and background) in the result (false positives, FPs). In order to address such a problem, in this study, we propose to apply 2.5-dimensional (2.5D) networks called CasDetNet_CLSTM, which can accurately detect mitotic events in 4D microscopic images. This CasDetNet_CLSTM involves a 2.5D faster region-based convolutional neural network (Faster R-CNN) as the first network, and a convolutional long short-term memory (CLSTM) network as the second network. The first network is used to select candidate cells using the information from nearby slices, whereas the second network uses temporal information to eliminate FPs and refine the result of the first network. Our experiment shows that the precision and recall of our networks yield better results than those of other state-of-the-art methods.

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.

Modulation of lipid fluidity likely contributes to the fructose/xylitol-induced acceleration of epidermal permeability barrier recovery.

We previously showed that topical application of hexoses such as fructose accelerates barrier recovery after disruption. We also showed that various hexoses and polyols interact with phospholipid and alter the phase transition temperature. Thus, we hypothesized that the improvement of barrier recovery by hexoses and polyols might be related to the interaction with phospholipid. Here, we tested this idea by examining the effects of xylitol (a component of some skin-care products) and fructose on lipid dynamics in an epidermal-equivalent model at the single-cell level by means of two-photon microscopy after staining with Laurdan, a fluorescent dye sensitive to the physical properties of its membrane environment. First, we confirmed that topical application of xylitol aqueous solution on tape-stripped human skin accelerated barrier recovery. Then, we examined changes of lipid fluidity in the epidermal-equivalent model after application of water or an aqueous solution of xylitol or fructose. Application of xylitol and/or fructose increased the lipid fluidity in the uppermost part of the stratum granulosum layer, compared to treatment with water alone, and accelerated the exocytosis of lamellar bodies to the intercellular domain between stratum corneum and stratum granulosum. Our results support the idea that the improvement of epidermal barrier homeostasis upon topical application of xylitol or fructose is due to increased lipid fluidity in the uppermost layer of the stratum granulosum, which enables accelerated release of lipid from the stratum granulosum, thereby improving the lamellar structure and accelerating epidermal permeability barrier recovery.

Development of 3D imaging technique of reconstructed human epidermis with immortalized human epidermal cell line.

The epidermis, the outermost layer of the skin, retains moisture and functions as a physical barrier against the external environment. Epidermal cells are continuously replaced by turnover, and thus to understand in detail the dynamic cellular events in the epidermis, techniques to observe live tissues in 3D are required. Here, we established a live 3D imaging technique for epidermis models. We first obtained immortalized human epidermal cell lines which have a normal differentiation capacity and fluorescence-labelled cytoplasm or nuclei. The reconstituted 3D epidermis was prepared with these lines. Using this culture system, we were able to observe the structure of the reconstituted epidermis live in 3D, which was similar to an in vivo epidermis, and evaluate the effect of a skin irritant. This technique may be useful for dermatological science and drug development.

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

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.

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 ?2.4×. The device can provide a simple approach to acquiring higher-quality images of biospecimens.

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 25× 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.

Improvement of lateral resolution and extension of depth of field in two-photon microscopy by a higher-order radially polarized beam.

The spatial resolution of laser scanning microscopes depends on the focal spot size. As previously reported, we successfully improved the lateral spatial resolution in confocal microscopy using liquid crystal devices (LCDs) to convert a linearly polarized (LP) beam into a higher-order radially polarized (HRP) beam. Taking advantage of the fact that those LCDs can be utilized at various wavelengths, including near-infrared, we employed a near-infrared HRP beam to improve the resolution in two-photon microscopy. Point-spread functions estimated from fluorescent beads embedded in agarose gel showed that an HRP beam at 800-nm excitation improved lateral resolution to 230 nm from 294 nm, which was obtained using an LP beam at the same wavelength. Furthermore, at the glass-water interface, the lateral resolution was considerably improved to 188 nm using the HRP beam, whereas it degraded to 510 nm while using the LP beam. The HRP beams visualized fine intracellular structures not only in fixed cells stained with various dyes but also in living cells. Moreover, the HRP beam significantly extended the depth of field, which facilitated obtaining in-focus images, especially during time-lapse observations of living cells. These results indicate that our method is applicable to various biological applications.