Reflectance Confocal Laser Scanning Microscopy Research Articles

Dissolvable microneedles in the skin: Determination the impact of barrier disruption and dry skin on dissolution

Dissolvable microneedles (DMNs), fabricated from biocompatible materials that dissolve in both water and skin have gained popularity in dermatology. However, limited research exists on their application in compromised skin conditions. This study compares the hyaluronic acid-based DMNs penetration, formation of microchannels, dissolution, and diffusion kinetics in intact, barrier-disrupted (tape stripped), and dry (acetone-treated) porcine ear skin ex vivo. After DMNs application, comprehensive investigations including dermoscopy, stereomicroscope, skin hydration, transepidermal water loss (TEWL), optical coherence tomography (OCT), reflectance confocal laser scanning microscopy (RCLSM), confocal Raman micro-spectroscopy (CRM), two-photon tomography combined with fluorescence lifetime imaging (TPT-FLIM), histology, and scanning electron microscopy (SEM) were conducted. The 400 µm long DMNs successfully penetrated the skin to depths of ≈200 µm for dry skin and ≈200–290 µm for barrier-disrupted skin. Although DMNs fully inserted into all skin conditions, their dissolution rates were high in barrier-disrupted and low in dry skin, as observed through stereomicroscopy and TPT-FLIM. The dissolved polymer exhibited a more significant expansion in barrier-disrupted skin compared to intact skin, with the smallest increase observed in dry skin. Elevated TEWL and reduced skin hydration levels were evident in barrier-disrupted and dry skins compared to intact skin. OCT and RCLSM revealed noticeable skin indentation and pronounced microchannel areas, particularly in barrier-disrupted and dry skin. Additional confirmation of DMN effects on the skin and substance dissolution was obtained through histology, SEM, and CRM techniques. This study highlights the impact of skin condition on DMN effectiveness, emphasizing the importance of considering dissolvability and dissolution rates of needle materials, primarily composed of hyaluronic acid, for optimizing DMN-based drug delivery.

Real Time In Vivo Confocal Microscopic Analysis of the Enamel Remineralization by Casein Phosphopeptide-Amorphous Calcium Phosphate (CPP-ACP): A Clinical Proof-of-Concept Study

Enamel defects (EDs) are qualitative and/or quantitative disturbances of the dental surface. To date, the responsiveness to remineralizing treatments has been studied ex vivo, on dental sections from extracted teeth. The present research aims to establish if in vivo reflectance confocal laser scanning microscopy is able to visualize the changes in the enamel architecture on living teeth, before, during and after remineralizing treatments by casein phosphopeptide-amorphous calcium phosphate (CPP-ACP). As proof-of-concept study, 17 consecutive children affected by EDs were enrolled and 38 EDs were considered. A CPP-ACP mousse was applied twice a week for 6 weeks and clinical and microscopic images were collected before, during and after the treatment for evaluating the changes occurred. For in vivo microscopic imaging, a reflectance confocal laser scanning microscope (RCM) for in vivo use was adopted. In this study RCM was proven to be able to visualize in vivo and at microscopic resolution the changes occurred during the remineralizing processes without needing for dental extractions and histopathological procedures. This in vivo RCM capability could encourage its clinical application in monitoring responsiveness to enamel therapies.

A high rigor temperature, not sarcomere length, determines light scattering properties and muscle colour in beef M. sternomandibularis meat and muscle fibres

Beef meat colour is impacted by both myoglobin status and the light scattering properties of the muscle, and the specific causative scattering elements of the latter are still unknown. We hypothesize that stretching muscles during rigor will generate a structure which favours light scattering, by increasing the length of the I-band (longer sarcomeres) and that a high rigor temperature will cause protein reconfiguration, changing the muscle structure and promoting light scattering. Muscle fibre fragments were isolated from four beef M. sternomandibularis and subjected to stretching (plus, minus) and three incubation temperatures (5, 15, 35 °C). Reflectance confocal laser scanning microscopy (rCLSM) revealed sarcomere stretching alone was not solely responsible for light scattering development. A high rigor temperature (35 °C) was more favourable for light scattering. Stretching and taking muscle into rigor at 35 °C promoted transverse shrinkage of muscle fibres and increased light scattering and could be applied post-mortem (PM) to reduce the occurrence of problematic dark meat.

High pH in beef longissimus thoracis reduces muscle fibre transverse shrinkage and light scattering which contributes to the dark colour

Most studies have focused on myoglobin regarding meat colour development, with little focus on the contribution of muscle structure and light scattering. Our aim was to investigate the pH-dependent changes in muscle structure, on the light scattering properties of the meat. Beef longissimus thoracis muscles were segregated into light, medium or dark colour groups (n=18). ‘Dark’ muscles had a high ultimate pH (pHu), lower lightness (L*), redness (a*), higher myoglobin concentration and deoxymyoglobin content compared to other muscles. Reflectance confocal laser scanning microscopy (rCLSM) revealed ‘dark’ muscles had decreased global brightness (indicator of light scattering) and increased fibre width. In a secondary experiment, decreasing the homogenisation buffer pH from 6.10 to 5.40 induced a 17% shrinkage in high pHu muscle fibres, which increased light scattering by ~25%. In conclusion, rCLSM demonstrated that muscle structure contributes to the magnitude of light scattering by a pH dependent mechanism.

Potential of confocal laser scanning microscopy for non-invasive diagnostics of malignant epithelial skin tumors in the course of dermatoheliosis progression

Most cases of malignant epithelial skin neoplasms including actinic keratosis and basal cell carcinoma, which are characterized by the most complicated course and numerous clinical and morphological options, involve dermatoheliosis progression. The risk of actinic keratosis transformation into basal cell carcinoma varies from 0.1% to 20% and up to 80% in cases of multiple AK lesion foci. A non-invasive method known as reflectance confocal laser scanning microscopy is the most promising one for the purposes of early diagnostics of signs pointing at epithelial skin neoplasm development and makes it possible to monitor the tumor in progress in vivo to diagnose the presence of a pool of squamous cells on a timely basis. The confocal laser scanning microscopy method provides high-contrast images of for any horizontal-oriented morphologic structures in the epidermis and upper dermis with a resolution comparable to those characteristic of traditional optical microscopy of skin tissue samples. According to our data obtained as a result of studying dynamic changes and morphologic structures in actinic keratosis foci (50 cases) using the confocal laser scanning microscopy method, we discovered a number of morphologic features, and their further analysis will distinguish the signs of progressing carcinogenesis in case of dermatoheliosis.

Comparison of in vivo and ex vivo laser scanning microscopy and multiphoton tomography application for human and porcine skin imaging

Two state-of-the-art microscopic optical methods, namely, confocal laser scanning microscopy in the fluorescence and reflectance regimes and multiphoton tomography in the autofluorescence and second harmonic generation regimes, are compared for porcine skin ex vivo and healthy human skin in vivo. All skin layers such as stratum corneum (SC), stratum spinosum (SS), stratum basale (SB), papillary dermis (PD) and reticular dermis (RD) as well as transition zones between these skin layers are measured noninvasively at a high resolution, using the above mentioned microscopic methods. In the case of confocal laser scanning microscopy (CLSM), measurements in the fluorescence regime were performed by using a fluorescent dye whose topical application on the surface is well suited for the investigation of superficial SC and characterisation of the skin barrier function. For investigations of deeply located skin layers, such as SS, SB and PD, the fluorescent dye must be injected into the skin, which markedly limits fluorescence measurements using CLSM. In the case of reflection CLSM measurements, the obtained results can be compared to the results of multiphoton tomography (MPT) for all skin layers excluding RD. CLSM cannot distinguish between dermal collagen and elastin measuring their superposition in the RD. By using MPT, it is possible to analyse the collagen and elastin structures separately, which is important for the investigation of anti-aging processes. The resolution of MPT is superior to CLSM. The advantages and limitations of both methods are discussed and the differences and similarities between human and porcine skin are highlighted.

Application of Confocal Laser Scanning Microscopy in Biology and Medicine

Fluorescence confocal laser scanning microscopy and reflectance confocal laser scanning microscopy are up-to-date highend study methods. Confocal microscopy is used in cell biology and medicine. By using confocal microscopy, it is possible to study bioplasts and localization of protein molecules and other compounds relative to cell or tissue structures, and to monitor dynamic cell processes. Confocal microscopes enable layer-by-layer scanning of test items to create demonstrable 3D models. As compared to usual fluorescent microscopes, confocal microscopes are characterized by a higher contrast ratio and image definition.

KT-Scale interactions between supported lipid bilayers

We use total internal reflection microscopy (TIRM) and confocal laser scanning microscopy (CSLM) to study supported lipid bilayer (SLB)-modified silica colloids with various SLB compositions (e.g., PEGylated vs. non-PEGylated) that control colloidal and bilayer stability. Measured and predicted potentials accurately capture stable configurations. For unstable conditions when SLBs adhere, fuse, or spread between surfaces, SLB structures are connected to effective potentials as well as time-dependent behavior. In all cases, directly measured and inferred interactions are well described by steric interactions between PEG brushes and van der Waals weakened by substrate roughness. Our findings quantify non-specific kT-scale interactions between SLB-modified colloids and surfaces, which enables the design of such systems for use in biomedical applications and studies of biomolecular interactions.

Reaction patterns of wheat starch granules substituted with an anionic propylene oxide analog (POA) revealed by confocal microscopy and 3D anaglyph imaging

A technique was developed for investigating granular reaction patterns of anionic starch derivatives, utilizing the optical sectioning capability of reflectance confocal laser scanning microscopy in combination with 3D anaglyphic imaging (3D glasses required for visualization). The method facilitated improved visualization of internal granular reaction patterns by discarding optical sections comprising the highly-reacted external granule surface (generating 3D image constructs from sections constituting exclusively internal regions of granules) and minimizing confounding background signal associated with non-starch constituents (e.g., proteins). For wheat starch A-type granules substituted with a propylene oxide analog (POA), granular reaction patterns revealed that reagent reached the hilum region of granules through channels, and primarily entered the granule matrix via lateral diffusion from channel surfaces, producing relatively homogeneous reaction patterns. The homogeneous nature of POA granular reaction patterns was attributed to the relatively low reactivity of the reagent (rate of diffusion>rate of reaction).

Applicability of confocal laser scanning microscopy for evaluation and monitoring of cutaneous wound healing

There is a high demand for noninvasive imaging techniques for wound assessment. In vivo reflectance confocal laser scanning microscopy (CLSM) represents an innovative optical technique for noninvasive evaluation of normal and diseased skin in vivo at near cellular resolution. This study was designed to test the feasibility of CLSM for noninvasive analysis of cutaneous wound healing in 15 patients (7 male/8 female), including acute and chronic, superficial and deep dermal skin wounds. A commercially available CLSM system was used for the assessment of wound bed and wound margins in order to obtain descriptive cellular and morphological parameters of cutaneous wound repair noninvasively and over time. CLSM was able to visualize features of cutaneous wound repair in epidermal and superficial dermal wounds, including aspects of inflammation, neovascularisation, and tissue remodelling in vivo. Limitations include the lack of mechanic fixation of the optical system on moist surfaces restricting the analysis of chronic skin wounds to the wound margins, as well as a limited optical resolution in areas of significant slough formation. By describing CLSM features of cutaneous inflammation, vascularisation, and epithelialisation, the findings of this study support the role of CLSM in modern wound research and management.

A new diagnostic technique for tinea incognito: In vivo reflectance confocal microscopy. Report of five cases

In vivo confocal laser scanning microscopy (CLSM) is a modern non-invasive method for investigation of the skin that allows real-time visualization of individual cells and subcellular structures with the highest resolution imaging comparable to the routine histopathology. Our aim was to demonstrate the potential of CLSM for non-invasive diagnosis of difficult tinea incognito cases. Clinically atypical lesions in five cases of tinea incognito due to dermatophyte spp. were demonstrated using reflectance confocal laser scanning microscopy (RCM), parallel to KOH preparation and fungal culture of skin scrapings performed in the same patients. The morphological features characteristic for tinea incognito, namely linear branched hyphae in the intercellular area of the stratum corneum, were readily detectable by means of CLSM. In vivo tissue imaging were performed at three different wavelengths (785, 658, 445 nm) and the best images of fungal elements were obtained at 445 nm. All of our five cases had similar reflectance confocal microscopical findings. Our findings suggest the potential of CLSM as a non-invasive tool for the diagnosis of tinea incognito having atypical clinical appearance. Although at present the reflectance confocal microscopy cannot replace the current diagnostic standards for tinea incognito, it may be successfully used as in vivo non-invasive screening tool to facilitate the diagnosis and point to the need for further investigation of the patient.

Fluorescence microscopy investigations of ligand propagation and accessibility under adherent cells

Fluorescence microscopy methods including total internal reflection fluorescence and confocal laser scanning microscopy have played a major role in modern cell biology research by permitting imaging of fluorescently tagged macromolecules in living cells. These methods are often used to examine the initial events in signal transduction, which involve interactions occurring between membrane receptors and ligands such as antibodies and growth factors. Most quantitative biophysical applications using these fluorescence imaging methods, including ligand binding assays, are based on the assumption that the fluorophore label of interest has equal access to all areas of the membrane on the cell. Our findings suggest that there is limited accessibility of fluorophores (25±2%)(-) under the basal membrane of adherent CHO-K1 cells expressing epidermal growth factor receptor plated on a bare glass in standard two-dimensional tissue cultures. The authors present a detailed study of the extent to which a small fluorescent dye molecule (Alexa 647) is able to propagate under the basal membrane of cells plated on a variety of biologically compatible substrates: fibronectin, bovine serum albumin, poly-d-lysine, collagen I, collagen IV, Geltrex™, and fibronectin such as binding polymer. For nonspecific dye propagation the best overall accessibility was achieved using a thin layer preparation of a commercially available basement membrane matrix, Geltrex™ (67±8%). Coupling of a specific high affinity ligand (epidermal growth factor) to the dye did result in a moderate increase in propagation for most substrates examined. Despite the overall increase in propagation for most substrates (60%-80%), large areas under the central regions of the adherent cells still remained inaccessible to the fluorescently labeled ligand. More importantly, the presence of the specific ligand did not result in consistent increase in ligand propagation. Taken together these results suggest that the reduced accessibility is not exclusively due to steric effects, and the chemistry of both the ligand and the substrate may be important when working under conditions of reduced dimensionality.

Monitoring of Microcirculation in Free Transferred Musculocutaneous Lattisimus Dorsi Flaps by Confocal-Laser-Scanning Microscopy – A Promising Noninvasive Methodical Approach

Confocal laser scanning microscopy (CLSM) may be used either as fluorescence or reflectance microscopy,1 with much of the previous work in human skin focusing on the investigation of pigmented2 and non-pigmented skin malignancies.3,4 Until now there has been little published on CLSM analysis of skin microvasculature in vivo, although there has been some work assessing dermal capillaries in fluorescent antibody-labelled skin biopsy specimens.5 Therefore the authors are to be congratulated on carrying out this early study demonstrating that reflectance CLSM has the potential to be a valuable bedside analysis tool for free flap monitoring.

In vivo reflectance confocal microscopy: usefulness for diagnosing hair diseases

Reflectance confocal laser scanning microscopy (R-CSLM) is a new diagnostic technique which allows visualization of "optical intersections" within the epidermis and superficial layers of the dermis. Outlines of cells and their architecture are imaged and may be analyzed both horizontally and vertically to the skin surface. The method proved useful in early melanoma detection. We evaluated the potential usefulness of this method in a short series of patients with hair diseases. Two healthy persons and 6 patients with hair diseases (1 with alopecia areata, 1 with androgenic alopecia and 4 with genetic hair shaft abnormalities) were examined with the use of Vivascope 1500. In all patients one scalp location and one location in the mid forearm were evaluated. R-CSLM examination gave in all cases high quality images of the hair shaft intersections, at 1µm intervals, which allowed detailed analysis of the hair structure. Hair follicles could be partly visualized at a depth of up to 200µm, which allowed analysis of only superficial parts of the hair follicles. An additional hurdle was bright reflection within the follicular ostia, which decreased the perception of details in these images. Hair could be best visualized, when analyzed on flat surfaces. Receiving good quality images from convex surfaces on the scalp required additional effort from the patient (to not move) and from the physician (to obtain best possible fit of the "optic window" to the scalp). These preliminary data show that R-CSLM may develop into a valuable tool in evaluation of hair shaft diseases. Further development is needed to apply this technique in abnormalities of the hair follicle and the perifollicular area.

Reflectance confocal‐laser‐scanning microscopy in vivo assessments of cigarette‐induced dynamic alterations of cutaneous microcirculation on histomorphological level

Until now, high resolution reflectance confocal-laser-scanning microscopy (CLSM) was used for observation of cutaneous morphology in vivo and in real time. We hypothesized that CLSM also allows observation of dynamic processes of cutaneous microcirculation. Reflectance CLSM (Vivascope1500; Lucid, Rochester, NY) was performed in 24 young male habitual smokers (23 years, range: 19-26, body mass index 23.9 +/- 4.04) with relatively limited cigarette exposure (mean: 3.1 +/- 2.4 pack-years). Eight matched nonsmokers served as controls. The quantitative blood cell flow and the diameter of capillary loops were determined prior (baseline), during, as well as 5 and 10 min after smoking. Baseline value for blood cell flow was 55.50 +/- 2.33 cells/min, and decreased over 45% during smoking (30.43 +/- 3.76/min; P = 0.02). They were still 22% lower (43.33 +/- 2.45/min; P = 0.01) 5 min after smoking and exceeded baseline values 10 min after smoking by 13% (63.00 +/- 3.10/min; P > 0.05). The baseline values for capillary loop diameter (9.03 +/- 0.22 microm) decreased by 21% (7.18 +/- 0.28 microm; P = 0.03) during smoking, remained about 9% (8.23 +/- 0.18 microm; P = 0.01) lower 5 min after smoking and exceeded baseline values insignificantly by 4% (9.38 +/- 0.28 microm; P > 0.05) 10 min after smoking. There were no significant differences to the controls. Reflectance CLSM enables qualitative and quantitative observation of dynamic processes of cutaneous microcirculation on histomorphological level.

Fluorescence confocal laser scanning microscopy for in vivo imaging of epidermal reactions to two experimental irritants

Fibre-optic fluorescence confocal laser scanning microscopy (CLSM) is a novel non-invasive technique for in vivo imaging of skin. The cellular structure of the epidermis can be studied. A fluorophore, e.g. fluorescein sodium, is introduced by an intradermal injection or applied to the skin surface before scanning. Images are horizontal optical sections parallel to the skin surface. Fluorescence CLSM has hitherto not been applied to experimental contact dermatitis. The aim was to study the applicability of fluorescence CLSM for in situ imaging of irritant contact dermatitis reactions caused by established model irritants, e.g. sodium lauryl sulphate (SLS) and pelargonic acid (PA). Twelve healthy individuals volunteered. The flexor aspect of the right and the left forearm was exposed to SLS in water and PA in isopropanol and occluded under Finn Chambers for 24 h. The reactions were rated clinically and, following epicutaneous and intra-dermal application of fluorescein sodium, studied by fluorescence CLSM, magnification x 1000. Both irritants disturbed the epidermal intercellular borders, which became blurred, thickened and variably altered. This was interpreted as being a result of chemical damage to cellular membranes. Cell borders might show a double contour as a result of inter-cellular oedema. PA might increase the size of individual keratinocytes interpreted as a result of intra-cellular disturbance with oedema. SLS-exposed sites showed clusters of keratinocytes with visible nuclei in the outer layers of the epidermis, e.g. a parakeratotic shift supposed to be due to increased cell proliferation elicited by SLS. The isopropanol vehicle and PA did not interfere with the CLSM imaging technique or the experimental procedures. SLS, being a detergent, however, modified the physico-chemical properties of the skin surface and both disturbed epicutaneous labelling with the flurophore and immersion oil coupling between the skin surface and the optical system. Thus, SLS was technically more difficult to study by CLSM than PA. This preliminary study demonstrated the applicability of fluorescence CLSM for a detailed study of experimental skin irritants in vivo. Essential findings were disturbed and widened cell borders, swelling of keratinocytes by PA and induction of a parakeratotic shift by SLS with clusters of keratinocytes holding nuclei in the epidermis. Fluorescence CLSM offers a unique opportunity to study the inter- and intracellular water compartments directly in the epidermis in situ and an opportunity to visualize cell proliferation manifested as parakeratosis. Fibre-optic fluorescence CLSM of irritant reactions is, however, technically more complicated than reflectance CLSM and may not be applicable to any irritant. SLS applied epicutaneously interacted with the skin surface and coupling to the microscope and was thus found to be more difficult to study technically than PA. PA dissolved in isopropanol is for technical reasons, and with SLS as alternative, considered the preferred model irritant.

Cellular Resolution Expression Profiling Using Confocal Detection of NBT/BCIP Precipitate by Reflection Microscopy

The determination of gene expression patterns in three dimensions with cellular resolution is an important goal in developmental biology. However the most sensitive, efficient, and widely used staining technique for whole-mount in situ hybridization (WMISH), nitroblue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP) precipitation by alkaline phosphatase, could not yet be combined with the most precise, high-resolution detection technique, confocal laser-scanning microscopy (CLSM). Here we report the efficient visualization of the NBT/BCIP precipitate using confocal reflection microscopy for WMISH samples of Drosophila, zebrafish, and the marine annelid worm, Platynereis dumerilii. In our simple WMISH protocol for reflection CLSM, NBT/BCIP staining can be combined with fluorescent WMISH, immunostainings, or transgenic green fluorescent protein (GFP) marker lines, allowing double labeling of cell types or of embryological structures of interest. Whole-mount reflection CLSM will thus greatly facilitate large-scale cellular resolution expression profiling in vertebrate and invertebrate model organisms.

Monitoring neurotransmitter release using surface-enhanced Raman spectroscopy

Surface-enhanced Raman spectroscopy (SERS) is a promising tool to monitor neurotransmitter release at the single-cell level: it is a sensitive technique that provides structural information of the released compounds and spatial information about their release sites. In this study we demonstrate that depolarization-evoked catecholamine secretion by rat phaeochromocytoma (PC12) cells can be spatially resolved by SERS using silver colloids. A suitable SERS substrate was created by adding silver colloids to the cell culture medium. Nomarski-DIC microscopy combined with reflection confocal laser scanning microscopy showed that the colloids were primarily present on top of the cell membrane. The SERS spectra were successfully corrected for the contribution of cell constituents. Dopamine and noradrenaline were localized by examining the correlation coefficient between spectra and reference catecholamine spectra. Potential improvements of the temporal resolution of the technique are discussed.

A novel method to immobilize collagen on polypropylene film as substrate for hepatocyte culture

To improve the biocompatibility of polypropylene membrane with hepatocytes, film was firstly allowed to generate peroxide groups by ammonium peroxydisulfate (APS) thermal decomposition and was then carboxylated by free radical grafting polymerization using acrylic acid as the monomer and ferrous(II) ions as redox initiator. Collagen was then covalently immobilized through amide bonds between the residue amine groups and carboxylic ones in the presence of water-soluble carbodiimide. The film was characterized by Fourier transformed infrared spectroscopy in attenuated total reflection mode (ATR–FTIR), X-photoelectron spectroscopy (XPS) and confocal laser scanning microscopy (CLSM), etc. The experimental results show that there exist hydroperoxide groups after APS aqueous thermal decomposition, which are subsequently converted into the polymeric free radicals initiating the vinyl monomers to subject grafting polymerization. The maximum collagen immobilization content is about 10.5 μg/cm 2 by ninhydrin assay. Hepatocytes cultured on collagen immobilized film show stable phenotype and normal liver-specific functions more than 20 days as observed by scanning electronic microscopy (SEM) and biochemical parameters determination.

Investigation of cell adhesion to structured surfaces using total internal reflection fluorescence and confocal laser scanning microscopy

Adhesion of adherent cells on structured surfaces is influenced by the surface pattern given. Here, we designed a structured gold relief surface based on cell adhesion patterns we had previously observed. We analysed the geometric parameters and the overall distribution of focal adhesion kinase in focal adhesions on unstructured glass surfaces using optical microscopy. The basic structural elements obtained from this analysis were arranged in regular clusters that resembled the shape of a polarised migratory cell. In time-lapse studies we observed that the cells adhere preferentially to the gold pads and adopt the shape of the clusters. Staining of the actin cytoskeleton revealed that the actin filaments are aligned to the gold pads of the elementary structure.