Nanoscale Structural Comparison of Fibrillin-1 Microfibrils Isolated from Marfan and Non-Marfan Syndrome Human Aorta.

Fibrillin-1 microfibrils are essential elements of the extracellular matrix serving as a scaffold for the deposition of elastin and endowing connective tissues with tensile strength and elasticity. Mutations in the fibrillin-1 gene (FBN1) are linked to Marfan syndrome (MFS), a systemic connective tissue disorder that, besides other heterogeneous symptoms, usually manifests in life-threatening aortic complications. The aortic involvement may be explained by a dysregulation of microfibrillar function and, conceivably, alterations in the microfibrils' supramolecular structure. Here, we present a nanoscale structural characterization of fibrillin-1 microfibrils isolated from two human aortic samples with different FBN1 gene mutations by using atomic force microscopy, and their comparison with microfibrillar assemblies purified from four non-MFS human aortic samples. Fibrillin-1 microfibrils displayed a characteristic "beads-on-a-string" appearance. The microfibrillar assemblies were investigated for bead geometry (height, length, and width), interbead region height, and periodicity. MFS fibrillin-1 microfibrils had a slightly higher mean bead height, but the bead length and width, as well as the interbead height, were significantly smaller in the MFS group. The mean periodicity varied around 50-52 nm among samples. The data suggest an overall thinner and presumably more frail structure for the MFS fibrillin-1 microfibrils, which may play a role in the development of MFS-related aortic symptomatology.

Effect of Air-Polishing and Different Post-Polishing Methods on Surface Roughness of Nanofill and Microhybrid Resin Composites.

Air-abrasion is a popular prophylactic procedure to maintain oral hygiene. However, depending on the applied air-abrasive powder, it can damage the surface of the tooth and restorations, making it susceptible to plaque accumulation. The purpose of this study was to investigate the effect of 5 s and 10 s air-abrasion of calcium carbonate on surface roughness (Ra) of enamel, nanofill, and microhybrid resin-composites and the effect of post-polishing with two-step rubber- (RP) or one-step brush polisher (BP) to re-establish the surface smoothness. Surface topography was visualized by scanning-electron-microscopy. The quantitative measurement of the Ra was carried out with atomic-force-microscopy. Air-abrasion for 10 s decreased the Ra of enamel as a result of abrasion of the natural surface texture. Post-polishing with RP after 10 s air-abrasion did not change the Ra or BP; however, Ra was increased significantly by scratching the surface. Air-abrasion increased the Ra of resin composites significantly, irrespective of the application time. While RP provided a similarly smooth surface to the control in the case of microhybrid resin composite, BP increased the Ra significantly. The Ra for the control group of the nanofill-resin composite was initially high, which was further increased by air-abrasion. RP and BP re-established the initial Ra with deeper and shallower scratches after BP. Both the material and treatment type showed a large effect on Ra.

Quantitative Analysis on Ex Vivo Nonlinear Microscopy Images of Basal Cell Carcinoma Samples in Comparison to Healthy Skin.

Basal cell carcinoma (BCC) is the most frequent malignant neoplasm in the Caucasian population. There are several therapeutic options for BCC, but surgical excision is considered gold standard treatment. As BCCs often have poorly defined borders, the clinical assessment of the tumor margins can be challenging. Therefore, there is an increasing demand for efficient in vivo imaging techniques for the evaluation of tumor borders prior to and during surgeries. In the near future, nonlinear microscopy techniques might meet this demand. We measured the two-photon excitation fluorescence (TPEF) signal of nicotinamide adenine dinucleotide hydride (NADH) and elastin and second harmonic generation (SHG) signal of collagen on 10 ex vivo healthy control and BCC skin samples and compared the images by different quantitative image analysis methods. These included integrated optical density (IOD) measurements on TPEF and SHG images and application of fast Fourier transform (FFT), CT-FIRE and CurveAlign algorithms on SHG images to evaluate the collagen structure. In the BCC samples, we found significantly lower IOD of both the TPEF and SHG signals and higher collagen orientation index utilizing FFT. CT-FIRE algorithm revealed increased collagen fiber length and decreased fiber angle while CurveAlign detected higher fiber alignment of collagen fibers in BCC. These results are in line with previous findings which describe pronounced changes in the collagen structure of BCC. In the future, these novel image analysis methods could be integrated in handheld nonlinear microscope systems, for sensitive and specific identification of BCC.

Ex vivo nonlinear microscopy imaging of Ehlers-Danlos syndrome-affected skin.

Ehlers-Danlos syndrome (EDS) is the name for a heterogenous group of rare genetic connective tissue disorders with an overall incidence of 1 in 5000. The histological characteristics of EDS have been previously described in detail in the late 1970s and early 1980s. Since that time, the classification of EDS has undergone significant changes, yet the description of the histological features of collagen morphology in different EDS subtypes has endured the test of time. Nonlinear microscopy techniques can be utilized for non-invasive in vivo label-free imaging of the skin. Among these techniques, two-photon absorption fluorescence (TPF) microscopy can visualize endogenous fluorophores, such as elastin, while the morphology of collagen fibers can be assessed by second-harmonic generation (SHG) microscopy. In our present work, we performed TPF and SHG microscopy imaging on ex vivo skin samples of one patient with classical EDS and two patients with vascular EDS and two healthy controls. We detected irregular, loosely dispersed collagen fibers in a non-parallel arrangement in the dermis of the EDS patients, while as expected, there was no noticeable impairment in the elastin content. Based on further studies on a larger number of patients, in vivo nonlinear microscopic imaging could be utilized for the assessment of the skin status of EDS patients in the future.

Voluntary exercise improves murine dermal connective tissue status in high-fat diet-induced obesity.

Obesity is a risk factor for several cardiovascular and metabolic diseases. Its influence on the skin is less obvious, yet certain negative effects of adipose tissue inflammation on the dermis have been suggested. Excess weight is closely associated with sedentary behavior, so any increase in physical activity is considered beneficial against obesity. To investigate the effects of obesity and physical exercise on the skin, we established a mouse model in which mice were kept either on a high-fat diet or received standard chow. After the two groups achieved a significant weight difference, physical exercise was introduced to both. Animals were given the opportunity to perform voluntary exercise for 40 min daily in a hamster wheel for a period of 8 weeks. We evaluated the status of the dermis at the beginning and at the end of the exercise period by in vivo nonlinear microscopy. Obese mice kept on high-fat diet lost weight steadily after they started to exercise. In the high-fat diet group, we could detect significantly larger adipocytes and a thicker layer of subcutaneous tissue; both changes started to normalize after exercise. Nonlinear microscopy revealed an impaired collagen structure in obese mice that improved considerably after physical activity was introduced. With the ability to detect damage on collagen structure, we set out to address the question whether this process is reversible. With the use of a novel imaging method, we were able to show the reversibility of connective tissue deterioration as a benefit of physical exercise.

Diet-induced obesity skin changes monitored by in vivo SHG and ex vivo CARS microscopy.

Obesity related metabolic syndrome and type 2 diabetes have severe consequences on our skin. Latest developments in nonlinear microscopy allow the use of noninvasive, label free imaging methods, such as second harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS), for early diagnosis of metabolic syndrome-related skin complications by 3D imaging of the skin and the connective tissue. Our aim was to study effects of various types of diet-induced obesity in mice using these methods. We examined mice on different diets for 32 weeks. The collagen morphology was evaluated four times in vivo by SHG microscopy, and adipocytes were examined once at the end of experiment by ex vivo CARS method. A strong correlation was found between the body weight and the adipocyte size, while we found that the SHG intensity of dermal collagen reduces considerably with increasing body weight. Obese mice on high-fat diet showed worse results than those on high-fat - high-fructose diet. Animals on high-fructose diet did not gain more weight than those on ordinary diet despite of the increased calorie intake, but their collagen damage was nonetheless significant. Obesity and high sugar intake damages the skin, mainly the dermal connective tissue and subcutaneous adipose tissue, which efficiently can be monitored by in vivo SHG and ex vivo CARS microscopy.

Handheld nonlinear microscope system comprising a 2 MHz repetition rate, mode-locked Yb-fiber laser for in vivo biomedical imaging.

A novel, Yb-fiber laser based, handheld 2PEF/SHG microscope imaging system is introduced. It is suitable for in vivo imaging of murine skin at an average power level as low as 5 mW at 200 kHz sampling rate. Amplified and compressed laser pulses having a spectral bandwidth of 8 to 12 nm at around 1030 nm excite the biological samples at a ~1.89 MHz repetition rate, which explains how the high quality two-photon excitation fluorescence (2PEF) and second harmonic generation (SHG) images are obtained at the average power level of a laser pointer. The scanning, imaging and detection head, which comprises a conventional microscope objective for beam focusing, has a physical length of ~180 mm owing to the custom designed imaging telescope system between the laser scanner mirrors and the entrance aperture of the microscope objective. Operation of the all-fiber, all-normal dispersion Yb-fiber ring laser oscillator is electronically controlled by a two-channel polarization controller for Q-switching free mode-locked operation. The whole nonlinear microscope imaging system has the main advantages of the low price of the fs laser applied, fiber optics flexibility, a relatively small, light-weight scanning and detection head, and a very low risk of thermal or photochemical damage of the skin samples.

Photosensitivity of murine skin greatly depends on the genetic background: clinically relevant dose as a new measure to replace minimal erythema dose in mouse studies.

Artificial UV irradiation of murine skin is a frequently used method for testing photosensitivity, study carcinogenesis and photoprotective effects of different compounds. However, doses of UV radiation and mouse strains used in experiments vary greatly. The genetic background of mice may influence the photosensitivity as melanin content, pigmentation and hair cycle parameters are dissimilar. Doses of UV are often expressed in relation to the minimal erythema dose (MED) that was not necessarily determined for the given strain. We set out to standardize the method of measuring photosensitivity in three commonly used mouse strains, C57BL/6N, Balb/c and SKH-1. We found that MED may not be determined for some strains as erythema development in mice with diverse genotypes differs greatly. We measured the oedema response in vivo and ex vivo by using OCT. Given the strain-specific variability of erythema, we introduced Clinically Relevant Dose (CRD) as a new term to replace MED in experiments, to describe the lowest dose that triggers a perceptible skin reaction in mice. Not only the CRD but the proportion of erythema and oedema were different in strains examined. C57BL/6N mice display skin reactions at the lowest UVB dose, while SKH-1 hairless mice show changes, mostly oedema, after higher doses of UVB. The cellular composition and skin thickness were examined by histopathology. IL-1beta and IL-6 levels in skin correlated with the increasing doses of UVB. Despite the variations in the degree of erythema and oedema, no major differences in cytokine expressions were seen among various strains of mice.

In vivo second-harmonic generation and ex vivo coherent anti-stokes raman scattering microscopy to study the effect of obesity to fibroblast cell function using an Yb-fiber laser-based CARS extension unit.

Nonlinear microscopy techniques are being increasingly used to perform in vivo studies in dermatology. These methods enable us to investigate the morphology and monitor the physiological process in the skin by the use of femtosecond lasers operating in the red, near-infrared spectral range (680-1,300 nm). In this work we used two different techniques that require no labeling: second harmonic generation (SHG) for collagen detection and coherent anti-Stokes Raman scattering (CARS) to assess lipid distribution in genetically obese murine skin. Obesity is one of the most serious public health problems due to its high and increasing prevalence and the associated risk of type 2 diabetes and cardiovascular diseases. Other than these diseases, nearly half of patients with diabetes mellitus suffer from dermatological complications such as delayed wound healing, foot ulcers and several other skin changes. In our experiment we investigated and followed the effects of obesity on dermal collagen alterations and adipocyte enlargement using a technique not reported in the literature so far. Our results indicate that the in vivo SHG and ex vivo CARS imaging technique might be an important tool for diagnosis of diabetes-related skin disorders in the near future.

In vivo study of targeted nanomedicine delivery into Langerhans cells by multiphoton laser scanning microscopy.

Epidermal Langerhans cells (LCs) function as professional antigen-presenting cells of the skin. We investigated the LC-targeting properties of a special mannose-moiety-coated pathogen-like synthetic nanomedicine DermaVir (DV), which is capable to express antigens to induce immune responses and kill HIV-infected cells. Our aim was to use multiphoton laser microscopy (MLM) in vivo in order to visualize the uptake of Alexa-labelled DV (AF546-DV) by LCs. Knock-in mice expressing enhanced green fluorescent protein (eGFP) under the control of the langerin gene (CD207) were used to visualize LCs. After 1 h, AF546-DV penetrated the epidermis and entered the eGFP-LCs. The AF546-DV signal was equally distributed inside the LCs. After 9 h, we observed AF546-DV signal accumulation that occurred mainly at the cell body. We demonstrated in live animals that LCs picked up and accumulated the nanoparticles in the cell body.

Reduced inflammatory threshold indicates skin barrier defect in transglutaminase 3 knockout mice.

Recently, a transglutaminase 3 knockout (TGM3/KO) mouse was generated that showed impaired hair development, but no gross defects in the epidermal barrier, although increased fragility of isolated corneocytes was demonstrated. Here we investigated the functionality of skin barrier in vivo by percutaneous sensitization to FITC in TGM3/KO (n=64) and C57BL/6 wild-type (WT) mice (n=36). Cutaneous inflammation was evaluated by mouse ear swelling test (MEST), histology, serum IgE levels, and by flow cytometry from draining lymph nodes. Inflammation-induced significant MEST difference (P<0.0001) was detected between KO and WT mice and was supported also by histopathology. A significant increase of CD4+ CD25+-activated T cells (P<0.01) and elevated serum IgE levels (P<0.05) in KO mice indicated more the development of FITC sensitization than an irritative reaction. Propionibacter acnes-induced intracutaneous inflammation showed no difference (P=0.2254) between the reactivity of WT and KO immune system. As in vivo tracer, FITC penetration from skin surface followed by two-photon microscopy demonstrated a more invasive percutaneous penetration in KO mice. The clinically uninvolved skin in TGM3/KO mice showed impaired barrier function and higher susceptibility to FITC sensitization indicating that TGM3 has a significant contribution to the functionally intact cutaneous barrier.