Evaluation of histochemical staining techniques for visualization of bacterial biofilm-associated extracellular matrix in skin

Biofilms play a significant role in histopathology and are complex structures consisting of bacterial cells embedded in an extracellular matrix that contains polysaccharides, proteins, and deoxyribonucleic acid (DNA). The biofilm matrix limits the effectiveness of topical antibiotic treatment in infected wounds and impedes wound healing and immune responses. The purpose of this study was to visualize the biofilm-associated extracellular matrix using standard histological techniques. The commercially available MatTek epidermal full-thickness skin tissue model (EFT-400) was injured and infected for 24 h with biofilm-forming Staphylococcus aureus. Tissue for paraffin sections was fixed in formalin, microwave-processed, and embedded in paraffin. Serial sections cut to 5 microns were stained with Periodic acid-Schiff reagent, Calcofluor, modified Congo Red/Ziehl carbol fuchsin stain and Feulgen reaction. Stained tissues were evaluated using light and fluorescent microscopy. A detailed analysis of the application of the different staining techniques in demonstration of biofilm-associated extracellular matrix revealed that both carbohydrates and DNA were present. Discussion of the value of each staining technique is presented.     

Isoantigens A, B, H in normal skin and tumors of the epidermal appendages.

Isoantigens A, B, H(O) in biopsy specimens of 26 normal skin and in 35 adnexal tumors were studied by the red cell adherence (RCA) test of Davidsohn. Isoantigens were detected in the stratum corneum, stratum granulosum, stratum spinosum, acrosyringium, keratogenous zone of hair follicle, eccrine duct, and eccrine gland. The apocrine gland and duct consistently had negative test results for isoantigens. Seventy percent of all tumors tested contained isoantigens and included syringoma, hidrocystoma, syringocystadenoma papilliferum, spiradenoma, eccrine poroma, dermal duct tumor, clear cell hidradenoma, and cylindroma. We interpreted the presence of isoantigens in adnexal tumors as being evidence of immunologic maturity with differentiation toward eccrine structures. The RCA test is a new, sensitive, immunologic technique that can complement enzyme histochemical stains and electron microscopy in the study of the histogenesis of adnexal skin tumors.     

3D-confocal structural analysis of bone marrow-derived renal tubular cells during renal ischemia/reperfusion injury

Bone marrow cells (BMC) have been shown to migrate into injured sites for parenchymal repair. However, the extent of BMC involvement is controversial. To determine whether and to what extent BMC contribute to renal parenchymal repair, we employed three-dimensional (3D) fluorescent confocal microscopy/video in renal warm and cold ischemia/reperfusion (I/R) injury using enhanced green fluorescent protein transgenic rats and their radiation chimeras. After induction of renal warm I/R injury in chimeras, BM-derived renal tubular cells were found in 2D microscopy as isolated single cells or clusters of 2-3 cells. Likewise, cold I/R injury resulted in host-derived tubular cells with frequencies approximately 0.2%. However, stringent confocal microscopic analysis and 3D image construction revealed that BM-derived tubules identified in 2D images were frequently artifacts of overlapping cells separately stained with different markers. The actual frequency in 3D analysis was approximately one-fourth of that seen in 2D analysis. 3D confocal imaging precisely detected BM-derived tubular epithelial cells and could be useful to study BMC contribution to tissue repair.     

In vivo confocal scanning laser microscopy: comparison of the reflectance and fluorescence mode by imaging human skin

Optical, noninvasive methods have become efficient in vivo tools in dermatological diagnosis and research. From these promising imaging techniques, only the confocal scanning laser microscopy (CSLM) provides visualization of subsurface skin structures with resolutions similar to those of light microscopy. Skin annexes, as well as cutaneous cells from different epidermal layers, can be distinguished excellently. Currently, two forms of application have been established in dermatological practice: the reflectance mode, predominantly in the clinical field, and the fluorescence mode in dermatological research. Differences in both methods exist in the preparative protocol, in maximum imaging depth and, particularly, in the gain of contrast extraction. The reflectance mode demonstrates naturally occurring tissue components, whereas the fluorescent CSLM achieves contrast by administering fluorescence dye, representing the dynamic distribution pattern of the dye's fluorescent emission. Therefore, the reflectance and fluorescent modes highlight various skin microstructures, providing dissimilar in vivo confocal images of the skin. This permits different predications and information on the state of the tissue. We report the advantages and disadvantages of both optical imaging modes. The comparison was drawn by scanning human skin in vivo. Representative images in varying depths were obtained and analyzed; preparation procedures are shown and discussed.     

High‐resolution three‐dimensional digital imaging of the human renal microcirculation: An aid to evaluating microvascular alterations in chronic kidney disease in humans

We have developed a new virtual microscopy method, with two‐ and three‐dimensional (2D, 3D) synchronization, that enables visualization of the human renal microvasculature. The method was used to evaluate 120–150 serially cut sections of paraffin‐embedded human renal tissue from nephrectomized samples. Virtual microscopy images of sections double‐immunostained with antibodies against CD34 (an endothelium marker) and smooth muscle actin (an arterial media marker) and stained with periodic acid‐Schiff were processed using digital imaging analysis software. Image registration was conducted to generate 3D displays with red–green–blue color segmentation. The reconstructed images of the microvasculature, including the interlobular arteries and the glomeruli, allowed visualization of 3D structures and direct glomerular connections. Synchronizing these 3D images with the corresponding 2D images revealed the relationships between arteriosclerotic lesions and downstream glomeruli. Thus, interlobular arteries with moderate intimal thickening and afferent arterioles with segmental hyalinosis/sclerosis, as seen on the 2D images, exhibited wall irregularities on the corresponding 3D images. However, these lesions were not directly influenced by lesions in downstream glomeruli, such as sclerotic lesions. Our virtual‐slide method based on 2D and 3D image synchronization provides a comprehensive view of the renal microcirculation and therefore novel insights into the pathogenesis of vascular‐associated renal diseases.     

Antibodies to intermediate filament proteins as molecular markers in clinical tumor pathology. Differentiation of carcinomas by their reaction with different cytokeratin antibodies

Antibodies to human and bovine epidermal prekeratin and antibodies to mouse liver cytokeratin component D (Mr 49 000) have been applied in indirect immunofluorescence microscopy on sections of human tumors of mammary gland and liver. In non-neoplastic mammary gland all epithelial cells were stained with these antibodies. In pre-invasive and invasive ductal and lobular carcinomas a cell population was observed which was not significantly stained with antibodies to epidermal prekeratin but did strongly react with antibodies to liver cytokeratin D. In the liver, the antibodies to epidermal prekeratin as well as those directed against liver cytokeratin D strongly decorated bile duct epithelia. In contrast, significant staining of the hepatocytes was only achieved with antibodies to liver cytokeratin D. This different staining reaction was maintained in liver tumors of hepatocellular and cholangiocellular origin. Antibodies to vimentin stained mesenchymal cells and tumors of mesenchymal derivation but reacted not significantly with any of the epithelial and carcinoma cells examined. The difference is of practical importance for the discrimination between anaplastic carcinomas and sarcomas of unknown origin. Cytokeratin could also be detected by antibody staining using the peroxidase-antiperoxidase (PAP) technique in formaldehyde-fixed and paraffin-embedded material of skin, gastrointestinal, respiratory, urinary and genital tract as well as various glands, liver and kidney. Examples of positive reactions were shown in a squamous cell carcinoma, a basalioma and a pleomorphic adenoma of the parotis. It is concluded that the immunohistochemical analysis of intermediate filament proteins has diagnostic potential in clinical pathology and may help to elucidate histogenesis and differentiation of tumors and possibly also prognosis of tumor growth. It is further suggested to use antibodies recognizing different subsets of proteins of the cytokeratin family in order to distinguish between different types of carcinomas.     

Three-dimensional imaging of xenograft tumors using optical computed and emission tomography

The physical basis and preliminary applications of optical computed tomography (optical-CT) and optical emission computed tomography (optical-ECT) are introduced, as new techniques with potential to provide unique 3D information on a variety of aspects of tumor structure and function. A particular focus here is imaging tumor micro-vasculature, and the spatial distribution of viable tumor cells, although the techniques have the potential for much wider application. The principle attractiveness of optical-CT and optical-ECT are that high resolution (<20 microm) and high contrast co-registered 3D images of structure and function can be acquired for relatively large intact samples. The unique combination of high contrast and resolution offers advantages over micro-CT and micro-MRI, and the lack of requirement for sectioning offers advantages over confocal microscopy, conventional microscopy, and histological sectioning techniques. Optical-CT/ECT are implemented using in-house custom apparatus and a commercial dissecting microscope capable of both transmission and fluorescence imaging. Basic studies to characterize imaging performance are presented. Negligible geometrical distortion and accurate reconstruction of relative attenuation coefficients was observed. Optical-CT and optical-ECT are investigated here by application to high resolution imaging of HCT116 xenograft tumors, about 1 cc in dimension, which were transfected with constitutive red fluorescent protein (RFP). Tumor microvasculature was stained in vivo by tail vein injection of either passive absorbing dyes or active fluorescent markers (FITC conjugated lectin). Prior to imaging, the tumors were removed (ex vivo) and optically cleared in a key process to make the samples amenable to light transmission. The cleared tumors were imaged in three modes (i) optical-CT to image the 3D distribution of microvasculature as indicated by absorbing dye, (ii) optical-ECT using the FITC excitation and emission filter set, to determine microvasculature as indicated by lectin-endothelial binding, and (iii) optical-ECT using the DSRed2 filter set to determine the 3D distribution of viable tumor as indicated by RFP emission. A clear correlation was observed between the independent vasculature imaging modes (i) and (ii) and postimaging histological sections, providing substantial validation of the optical-CT and optical-ECT techniques. Strong correlation was also observed between the RFP imaging of mode iii, and modes i and ii, supporting the intuitive conclusion that well-perfused regions contain significant viable tumor. In summary, optical-CT and optical-ECT, when combined with new optical clearing techniques, represent powerful new imaging modalities with potential for providing unique information on the structure and function of tumors.     

Three-dimensional reconstruction of medieval child mummy in Yangju, Korea, using multi-detector computed tomography

Recently reported studies on the medieval mummies in Korea have been regarded as an invaluable source for studying the physical characteristics of medieval Koreans. However, since the reported medieval mummies were re-buried by their descendants without any scientific investigations, the development of a brief, non-invasive investigation technique was desperately needed among the researchers in Korea. In this regard, we tried to apply high-quality multi-detector computed tomography (MDCT) with three-dimensional (3D) reconstructions and multi-planar reformat (MPR) to investigate Korean mummies.

In our study, after 1.25 or 2.5 mm thin slice axial images were taken, 3D reconstruction and MPR were performed to get more accurate information about internal organs. In this trial, we successfully showed high-quality images for the brain, muscles, bones, heart and liver. During various trials for getting selected organs, we could make the 3D reconstructed images of them. Since we could show that the current MDCT technique could be useful for obtaining high-quality 3D reconstructed images of the internal organs of Korean mummies, this technique will be used in forthcoming similar cases, which could not be investigated using invasive techniques.     

小鼠表皮干细胞分化潜能的体内植入研究

目的:将小鼠表皮干细胞(epidermal stem cells,ESCs)种植到可生物降解的载体内并移植到同基因受者小鼠皮下,研究其分化潜能。方法:在体外诱导ES细胞分化为ESC,经用Hoechst 33342荧光染色后,种植到含有胶原凝胶的聚乙醇酸(polyglycolic acid,PGA)小网内,作为细胞载体移植到与干细胞同基因的129/J小鼠皮下。结果:荧光显微镜观察发现ESC在移植物内至少能存活3周以上,并能分化形成毛囊样和腺样结构,形成与天然真皮相似的结构。在植入后10周内未观察到明显排斥或毒性反应。结论:ESC可作为种子细胞用于制备具有形成真皮附属结构潜能的皮肤替代物的实验研究。     

Fish cast NETs: neutrophil extracellular traps are released from fish neutrophils

Neutrophil extracellular traps (NETs), which are extracellular DNA structures released from neutrophils, are described and characterized for the first time in fish using fluorescent confocal microscopy. Confocal images of fish neutrophil suspensions stained with 6'-diamino-2-phenylindole, dihydrochloride DNA fluorescent stain (DAPI) revealed the presence of NETs which appeared as fibrous structures connecting several cells. Co-localization of NETs with neutrophil granular proteins and actin was investigated using specific antibodies and probes. Double staining of neutrophils with SYTOX green and DAPI revealed that SYTOX stain applied to living cells stained extracellular DNA, but not nuclei. NETs are actively released from stimulated living cells, associated with granular proteins, but not with cytoskeleton, and are not a product of nuclear degradation seen in late apoptotic stages. Additionally, a fluorometric microtiter plate assay to quantify the release of NETs was adopted for use with fish neutrophils, and the effect of stress on NETs release was studied. This assay detected the inhibition of DNA release during stress conditions. In summary, NETs were released from living fish kidney neutrophils upon stimulation, characterized using fluorescence DNA-binding dyes, specific antibodies and probes, and quantified using a microtiter plate fluorometric assay that can rapidly measure a large number of samples. Detection of NETs can be used as an additional assay to an existing battery of functional tests, and as a new research model to study the effects of stress, immunomodulators, and diseases.     

Three-dimensional volume rendering of the ankle based on magnetic resonance images enables the generation of images comparable to real anatomy

We have applied high-quality medical imaging techniques to study the structure of the human ankle. Direct volume rendering, using specific algorithms, transforms conventional two-dimensional (2D) magnetic resonance image (MRI) series into 3D volume datasets. This tool allows high-definition visualization of single or multiple structures for diagnostic, research, and teaching purposes. No other image reformatting technique so accurately highlights each anatomic relationship and preserves soft tissue definition. Here, we used this method to study the structure of the human ankle to analyze tendon–bone–muscle relationships. We compared ankle MRI and computerized tomography (CT) images from 17 healthy volunteers, aged 18–30 years (mean 23 years). An additional subject had a partial rupture of the Achilles tendon. The MRI images demonstrated superiority in overall quality of detail compared to the CT images. The MRI series accurately rendered soft tissue and bone in simultaneous image acquisition, whereas CT required several window-reformatting algorithms, with loss of image data quality. We obtained high-quality digital images of the human ankle that were sufficiently accurate for surgical and clinical intervention planning, as well as for teaching human anatomy. Our approach demonstrates that complex anatomical structures such as the ankle, which is rich in articular facets and ligaments, can be easily studied non-invasively using MRI data.     

New technical approaches for 3D morphological imaging and quantification of measurements

3D imaging is becoming more and more popular, as it allows us to identify interactions between structures in organs. Furthermore, it gives the possibility to quantify and size these structures. To allow 3D imaging, the tissue sample has to be transparent. This is usually achieved by using optical tissue clearing protocols. Although using optical tissue clearing often results in perfect 3D images, these protocols have some pitfalls, like long duration of sample preparation (up to several weeks), use of toxic substances, damage to antibody staining, fluorescent proteins or dyes, high refractive indices, and high costs of sample processing.Recently we described [Huang et al., Scientific Reports 9(1): 521 (2019)] a fast, safe, and inexpensive ethyl cinnamate (ECi) based optical tissue clearing protocol. Here, we present extensions of our protocol with respect to the deparaffinization of old paraffin-embedded samples allowing 3D imaging of the blocks. In addition, we learned to remove ECi from the samples allowing the use of routine immunolabeling protocols. Furthermore, we demonstrate new pictures of lungs after expansion microscopy and adaptation of already existing protocols. The aim of our work is, in summary, to describe the advances in these methodologies, focusing on the morphological imaging of kidneys and lungs.     

Epidermal Dendritic Cell Populations in the Flaky Skin Mutant Mouse

Flaky skin (gene symbol: fsn) is an autosomal recessive mouse mutation that causes pathologic changes in the skin yielding a papulosquamous disease resembling human psoriasis. Preliminary studies of epidermal sheets from foot pads of fsn/fsn mice stained for Ia⁺ Langerhans cells (LC) or Thy-1⁺ dendritic epidermal cells (Thy-1⁺ DEC) indicated a rise in LC numbers at the time of weaning, when the skin lesion becomes clinically evident. To further investigate this observation, epidermal sheets were obtained from the ear, dorsal skin, and foot pads from replicates of 6 female mice (both mutants and normal littermates) on weekly intervals from birth to 8 weeks of age. Dorsal skin epidermal thickness was quantitated by computer assisted image analysis and found to be significantly thickened from one week onward in the mutant mice. Using immunofluorescence microscopy, epidermal dendritic cell numbers were determined following staining with antibodies for the following markers: Ia, NLDC-145, and S-100 (for LC) or Thy 1.2 and asialc-GM1 (for Thy-1⁺ DEC). Use of all 5 markers to evaluate skin from 3 different locations yielded a subtle but significant increase in LC and Thy-1⁺ DEC in flaky skin mice. Of the three sites evaluated, the dorsal skin and ear epidermal sheets were most informative, which corresponded to the degree of pathological involvement. Mice doubly homozygous for fsn and for the severe combined immunodeficiency (scid) mutation developed the psoriasiform dermatitis. Bone marrow grafts from fsn/fsn homozygotes to homozygous scid/scid mice reproduce the skin lesion. These studies suggest that the psoriasiform dermatitis in the flaky skin mouse mutation is associated with abnormalities at the level of hematopoietic progenitor cells.     

Three-dimensional reconstruction of cytoskeletons in rat salivary glands using confocal fluorescence microscopy and volume-rendering computer graphics]

A new reconstruction technique for the demonstration of three-dimensional architectural details from biological fluorescent specimens was used to determine the precise spacial organization of F-actin in rat sublingual glands. F-actin was stained with NBD-phallacidin in thick (20-30 microns) frozen sections and observed with a confocal laser scanning microscope to obtain thin (-1 micron) optically sectioned images. A series of optical sections taken successively at different focal levels in steps of 1 micron was then reconstructed with volume-rendering computer graphics. The rendered images viewed from several angles (e.g. top, bottom, sides) clearly revealed the presence of F-actin fluorescence along the hexagonal framework of the junctional complex within the acini and in the cytoplasm of star-shaped myoepithelial cells encircling the acini. The detailed structures and the topographical relations between the junctional complex and myoepithelial cells were more impressively observed when the rendered images were displayed by motion picture. These images are free from any artifacts caused by the mechanical sectioning and revealed well the delicate and complicated profiles of cellular structures that previously have been almost impossible to demonstrate by conventional reconstruction techniques. We expect that the combination of confocal microscopy and volume rendering will permit the dynamic observation of previously unseen phenomena in three dimensions, such as the behavior of biologically active molecules in living cells.     

Imaging the vascular wall using confocal microscopy

Blood vessels are capable of structural changes in a dynamic process called 'vascular remodelling', which involves cell growth, death, phenotypic change and migration, as well as extracellular matrix synthesis and degradation. An integrated view of the interrelationships of the different elements of the arterial wall is made possible by fluorescence confocal microscopy which enables collection of serial optical sections of relatively thick specimens without the need to cut them as with conventional histology. With the aid of image analysis software, these serial sections can be further reconstructed to obtain 3-D images, where the structures of interest are localized and quantified. Confocal microscopy can be combined with pressure myography to obtain, simultaneously, information on vascular function and 3-D structure at near-to-physiological conditions. There are a vast number of fluorescent compounds useful for imaging vessel structure and function. Nuclear dyes allow the identification of the different types of vascular cells and the quantification of their number, shape and orientation. The speed of confocal image acquisition and processing makes it possible to scan entire intact arteries stained with fluorescent kits or antibodies to locate infrequent events such as cell apoptosis, proliferation or migration. Confocal microscopy is not only useful for imaging vascular wall structure, but also to visualize and quantify, by the intensity of fluorescence, the generation of vascular cell factors such as nitric oxide or superoxide anion. In conclusion, confocal microscopy and image analysis software provide insight into vascular wall structure and function and the active process of vascular remodelling in physiological and pathological situations.     

Expression of class II HLA antigens (HLA-DR, DQW1, DQW3) in normal skin and cutaneous pathology

We report the cutaneous expression of class II HLA antigens disclosed by immunohistochemical staining of normal and lesional skin with monoclonal antibodies (MoAbs) reacting with HLA-DR, DQW1 and DQW3 antigens. Briefly, we disclosed: on normal human skin: 1) The Langerhans cells (LC) HLA-DR+, DQW1+; 2) the acrosyringium HLA-DR+, DQW1+, DQW3+; 3) the dermal vessel endothelial cells HLA-DR+. On lesional skin: 1) The LC were found HLA-DQW3+ in the lesional skin of some cutaneous diseases; this expression was never shown on LC of normal human skin; 2) the epidermal keratinocytes disclosed an uniform membrane expression of HLA-DR antigens in some cutaneous diseases; this kind of expression was not found by immunostaining with MoAbs directed against HLA-DQ antigens; 3) in psoriatic lesions some keratinocytes disclosed an heterogeneous expression of HLA-DR, DQW1 and DQW3 antigens; 4) tumoral cells from cutaneous malignant melanomas were shown to be HLA-DR+, DQW1+, DQW3+. The HLA-DQW3 expression on the LC of lesional skin is in favour with a modulation of HLA-DQW3 expression by unknown factors present in pathological skin. The HLA-DR expression on epidermal keratinocytes suggests a functional collaboration of keratinocytes with LC in the genetic restriction of cutaneous immune reactions.     

In Vitro Models Mimicking Immune Response in the Skin

The skin is the first line of defense of our body, and it is composed of the epidermis and dermis with diverse immune cells. Various in vitro models have been investigated to recapitulate the immunological functions of the skin and to model inflammatory skin diseases. The simplest model is a two-dimensional (2D) co-culture system, which helps understand the direct and indirect cell-to-cell interactions between immune and structural cells; however, it has limitations when observing three-dimensional (3D) interactions or reproducing skin barriers. Conversely, 3D skin constructs can mimic the human skin characteristics in terms of epidermal and dermal structures, barrier functions, cell migration, and cell-to-cell interaction in the 3D space. Recently, as the importance of neuro-immune-cutaneous interactions in the inflammatory response is emerging, 3D skin constructs containing both immune cells and neurons are being developed. A microfluidic culture device called “skin-on-a-chip,” which simulates the structures and functions of the human skin with perfusion, was also developed to mimic immune cell migration through the vascular system. This review summarizes the in vitro skin models with immune components, focusing on two highly prevalent chronic inflammatory skin diseases: atopic dermatitis and psoriasis. The development of these models will be valuable in studying the pathophysiology of skin diseases and evaluating the efficacy and toxicity of new drugs.     

New methodology for assessment of the Langerhans cell network

A new procedure is described for staining Langerhans cells (LCs) based on the ability of anti-S-100 antibody to stain both epidermal LCs and melanocytes, while L-Dopa stains only melanocytes. This procedure can be used on paraffin-embedded skin sections and is therefore advantageous for examination of pathological skin specimens. In order to determine how best to quantitate LCs in skin sections the distribution of LCs has been investigated using an improved method for preparation of epidermal sheets from mouse skin. Epidermal LCs stained for their surface membrane-bound enzyme adenosine triphosphatase were observed to link with each other via their dendrites, forming a single cell layer which undulates throughout the epidermis. It is therefore proposed that LCs in skin sections should be enumerated per unit length, after identification in paraffin-embedded sections double stained with anti-S-100 antibody and L-Dopa.     

Characterization of powdered epidermal vaccine delivery with multiphoton microscopy

Multiphoton laser scanning microscopy (MPLSM) has been adapted to non-invasively characterize hand-held powdered epidermal vaccine delivery technology. A near infrared femtosecond pulsed laser, wavelength at approximately 920 nm, was used to evoke autofluorescence of endogenous fluorophores within ex vivo porcine and human skin. Consequently, sub cellular resolution three-dimensional images of stratum corneum and viable epidermal cells were acquired and utilized to observe the morphological deformation of these cells as a result of micro-particle penetration. Furthermore, the distributional pattern of micro-particles within the specific skin target volume was quantified by measuring the penetration depth as revealed by serial optical sections in the axial plane obtained with MPLSM. Additionally, endogenous fluorescence contrast images acquired at the supra-basal layer reveal cellular structures that may pertain to dendritic Langerhans cells of the epidermis. These results show that MPLSM has advantages over conventional histological approaches, since three-dimensional functional images with sub-cellular spatial resolution to depths beyond the epidermis can be acquired non-invasively. Accordingly, we propose that MPLSM is ideal for investigations of powdered epidermal vaccine delivery.