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Information on the penetration depth, pathways, metabolization, storage of vehicles, active pharmaceutical ingredients (APIs), and functional cosmetic ingredients (FCIs) of topically applied formulations or contaminants (substances) in skin is of great importance for understanding their interaction with skin targets, treatment efficacy, and risk assessment—a challenging task in dermatology, cosmetology, and pharmacy. Non-invasive methods for the qualitative and quantitative visualization of substances in skin in vivo are favored and limited to optical imaging and spectroscopic methods such as fluorescence/reflectance confocal laser scanning microscopy (CLSM); two-photon tomography (2PT) combined with autofluorescence (2PT-AF), fluorescence lifetime imaging (2PT-FLIM), second-harmonic generation (SHG), coherent anti-Stokes Raman scattering (CARS), and reflectance confocal microscopy (2PT-RCM); three-photon tomography (3PT); confocal Raman micro-spectroscopy (CRM); surface-enhanced Raman scattering (SERS) micro-spectroscopy; stimulated Raman scattering (SRS) microscopy; and optical coherence tomography (OCT). This review summarizes the state of the art in the use of the CLSM, 2PT, 3PT, CRM, SERS, SRS, and OCT optical methods to study skin penetration in vivo non-invasively (302 references). The advantages, limitations, possibilities, and prospects of the reviewed optical methods are comprehensively discussed. The ex vivo studies discussed are potentially translatable into in vivo measurements. The requirements for the optical properties of substances to determine their penetration into skin by certain methods are highlighted. [ABSTRACT FROM AUTHOR]

The aroma of vanilla pods is mainly derived from vanillin. Microbial biotransformation reactions of vanillin precursors yield "natural" vanillin-related aroma metabolites. In this study, we coated vanilla pods with three edible microorganisms and observed the changes in tissues with a laser scanning microscope during early curing. In addition, the conducted volatile components analysis using gas chromatography-mass spectrometry (GC-MS) with ethanol extracts to investigate the differences in the aroma components of coated and uncoated microbial vanilla pods and to identify the correlation between processing and the oily luster of pods. The results demonstrate that the oily luster on the surface of vanilla pods coated with Bacillus subtilis subsp. subtilis is one of the necessary conditions for a high-quality vanilla product. Eight categories of compounds were found in the ethanol extract of vanilla pods. A total of 69 volatile components were analyzed. Different microbial species significantly influenced the volatile components, with 31 compounds not found in the control group. Furthermore, 30 odor and aroma compounds were identified. This study reveals the role of edible microbial coatings in enhancing the natural aroma of vanilla pods and offers possibilities for the development of new and unique vanilla aroma profiles. [ABSTRACT FROM AUTHOR]

Glabrous skin is hair‐free skin with a high density of sweat glands, which is found on the palms, and soles of mammalians, covered with a thick stratum corneum. Dry hands are often an occupational problem which deserves attention from dermatologists. Urea is found in the skin as a component of the natural moisturizing factor and of sweat. We report the discovery of dendrimer structures of crystalized urea in the stratum corneum of palmar glabrous skin using laser scanning microscopy. The chemical and structural nature of the urea crystallites was investigated in vivo by non‐invasive techniques. The relation of crystallization to skin hydration was explored. We analysed the index finger, small finger and tenar palmar area of 18 study participants using non‐invasive optical methods, such as laser scanning microscopy, Raman microspectroscopy and two‐photon tomography. Skin hydration was measured using corneometry. Crystalline urea structures were found in the stratum corneum of about two‐thirds of the participants. Participants with a higher density of crystallized urea structures exhibited a lower skin hydration. The chemical nature and the crystalline structure of the urea were confirmed by Raman microspectroscopy and by second harmonic generated signals in two‐photon tomography. The presence of urea dendrimer crystals in the glabrous skin seems to reduce the water binding capacity leading to dry hands. These findings highlight a new direction in understanding the mechanisms leading to dry hands and open opportunities for the development of better moisturizers and hand disinfection products and for diagnostic of dry skin. [ABSTRACT FROM AUTHOR]

Introduction: Surgical resection remains the first-line treatment for gliomas. Several fluorescent dyes are currently in use to augment intraoperative tumor visualization, but information on their comparative effectiveness is lacking. We performed systematic assessment of fluorescein sodium (FNa), 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PpIX), and indocyanine green (ICG) fluorescence in various glioma models using advanced fluorescence imaging techniques. Methods: Four glioma models were used: GL261 (high-grade model), GB3 (lowgrade model), and an in utero electroporation model with and without red fluorescence protein (IUE +RFP and IUE -RFP, respectively) (intermediate-tolow-grade model). Animals underwent 5-ALA, FNa, and ICG injections and craniectomy. Brain tissue samples underwent fluorescent imaging using a wide-field operative microscope and a benchtop confocal microscope and were submitted for histologic analysis. Results: Our systematic analysis showed that wide-field imaging of highly malignant gliomas is equally efficient with 5-ALA, FNa, and ICG, although FNa is associated with more false-positive staining of the normal brain. In low-grade gliomas, wide-field imaging cannot detect ICG staining, can detect FNa in only 50% of specimens, and is not sensitive enough for PpIX detection. With confocal imaging of low-intermediate grade glioma models, PpIX outperformed FNa. Discussion: Overall, compared to wide-field imaging, confocal microscopy significantly improved diagnostic accuracy and was better at detecting low concentrations of PpIX and FNa, resulting in improved tumor delineation. Neither PpIX, FNa, nor ICG delineated all tumor boundaries in studied tumor models, which emphasizes the need for novel visualization technologies and molecular probes to guide glioma resection. Simultaneous administration of 5-ALA and FNa with use of cellular-resolution imaging modalities may provide additional information for margin detection and may facilitate maximal glioma resection. [ABSTRACT FROM AUTHOR]

Recently, photoluminescence tomography based on the confocal laser scanning microscopy with two-photon excitation has been developed to study the distribution of point and extended defects in the bulk of ZnSe laser crystals. This article presents the use of the tomography to investigate luminescent micro inclusions in transparent amorphous media such as silicate glass. Studies of CdZnSSe crystals synthesized in a silicate glass melt have been carried out using the tomography with both two-photon and single-photon excitation of luminescence. Zn-rich glass manufactured in the 19th century has been found to contain micron-sized crystals of Cd ξ Zn 1 − ξ S ζ Se 1 − ζ (ξ ≈ 0. 5 , ζ ≈ 0. 5) of hexagonal crystal system exhibiting intense photoluminescence. The photoluminescence band of these crystals has been found to peak at about 2.1 eV (∼ 590 nm) at 300 K. Minor shifts in the maximum of bands and changes in their shape in individual crystals or at tomogram points within the crystal are associated with variations in their composition. Changes in the photoluminescence band shape and maximum due to the excitation of luminescence in CdZnSSe crystallites by laser radiation of different energies have also been observed in this study. • Photoluminescence tomography of glass with micro crystallites is demonstrated. • Tomograms of historical Zn-rich silicate glass with CdZnSSe crystals are explored. • Tomograms were obtained with both two-photon and single-photon PL excitation. • CdZnSSe micro crystals exhibit bright photoluminescence peaked at about 2 eV. • Tomograms show changes in PL spectra obtained under different excitation modes. [ABSTRACT FROM AUTHOR]

ObjectivesNon-invasive visualization of hair follicles is important for proper diagnosis and management of alopecia; however, histological assessment remains the gold standard. Laser imaging technologies have made possible noninvasive in vivo evaluation of skin and hair follicle. The aim of this study was to evaluate the ability of multiphoton microscopy (MPM) to non-invasively identify morphological features that can distinguish scarring from non-scarring alopecia.MethodsMPM images were obtained from areas on the scalp affected by alopecia. Investigators blinded to the diagnosis analyzed hair follicle and shaft sizes. Patients were recruited and imaged at the UC Irvine Health Medical Center and the University of California, Irvine Beckman Laser Institute. Patients with androgenetic alopecia (AGA) and alopecia areata (AA), and scarring alopecia, in particular frontal fibrosing alopecia (FFA) were recruited and imaged from July 2016 to July 2017.ResultsWe imaged 5 normal scalp subjects and 12 patients affected by non-scarring (7 subjects) and scarring (5 subjects) alopecia. In normal and non-scarring alopecia patients, MPM identified presence of sebaceous glands associated with hair follicles. MPM images of scarring alopecia were characterized by the presence of inflammatory cells surrounding hair follicles. Measurements of hair follicle diameter sizes were found to be significantly smaller in scarring alopecia patients compared to normal (P

IntroductionSurgical resection remains the first-line treatment for gliomas. Several fluorescent dyes are currently in use to augment intraoperative tumor visualization, but information on their comparative effectiveness is lacking. We performed systematic assessment of fluorescein sodium (FNa), 5-aminolevulinic acid (5-ALA)–induced protoporphyrin IX (PpIX), and indocyanine green (ICG) fluorescence in various glioma models using advanced fluorescence imaging techniques.MethodsFour glioma models were used: GL261 (high-grade model), GB3 (low-grade model), and an in utero electroporation model with and without red fluorescence protein (IUE +RFP and IUE -RFP, respectively) (intermediate-to-low-grade model). Animals underwent 5-ALA, FNa, and ICG injections and craniectomy. Brain tissue samples underwent fluorescent imaging using a wide-field operative microscope and a benchtop confocal microscope and were submitted for histologic analysis.ResultsOur systematic analysis showed that wide-field imaging of highly malignant gliomas is equally efficient with 5-ALA, FNa, and ICG, although FNa is associated with more false-positive staining of the normal brain. In low-grade gliomas, wide-field imaging cannot detect ICG staining, can detect FNa in only 50% of specimens, and is not sensitive enough for PpIX detection. With confocal imaging of low-intermediate grade glioma models, PpIX outperformed FNa.DiscussionOverall, compared to wide-field imaging, confocal microscopy significantly improved diagnostic accuracy and was better at detecting low concentrations of PpIX and FNa, resulting in improved tumor delineation. Neither PpIX, FNa, nor ICG delineated all tumor boundaries in studied tumor models, which emphasizes the need for novel visualization technologies and molecular probes to guide glioma resection. Simultaneous administration of 5-ALA and FNa with use of cellular-resolution imaging modalities may provide additional information for margin detection and may facilitate maximal glioma resection.

To enable detailed studies of interactions between nanoparticles and their environment and the correlations between various nanoparticle properties, one must go beyond ensemble averages and toward single‐particle measurements. However, current methodologies for the single‐nanoparticle analysis of charge and size either lack the flexibility to study dynamic processes on the single‐particle level or are highly specific and require complex microfluidic devices. In addition, accurate measurements of the electrophoretic mobility (or zeta‐potential) based on the optical detection of single nanoparticles remain challenging due to the low photon budget, the required sampling frequency, and the fact that electroosmosis in typical microfluidic devices must be analyzed carefully. In this study, a method is investigated to accurately characterize the electrophoretic mobility of individual nanoparticles and estimate their size by simultaneously analyzing the electrokinetic‐ and Brownian motion in a simple microfluidic channel. Fast laser scanning excitation and sensitive detection of fluorescent photons enable single‐nanoparticle velocimetry experiments in an oscillating electric field at high frame rates. [ABSTRACT FROM AUTHOR]

Two-dimensional holographic structures based on photopolymer compositions with luminescent nanoparticles, such as quantum dots, are promising candidates for multiresponsive luminescence sensors. However, their applicability may suffer from the incompatibility of the components, and hence aggregation of the nanoparticles. We showed that the replacement of an organic shell at the CdSe/ZnS quantum dots' surface with monomer molecules of the photopolymerizable medium achieved full compatibility with the surrounding medium. The effect was demonstrated by luminescence spectroscopy, and steady-state and time-resolved luminescent laser scanning microscopy. We observed the complete spectral independence of local photoluminescence decay, thus proving the absence of even nanoscale aggregation, either in the liquid composition or in the nodes and antinodes of the grating. Therefore, nanostructured luminescent photopolymer gratings with monomer-covered quantum dots can act as hybrid diffractive–luminescent sensor elements. [ABSTRACT FROM AUTHOR]

Background: Modern methods of microscopy expand our capabilities to detail objects and move to the study of native tissue. The varieties of laser microscopy, which are becoming more and more popular, have broad prospects in the study of morphological properties, combining high resolution and minimal exposure to aggressive media during sample preparation. However, in the scientific literature, the aspects of the structure of individual structural components of the skin or morphofunctional changes in various pathological conditions are not well covered. In this regard, the purpose of our study was a multilevel analysis of structural components using both classical and modern morphological methods. Methods and Results: The material for this study was skin fragments obtained from laboratory male Wistar rats. The study of the structural components was carried out by the methods of light microscopy, scanning electron microscopy, and laser scanning microscopy. The results of our study indicate that the most effective way to obtain complete information is an integrated approach to the study of tissue morphology, where the researcher requires deep knowledge and the use of not only modern methods, but also the possibility of combining them with existing classical methods.

The aroma of vanilla pods is mainly derived from vanillin. Microbial biotransformation reactions of vanillin precursors yield “natural” vanillin-related aroma metabolites. In this study, we coated vanilla pods with three edible microorganisms and observed the changes in tissues with a laser scanning microscope during early curing. In addition, the conducted volatile components analysis using gas chromatography-mass spectrometry (GC-MS) with ethanol extracts to investigate the differences in the aroma components of coated and uncoated microbial vanilla pods and to identify the correlation between processing and the oily luster of pods. The results demonstrate that the oily luster on the surface of vanilla pods coated with Bacillus subtilis subsp. subtilis is one of the necessary conditions for a high-quality vanilla product. Eight categories of compounds were found in the ethanol extract of vanilla pods. A total of 69 volatile components were analyzed. Different microbial species significantly influenced the volatile components, with 31 compounds not found in the control group. Furthermore, 30 odor and aroma compounds were identified. This study reveals the role of edible microbial coatings in enhancing the natural aroma of vanilla pods and offers possibilities for the development of new and unique vanilla aroma profiles.

Current simulations of sintering processes work often with heavily idealized powder geometries. As sintering is mainly driven by gradients of chemical potential due to surface curvatures, a realistic description of the particle geometry is essential for achieving precise simulation results. In previous work, a model able to simulate sintering behavior of irregularly shaped particles was shown. Herein, a statistical approach for description of particles' morphology in a powder mixture is developed by fitting a simplified particle shape model on image data. The obtained data are analyzed and conditioned for input into sintering simulation. Instead of building an equivalent volume cell of particles, as common, a Monte Carlo approach is applied to describe the sintering behavior of the powder. The resulting distributions of sintering time curves are analyzed regarding the influence of the morphology description in comparison to circular particles. Using thumb rules of Cohen's effect size, a small effect on shrinkage and a medium effect on neck radii is observed. [ABSTRACT FROM AUTHOR]

In plants, water flows are the major driving force behind growth and play a crucial role in the life cycle. To study hydrodynamics, methods based on tracking small particles inside water flows attend a special place. Thanks to these tools, it is possible to obtain information about the dynamics of the spatial distribution of the flux characteristics. In this paper, using contrast-enhanced magnetic resonance imaging (MRI), we show that gadolinium chelate, used as an MRI contrast agent, marks the structural characteristics of the xylem bundles of maize stem nodes and internodes. Supplementing MRI data, the high-precision visualization of xylem vessels by laser scanning microscopy was used to reveal the structural and dimensional characteristics of the stem vascular system. In addition, we propose the concept of using prototype "Y-type xylem vascular connection" as a model of the elementary connection of vessels within the vascular system. A Reynolds number could match the microchannel model with the real xylem vessels. [ABSTRACT FROM AUTHOR]

Laser scanning microscopy is widely used to measure blood hemodynamics with line-scans in physiological and pathological vessels. With scans of broken lines, i.e., lines made of several segments with different orientations, it also allows simultaneous monitoring of vessel diameter dynamics or the activity of specific cells. Analysis of red blood cell (RBC) velocity from line-scans requires specific image-processing algorithms, as angle measurements, Line-Scanning Particle Image Velocimetry (LSPIV) or Fourier transformation of line-scan images. The conditions under which these image-processing algorithms give accurate measurements have not been fully characterized although the accuracy of measurements vary according to specific experimental parameters: the vessel type, the RBC velocity, the scanning parameters, and the image signal to noise ratio. Here, we developed mathematical models for the three previously mentioned line-scan image-processing algorithms. Our models predict the experimental conditions in which RBC velocity measurements are accurate. We illustrate the case of different vessel types and give the parameter space available for each of them. Last, we developed a software generating artificial line-scan images and used it to validate our models. [ABSTRACT FROM AUTHOR]

Laser scanning microscopy is widely used to measure blood hemodynamics with line-scans in physiological and pathological vessels. With scans of broken lines, i.e., lines made of several segments with different orientations, it also allows simultaneous monitoring of vessel diameter dynamics or the activity of specific cells. Analysis of red blood cell (RBC) velocity from line-scans requires specific image-processing algorithms, as angle measurements, Line-Scanning Particle Image Velocimetry (LSPIV) or Fourier transformation of line-scan images. The conditions under which these image-processing algorithms give accurate measurements have not been fully characterized although the accuracy of measurements vary according to specific experimental parameters: the vessel type, the RBC velocity, the scanning parameters, and the image signal to noise ratio. Here, we developed mathematical models for the three previously mentioned line-scan image-processing algorithms. Our models predict the experimental conditions in which RBC velocity measurements are accurate. We illustrate the case of different vessel types and give the parameter space available for each of them. Last, we developed a software generating artificial line-scan images and used it to validate our models.

The specialized pericellular matrix (PCM) surrounding chondrocytes within articular cartilage is critical to the tissue's health and longevity. Growing evidence suggests that PCM alterations are ubiquitous across all trajectories of osteoarthritis, a crippling and prevalent joint disease. The PCM geometry is of particular interest as it influences the cellular mechanical environment. Observations of asymmetrical PCM thickness have been reported, but a quantified characterization is lacking. To this end, a novel microscopy protocol was developed and applied to acquire images of the PCM surrounding live cells. Morphometric analysis indicated a statistical bias towards thicker PCM on the inferior cellular surface. The mechanical effects of this bias were investigated with multiscale modelling, which revealed potentially damaging, high tensile strains in the direction perpendicular to the membrane and localized on the inferior surface. These strains varied substantially between PCM asymmetry cases. Simulations with a thicker inferior PCM, representative of the observed geometry, resulted in strain magnitudes approximately half of those calculated for a symmetric geometry, and a third of those with a thin inferior PCM. This strain attenuation suggests that synthesis of a thicker inferior PCM may be a protective adaptation. PCM asymmetry may thus be important in cartilage development, pathology, and engineering. [ABSTRACT FROM AUTHOR]

Nonlinear nanoplasmonics is a largely unexplored research area that paves the way for many exciting applications, such as nanolasers, nanoantennas, and nanomodulators. In the field of nonlinear nanoplasmonics, it is highly desirable to characterize the nonlinearity of the optical absorption and scattering of single nanostructures. Currently, the common method to quantify optical nonlinearity is the z-scan technique, which yields real and imaginary parts of the permittivity by moving a thin sample with a laser beam. However, z-scan typically works with thin films, and thus acquires nonlinear responses from ensembles of nanostructures, not from single ones. In this work, we present an x-scan technique that is based on a confocal laser scanning microscope equipped with forward and backward detectors. The two-channel detection offers the simultaneous quantification for the nonlinear behavior of scattering, absorption and total attenuation by a single nanostructure. At low excitation intensities, both scattering and absorption responses are linear, thus confirming the linearity of the detection system. At high excitation intensities, we found that the nonlinear response can be derived directly from the point spread function of the x-scan images. Exceptionally large nonlinearities of both scattering and absorption are unraveled simultaneously for the first time. The present study not only provides a novel method for characterizing nonlinearity of a single nanostructure, but also reports surprisingly large plasmonic nonlinearities.

Stereology is the method of choice for the quantitative assessment of biological objects in microscopy. It takes into account the fact that, in traditional microscopy such as conventional light and transmission electron microscopy, although one has to rely on measurements on nearly two-dimensional sections from fixed and embedded tissue samples, the quantitative data obtained by these measurements should characterize the real three-dimensional properties of the biological objects and not just their "flatland" appearance on the sections. Thus, three-dimensionality is a built-in property of stereological sampling and measurement tools. Stereology is, therefore, perfectly suited to be combined with 3D imaging techniques which cover a wide range of complementary sample sizes and resolutions, e.g. micro-computed tomography, confocal microscopy and volume electron microscopy. Here, we review those stereological principles that are of particular relevance for 3D imaging and provide an overview of applications of 3D imaging-based stereology to the lung in health and disease. The symbiosis of stereology and 3D imaging thus provides the unique opportunity for unbiased and comprehensive quantitative characterization of the three-dimensional architecture of the lung from macro to nano scale. [ABSTRACT FROM AUTHOR]

By eliminating the photodamage and photobleaching induced by high intensity laser and fluorescent molecular, the label-free laser scanning microscopy shows powerful capability for imaging and dynamic tracing to biological tissues and cells. In this review, three types of label-free laser scanning microscopies: laser scanning coherent Raman scattering microscopy, second harmonic generation microscopy and scanning localized surface plasmon microscopy are discussed with their fundamentals, features and recent progress. The applications of label-free biological imaging of these laser scanning microscopies are also introduced. Finally, the performance of the microscopies is compared and the limitation and perspectives are summarized. [ABSTRACT FROM AUTHOR]

Methods and applications of light microscopy in the life sciences are compared with respect to 3D imaging, resolution, light exposure, sensitivity, and recording time. While conventional wide-field or laser scanning microscopy appear appropriate for smaller samples of only a few micrometers in size with a limited number of light exposures, light sheet microscopy appears to be an optimal method for larger 3D cell cultures, biopsies, or small organisms if multiple exposures or long measuring periods are desired. Super-resolution techniques should be considered in the context of high light exposure possibly causing photobleaching and photo-toxicity to living specimens. [ABSTRACT FROM AUTHOR]

Aim: The skin represents a drug delivery portal. The establishment of a skin model capable of distinguishing between the follicular and intercellular penetration pathways remains a challenge. The study described herein was aimed at showing the influence of two nail varnishes as closure material and four application techniques to spread the active pharmaceutical ingredient (API) on a successful follicular closure without inducing penetration-enhancing effects. Materials and Methods: For all experiments, ex vivo porcine ear skin was used. In study design A, a standard and a solvent-free nail varnish were compared. It was tested whether the different application techniques (spreading with pipette, careful finger massage, 5-Hz finger massage, 5-Hz automatic massage) potentially destroy an intact follicular closure. Laser scanning microscopy imaging was used to measure if the model drug (fluorescein sodium salt) penetrated into the hair follicles. Study design B investigated how the penetration is affected when applying standard nail varnish containing solvents to skin. It was tested if the varnish blocks the API (caffeine) on completely covered areas and if adjacent areas show increased penetration. Furthermore, lateral diffusion of the API was investigated. After 20 h, the skin layers were separated by tape stripping and heat separation. The tissue samples were homogenized. Caffeine was quantified by chromatography. Results: In study design A, the standard nail varnish showed a secure follicular closure, while the solvent-free nail varnish was not able to prevent follicular penetration. Moreover, rapid application techniques were found to destroy an intact follicular closure. Only the two most gentle application techniques kept the follicular closing intact. In study design B, no caffeine was detected in both skin areas that were completely covered. Since no significant difference in caffeine penetration between the two uncovered groups was found, any influence of the applied closure material on adjacent areas was excluded. Conclusion: This study clearly demonstrates that a standard nail varnish in combination with a gentle application technique of the API provides a secure follicular closure. The presented study only investigated the closure for the substances caffeine and fluorescein sodium salt. The results might not be transferable to all kinds of APIs. [ABSTRACT FROM AUTHOR]

To understand brain functions, it is important to study functional connectivity among stereoscopically distributed neurons. Since the brain is composed of 3D neuron networks, volumetric imaging with high spatiotemporal resolution is highly desirable. Two-photon microscopy (2PM) conveniently offers 3D tissue imaging with sub-micrometer resolution based on its intrinsic optical sectioning and deep penetration capabilities. However, the main challenge lies in the volumetric imaging speed and contrast reduction in deep tissue due to aberration. In this study, we integrate a tunable acoustic gradient lens and a plug-and-play adaptive-optics lens into 2PM. The former provides ∼100 kHz axial scan rate, achieving volumetric imaging rate in 1–10 Hz range, while the latter enhances image contrast by nearly two-fold in deep brain regions via correcting both systematic and sample aberrations. The combination offers a practical approach toward high-speed, high-contrast optical volumetric imaging of brain tissues.

Laser scanning microscopy (LSM) techniques are of paramount importance at this time for key domains such as biology, medicine, or materials science. Computer vision methods are instrumental for boosting the potential of LSM, providing reliable results for important tasks, such as image segmentation, registration, classification, or retrieval in a fraction of the time that a human expert would require (at similar or even higher accuracy levels). Image keypoint extraction and description represent essential building blocks of modern computer vision approaches, and the development of such techniques has gained massive interest over the past couple of decades. In this paper, we compare side-by-side five popular keypoint description techniques, scale invariant feature transform (SIFT), speeded-up robust features (SURF), binary robust invariant scalable keypoints (BRISK), fast retina keypoint (FREAK) and BLOCK, with respect to their capacity to represent in a reproducible manner image regions contained in LSM data sets acquired under different acquisition conditions. We evaluate this capacity in terms of descriptor matching performance, using data sets acquired in a principled manner and a thorough Precision-Recall analysis. We identify which of the five evaluated techniques is most robust to specific LSM image modifications associated to the laser beam power, photomultiplier gain, or pixel dwell, and show that certain pre-processing steps have the potential to enhance keypoint matching.

At industrial enterprises in building structures and equipment one can see corrosion damage, as well as damage accumulated during operation period. The areas of stress concentration are welded joints as their structure is heterogeneous. From the point of view of the scale hierarchy, the welded joint represents the welded and base metal zones at the meso-macrolevel, the weld zone, the thermal zone, the base metal at the micro-mesolevel, the grain constituents at the nano-microlevel. Borders are the stress concentrators at different scale levels, thus they becomes the most dangerous places of metal structure. Modeling by the molecular dynamics method at the atomic level has shown nanocracks initiation in triple junctions of grain boundaries and on the ledges of the grain boundaries. Due to active development of nanotechnology, it became possible to evaluate the state of the weld metal at the nanoscale, where irreversible changes take place from the very beginning. Existing methods of nondestructive testing can detect damage only at the meso- and macrolevel. Modern equipment makes it possible to use other methods of control and approaches. For example, according to GOST R55046-2012 and R57223-2016, the analysis of the parameters of the surface projection deformation performed by confocal laser scanning microscopy should be taken into account when the evaluation of state of metal pipelines is carried out. However, there is a problem to monitore it due to various factors affecting the surface during operation. The paper proposes an additional method to estimate the state of weld metal at any stage of deformation that uses 3D analysis of the parameters of the «artificial» corrosion relief of surface. During the operation period changes in the stress-strain state and structure of the metal take place, as the result the character and depth of etching of the grains of the structural components and their boundaries change too. Evaluation of the measurement results of the etching relief 3D surface reveals dangerous signs of the development of the critical state of metal on the nanoscale. The relationship between the method of corrosion surface relief and known methods studying the deformation relief of the surface and microhardness is presented.

Two-photon laser scanning microscopy is widely used to measure blood hemodynamics in brain blood vessels. Still, the algorithms used so far to extract red blood cell (RBC) size and velocity from line-scan acquisitions have ignored the extent to which scanning speed influences the measurements. Here, we used a theoretical approach that takes into account the velocity and direction of both scanning mirrors and RBCs during acquisition to provide an algorithm that measures the real RBC size and velocity. We validate our approach in brain vessels of anesthetized mice, and demonstrate that it corrects online measurement errors that can reach several 10s of percent as well as data previously acquired. To conclude, our analysis allows unbiased comparisons of blood hemodynamic parameters from brain capillaries and large vessels in control and pathological animal models.

A short overview on 3D live cell imaging is given. Relevant samples are described and various problems and challenges—including 3D imaging by optical sectioning, light scattering and phototoxicity—are addressed. Furthermore, enhanced methods of wide-field or laser scanning microscopy together with some relevant examples and applications are summarized. In the future one may profit from a continuous increase in microscopic resolution, but also from molecular sensing techniques in the nanometer range using e.g., non-radiative energy transfer (FRET).

Recent developments in laser scanning microscopy have greatly extended its applicability in cancer imaging beyond the visualization of complex biology, and opened up the possibility of quantitative analysis of inherently dynamic biological processes. However, the physics of image acquisition intrinsically means that image quality is subject to a tradeoff between a number of imaging parameters, including resolution, signal-to-noise ratio, and acquisition speed. We address the problem of geometric distortion, in particular, jaggedness artefacts that are caused by the variable motion of the microscope laser, by using a combination of image processing techniques. Image restoration methods have already shown great potential for post-acquisition image analysis. The performance of our proposed image restoration technique was first quantitatively evaluated using phantom data with different textures, and then qualitatively assessed using in vivo biological imaging data. In both cases, the presented method, comprising a combination of image registration and filtering, is demonstrated to have substantial improvement over state-of-the-art microscopy acquisition methods. [ABSTRACT FROM AUTHOR]

Two-photon laser scanning microscopy is widely used to measure blood hemodynamics in brain blood vessels. Still, the algorithms used so far to extract red blood cell (RBC) size and velocity from line-scan acquisitions have ignored the extent to which scanning speed influences the measurements. Here, we used a theoretical approach that takes into account the velocity and direction of both scanning mirrors and RBCs during acquisition to provide an algorithm that measures the real RBC size and velocity. We validate our approach in brain vessels of anesthetized mice, and demonstrate that it corrects online measurement errors that can reach several 10s of percent as well as data previously acquired. To conclude, our analysis allows unbiased comparisons of blood hemodynamic parameters from brain capillaries and large vessels in control and pathological animal models. [ABSTRACT FROM AUTHOR]

Background: Skin diseases can develop upon disadvantageous microclimate in relation to skin contact with textiles of supporting devices. Increased temperature, moisture, mechanical fracture, pressure, and inflammatory processes often occur mutually and enhance each other in their adverse effects. Therefore, the early prevention of skin irritations by improvement of microclimatic properties of skin in contact with supporting devices is important. Materials and Methods: In this study, the microclimate under occlusion with polyester, cotton, chloroprene rubber, and silicone textiles, used for supporting devices, was analyzed by determining several characteristic physiologic skin parameters in vivo, including temperature, moisture, and transepidermal water loss (TEWL). This is achieved by comparing a miniaturized in vivo detection device with several established optical and sensory methods in vivo. Results: A highly significant TEWL decrease was found after polyester, chloroprene rubber, and silicone application. The application of all materials showed highly significant decrease in skin surface temperature, with chloroprene rubber showing the lowest. Similarly, all materials showed highly significant increase in relative moisture, where the highest increase was found for chloroprene rubber and silicone and the lowest increase for cotton. The cutaneous carotenoid concentration of chloroprene rubber, silicone, and polyester decreased. A manipulation of the surface structure of the stratum corneum was recognized for all materials except for cotton by laser scanning microscopy. Conclusion: The skin parameters temperature, relative moisture, antioxidant status, and TEWL can effectively characterize the microclimatic environment during occlusion with medical supporting materials. These parameters could potentially be used to develop standardized testing procedures for material evaluation. [ABSTRACT FROM AUTHOR]

An experimental study was done in rabbits to characterize the bone-hydroxyapatite (HA) interface by the use of histochemical staining for mineralized tissue and laser scanning microscopy (LSM) on undemineralized sections. Twenty HA-coated Sustain implants were placed in rabbit femurs and retrieved after 6 months. The specimens were then processed to obtain thin ground sections. In all specimens, there was intimate contact between bone and HA. In some portions, mineralized bone was in tight, direct contact with the HA, while in other portions, a basophilic unmineralized material was present between bone and HA. This material was thicker in areas with active bone formation upon the HA surface and had staining characteristics similar to the material present around the osteocyte lacunae. LSM showed a fluorescence present in many areas of the interface, in osteocyte lacunae, and inside the coating. An organic bonding between bone and HA can probably be hypothesized. [ABSTRACT FROM AUTHOR]

Methods and applications of light microscopy in the life sciences are compared with respect to 3D imaging, resolution, light exposure, sensitivity, and recording time. While conventional wide-field or laser scanning microscopy appear appropriate for smaller samples of only a few micrometers in size with a limited number of light exposures, light sheet microscopy appears to be an optimal method for larger 3D cell cultures, biopsies, or small organisms if multiple exposures or long measuring periods are desired. Super-resolution techniques should be considered in the context of high light exposure possibly causing photobleaching and photo-toxicity to living specimens.

By eliminating the photodamage and photobleaching induced by high intensity laser and fluorescent molecular, the label-free laser scanning microscopy shows powerful capability for imaging and dynamic tracing to biological tissues and cells. In this review, three types of label-free laser scanning microscopies: laser scanning coherent Raman scattering microscopy, second harmonic generation microscopy and scanning localized surface plasmon microscopy are discussed with their fundamentals, features and recent progress. The applications of label-free biological imaging of these laser scanning microscopies are also introduced. Finally, the performance of the microscopies is compared and the limitation and perspectives are summarized.

Edema, i.e., fluid accumulation in the interstitial space, accompanies numerous pathological states of the human organism, including heart failure (HF), inflammatory response, and lymphedema. Nevertheless, techniques for quantitative assessment of the edema’s severity and dynamics are absent in clinical practice, and the analysis is mainly limited to physical examination. This fact stimulates the development of novel methods for fast and reliable diagnostics of fluid retention in tissues. In this work, we focused on the possibilities of two microscopic techniques, nailfold video capillaroscopy (NVC) and confocal laser scanning microscopy (CLSM), in the assessment of the short-term and long-term cutaneous edema. We showed that for the patients with HF, morphological parameters obtained by NVC—namely, the apical diameter of capillaries and the size of the perivascular zone—indicate long-term edema. On the other hand, for healthy volunteers, the application of two models of short-term edema, venous occlusion, and histamine treatment of the skin, did not reveal notable changes in the capillary parameters. However, a significant reduction of the NVC image sharpness was observed in this case, which was suggested to be due to water accumulation in the epidermis. To verify these findings, we made use of CLSM, which provides the skin structure with cellular resolution. It was observed that for the histamine-treated skin, the areas of the dermal papillae become hyporefractive, leading to the loss of contrast and the lower visibility of capillaries. Similar effect was observed for patients undergoing infusion therapy. Collectively, our results reveal the parameters can be used for pericapillary edema assessment using the NVC and CLSM, and paves the way for their application in a clinical set-up.

The cerebral microvasculature consists of pial vascular networks, parenchymal descending arterioles, ascending venules and parenchymal capillaries. This vascular compartmentalization is vital to precisely deliver blood to balance continuously varying neural demands in multiple brain regions. Optical imaging techniques have facilitated the investigation of dynamic spatial and temporal properties of microvascular functions in real time. Their combination with transgenic animal models encoding specific genetic targets have further strengthened the importance of optical methods for neurovascular research by allowing for the modulation and monitoring of neuro vascular function. Image analysis methods with three-dimensional reconstruction are also helping to understand the complexity of microscopic observations. Here, we review the compartmentalized cerebral microvascular responses to global perturbations as well as regional changes in response to neural activity to highlight the differences in vascular action sites. In addition, microvascular responses elicited by optical modulation of different cell-type targets are summarized with emphasis on variable spatiotemporal dynamics of microvascular responses. Finally, long-term changes in microvascular compartmentalization are discussed to help understand potential relationships between CBF disturbances and the development of neurodegenerative diseases and cognitive decline. [ABSTRACT FROM AUTHOR]

Frequency-doubled femtosecond Er-doped fiber laser is a low-cost and portable excitation source suitable for multiphoton endoscopy. The frequency-doubled wavelength at 780 nm is used to excite the intrinsic fluorescence signal. The frequency-doubling with a periodically poled MgO:LiNbO3 (PPLN) is integrated in the distal end of the imaging head to achieve fiber connection. The imaging speed is further improved by optimizing the excitation laser source. A 0.3-mm length of PPLN crystal is selected and the Er-doped fiber laser is manipulated to match its bandwidth with the acceptance bandwidth of the PPLN. Through this optimization, a reduced pulsewidth of 80 fs of the frequency-doubled pulse is achieved. All-fiber dispersion compensation and pulse compression by single mode fiber is conducted, which makes the fiber laser directly fiber-coupled to the imaging head. An imaging speed of 4 frames/s is demonstrated on ex vivo imaging of unstained biological tissues, which is 10 times faster than our previous study using a 1-mm-long PPLN. The results show that miniature multiphoton endoscopy using frequency-doubled Er-doped fiber laser has great potential for clinical applications. [ABSTRACT FROM AUTHOR]

The research aims to understand how heat and added ingredients influence the properties of egg yolk which in turn impact the stability of oil-in-water emulsions. Apparent viscosity and Zeta potential of egg yolk dispersion, interfacial tension between rice bran oil and water, oil droplet size and confocal microscope image of each emulsion were investigated. The confocal microscope images of emulsions clearly show that the adsorbed protein layer was thickest when the egg yolk was heated at 65°C for 9 min and a salt-sugar-vinegar mixture was used. Thus, this condition could stabilize an oil-in-water emulsion by preventing the coalescence of oil droplets. [ABSTRACT FROM AUTHOR]

Background/Aims: Airborne pollutants, such as nano-sized soot particles, are increasingly being released into the environment as a result of growing population densities and industrialization. They can absorb organic and metal compounds with potential biological activity, such as polycyclic aromatic hydrocarbons and airborne pollen allergens. Local and systemic toxicities may be induced in the skin if the particulates release their harmful components upon dermal contact. Methods: In the present study, skin pretreatments with serum and/or shield as barrier formulations prior to exposure and washing with a cleanser subsequent to exposure were evaluated as a protection and decontamination strategy using laser scanning microscopy. Results: The results indicate that while the application of serum and a cleanser was insufficient for decontamination, the pretreatment with shield prior to nanoparticle exposure followed by washing led to the removal of a considerable amount of the carbon black particles. The combined application of serum and shield before the administration of carbon black particles and subsequent washing led to their elimination from the skin samples. Conclusion: The application of barrier-enhancing formulations in combination with a cleanser may reduce the penetration of harmful airborne particulates by preventing their adhesion to the skin and facilitating their removal by subsequent washing with the cleanser. [ABSTRACT FROM AUTHOR]

Although high‐resolution three‐dimensional imaging of endoscopically resected gastrointestinal specimens can help elucidating morphological features of gastrointestinal mucosa or tumor, there are no established methods to achieve this without breaking specimens apart. We evaluated the utility of transparency‐enhancing technology for three‐dimensional assessment of gastrointestinal mucosa in porcine models. Esophagus, stomach, and colon mucosa samples obtained from a sacrificed swine were formalin‐fixed and paraffin‐embedded, and subsequently deparaffinized for analysis. The samples were fluorescently stained, optically cleared using transparency‐enhancing technology: ilLUmination of Cleared organs to IDentify target molecules method (LUCID), and visualized using laser scanning microscopy. After observation, all specimens were paraffin‐embedded again and evaluated by conventional histopathological assessment to measure the impact of transparency‐enhancing procedures. As a result, microscopic observation revealed horizontal section views of mucosa at deeper levels and enabled the three‐dimensional image reconstruction of glandular and vascular structures. Besides, paraffin‐embedded specimens after transparency‐enhancing procedures were all assessed appropriately by conventional histopathological staining. These results suggest that transparency‐enhancing technology may be feasible for clinical application and enable the three‐dimensional structural analysis of endoscopic resected specimen non‐destructively. Although there remain many limitations or problems to be solved, this promising technology might represent a novel histopathological method for evaluating gastrointestinal cancers. [ABSTRACT FROM AUTHOR]

The distribution of Ca2+-ATPase activity and Ca2+ in cortex cells of root distal elongation zone (DEZ) was investigated by an electron cytochemical method and laser scanning microscopy in Pisum sativum seedlings grown in the control and under adaptation to horizontal clinorotation (2 rpm), which partly imitated the biological effects of microgravity. The sensitivity of Ca2+-ATPase and Ca2+ to clinorotation conditions has been revealed. The study of Ca2+-ATPase localization in cells of DEZ showed the presence of this enzyme on plasma membrane (PM), vesicles, endoplasmic reticulum, and organelles envelope of the control and clinorotated samples. Clinorotation lead to the redistribution of Ca2+-ATPase on cell membranes. The analysis of the density of precipitate of cytochemical reaction is shown that clinorotation conditions led to decrease of density precipitate on PM and it significantly increase on endomembranes. The change of level free calcium in cells of clinorotated roots has been also established with using of laser scanning microscopy. The correlation of redistribution of Ca2+-ATPase activity on the membranes and the level calcium in cells under adaptation to clinorotation was revealed. It is discussed that the short-term clinorotation affects the plant cell as a stressor of calcium balance. [ABSTRACT FROM AUTHOR]