In vivo confocal laser scanning microscopy imaging of skin inflammation: Clinical applications and research directions.
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ABSTRACT:
SUBMITTER: Ilie MA
PROVIDER: S-EPMC6327452 | biostudies-literature | 2019 Feb
REPOSITORIES: biostudies-literature
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Project description:BackgroundDigitally stained ex vivo confocal laser scanning microscopy (CLSM) scans are a possible alternative to formalin-fixed and paraffin-embedded (FFPE) and hematoxylin-eosin (H&E) stained slides. This study explores the diagnostic accuracy of digitally-stained CLSM scans in comparison to H&E-stained slides in various dermatologic diseases in a real-life setting.MethodsSamples of patients out of one selected dermatologic office were primarily scanned via CLSM; a diagnosis was made afterwards using FFPE- and H&E-stained slides by two experienced dermatopathologists. Primary outcomes were sensitivity and specificity of diagnosis in digitally stained CLSM scans in three separate diagnostic groups.ResultsCLSM evaluation of epithelial tumors (n = 132) demonstrated a sensitivity of 64.3%/83.9% and a specificity of 84.2%/71.1%. Diagnosis of melanocytic tumors (n = 86) showed a sensitivity of 19.1%/85.1% and a specificity of 96.3%/66.7%. In the diagnosis of other tumors/cysts and inflammatory dermatoses (n = 42), a sensitivity of 96.4%/96.8% and a specificity of 57.1%/45.5% was reached.ConclusionsThis study shows the possibilities and limitations of a broad use of CLSM. Because of a partly low diagnostic accuracy, such an application does not seem to be recommendable at present for every indication.
| S-EPMC9777122 | biostudies-literature
Project description:A new approach to quantitative single-molecule imaging by confocal laser scanning microscopy (CLSM) is presented. It relies on fluorescence intensity distribution to analyze the molecular occurrence statistics captured by digital imaging and enables direct determination of the number of fluorescent molecules and their diffusion rates without resorting to temporal or spatial autocorrelation analyses. Digital images of fluorescent molecules were recorded by using fast scanning and avalanche photodiode detectors. In this way the signal-to-background ratio was significantly improved, enabling direct quantitative imaging by CLSM. The potential of the proposed approach is demonstrated by using standard solutions of fluorescent dyes, fluorescently labeled DNA molecules, quantum dots, and the Enhanced Green Fluorescent Protein in solution and in live cells. The method was verified by using fluorescence correlation spectroscopy. The relevance for biological applications, in particular, for live cell imaging, is discussed.
| S-EPMC2587633 | biostudies-literature
Project description:Two-dimensional (2D) materials such as graphene have become the focus of extensive research efforts in condensed matter physics. They provide opportunities for both fundamental research and applications across a wide range of industries. Ideally, characterization of graphene requires non-invasive techniques with single-atomic-layer thickness resolution and nanometer lateral resolution. Moreover, commercial application of graphene requires fast and large-area scanning capability. We demonstrate the optimized balance of image resolution and acquisition time of non-invasive confocal laser scanning microscopy (CLSM), rendering it an indispensable tool for rapid analysis of mass-produced graphene. It is powerful for analysis of 1-5 layers of exfoliated graphene on Si/SiO2, and allows us to distinguish the interfacial layer and 1-3 layers of epitaxial graphene on SiC substrates. Furthermore, CLSM shows excellent correlation with conventional optical microscopy, atomic force microscopy, Kelvin probe force microscopy, conductive atomic force microscopy, scanning electron microscopy and Raman mapping.
| S-EPMC6512973 | biostudies-literature
Project description:Chemical imaging, especially mid-infrared spectroscopic microscopy, enables label-free biomedical analyses while achieving expansive molecular sensitivity. However, its slow speed and poor image quality impede widespread adoption. We present a microscope that provides high-throughput recording, low noise, and high spatial resolution where the bottom-up design of its optical train facilitates dual-axis galvo laser scanning of a diffraction-limited focal point over large areas using custom, compound, infinity-corrected refractive objectives. We demonstrate whole-slide, speckle-free imaging in ~3 min per discrete wavelength at 10× magnification (2 μm/pixel) and high-resolution capability with its 20× counterpart (1 μm/pixel), both offering spatial quality at theoretical limits while maintaining high signal-to-noise ratios (>100:1). The data quality enables applications of modern machine learning and capabilities not previously feasible - 3D reconstructions using serial sections, comprehensive assessments of whole model organisms, and histological assessments of disease in time comparable to clinical workflows. Distinct from conventional approaches that focus on morphological investigations or immunostaining techniques, this development makes label-free imaging of minimally processed tissue practical.
| S-EPMC10457288 | biostudies-literature
Project description:No plant or cryptogam exists in nature without microorganisms associated with its tissues. Plants as microbial hosts are puzzles of different microhabitats, each of them colonized by specifically adapted microbiomes. The interactions with such microorganisms have drastic effects on the host fitness. Since the last 20 years, the combination of microscopic tools and molecular approaches contributed to new insights into microbe-host interactions. Particularly, confocal laser scanning microscopy (CLSM) facilitated the exploration of microbial habitats and allowed the observation of host-associated microorganisms in situ with an unprecedented accuracy. Here I present an overview of the progresses made in the study of the interactions between microorganisms and plants or plant-like organisms, focusing on the role of CLSM for the understanding of their significance. I critically discuss risks of misinterpretation when procedures of CLSM are not properly optimized. I also review approaches for quantitative and statistical analyses of CLSM images, the combination with other molecular and microscopic methods, and suggest the re-evaluation of natural autofluorescence. In this review, technical aspects were coupled with scientific outcomes, to facilitate the readers in identifying possible CLSM applications in their research or to expand their existing potential. The scope of this review is to highlight the importance of confocal microscopy in the study of plant-microbe interactions and also to be an inspiration for integrating microscopy with molecular techniques in future researches of microbial ecology.
| S-EPMC3945399 | biostudies-literature
Project description:Background/aimsThis systematic review critically evaluated peer-reviewed publications describing morphological features consistent with, or using terms related to, a 'neuroma' or 'microneuroma' in the human cornea using laser-scanning in vivo confocal microscopy (IVCM).MethodsThe review was prospectively registered on PROSPERO (CRD42020160038). Comprehensive literature searches were performed in Ovid MEDLINE, Ovid Embase and the Cochrane Library in November 2019. The review included primary research studies and reviews that described laser-scanning IVCM for examining human corneal nerves. Papers had to include at least one of a pre-specified set of keyword stems, broadly related to neuromas and microneuromas, to describe a corneal nerve feature.ResultsTwenty-five papers (20 original studies; 5 reviews) were eligible. Three original studies evaluated corneal nerve features in healthy eyes. Most papers assessed corneal nerves in ocular and systemic conditions; seven studies did not include a control/comparator group. There was overlap in terminology used to describe nerve features in healthy and diseased corneas (eg, bulb-like/bulbous, penetration, end/s/ing). Inspection of IVCM images within the papers revealed that features termed 'neuromas' and 'microneuromas' could potentially be physiological corneal stromal-epithelial nerve penetration sites. We identified inconsistent definitions for terms, and limitations in IVCM image acquisition, sampling and/or reporting that may introduce bias and lead to inaccurate representation of physiological nerve characteristics as pathological.ConclusionThese findings identify a need for consistent nomenclature and definitions, and rigorous IVCM scanning and analysis protocols to clarify the prevalence of physiological, as opposed to pathological, corneal nerve features.
| S-EPMC8353004 | biostudies-literature
Project description:HRT3 in vivo confocal microscopy (IVCM) images may indicate clinical outcome, but few studies have analysed this in fungal keratitis (FK). Adults with FK (diameter ≥3 mm) presenting to Aravind Eye Hospital, India from 2012-3 were enrolled prospectively. IVCM was performed at baseline, days 7, 14 and 21 post-enrolment (+/- 3 days where possible). Specific morphologies were identified in IVCM images by a grader masked to microbiology and clinical outcome (defined as good: healed/improving, or poor: enlarged ulcer, perforation or transplant/glue). Associations with final visit outcome assessed using logistic regression. 143 FK participants were enrolled; 87 had good outcome, 56 had poor outcome. Poor outcomes were associated with stellate interconnected cellular processes with no visible nuclei (OR 2.28, 95% CI: 1.03-5.06, p = 0.043) in baseline IVCM images, and fungal filaments (OR 6.48, 95% CI:2.50-16.78, p < 0.001) or honeycomb distribution of inflammatory cells (OR 5.24, 95% CI: 1.44-19.06, p = 0.012) in final visit images. Fungal filaments (OR 3.61, 95% CI:1.64-7.95, p = 0.001), stromal dendritiform cells (OR 2.88, 95% CI:1.17-7.11, p = 0.022), or stellate cellular processes with no visible nuclei (OR 2.09, 95% CI:1.14-3.82, p = 0.017) were associated with poor outcome if not in baseline but present in final visit images. IVCM can reveal morphological changes associated with clinical outcome.
| S-EPMC6554396 | biostudies-literature
Project description:Larval morphology of flies is traditionally studied using light microscopy, yet in the case of fine structures compound light microscopy is limited due to problems of resolution, illumination and depth of field, not allowing for precise recognition of sclerites' edges and interactions. Using larval instars of cyclorrhaphan Diptera, we show the usefulness of confocal laser scanning microscopy (CLSM) for studying the morphological characters of immature stages by taking advantage of the autofluorescent properties of cephaloskeleton structures. We compare data obtained from killed but unprepared larvae with those from larvae prepared by clearing according to two commonly used methods, either with potassium hydroxide or with Hoyer's medium. We also evaluated the CLSM application for examining already slide-mounted larvae stored in museum collections and those freshly prepared. Our results indicate that CLSM and 3D reconstruction are excellent for visualizing small, compound structures of cylrorrhaphan larvae cephaloskeleton, if appropriate clearing techniques, i.e. the application of KOH, are used. Maximum intensity projection of confocal data sets obtained from material freshly prepared and that stored in museum collection does not differ. Because of this and the fact that KOH is commonly used as a clearing method to examine the cephaloskeleton of Diptera larvae, it is possible, and highly recommended, to use slides already prepared with this method for re-examination by CLSM. We conclude that CLSM application can be an invaluable source of data for studies of larval morphology of Cyclorrhapha by way of taxonomic diagnoses, character identification and improvement in characters homologization.
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Project description:BACKGROUND: In clinical diagnostics, as well as in routine dermatology, the increased need for non-invasive diagnosis is currently satisfied by reflectance laser scanning microscopy. However, this technique has some limitations as it relies solely on differences in the reflection properties of epidermal and dermal structures. To date, the superior method of fluorescence laser scanning microscopy is not generally applied in dermatology and predominantly restricted to fluorescein as fluorescent tracer, which has a number of limitations. Therefore, we searched for an alternative fluorophore matching a novel skin imaging device to advance this promising diagnostic approach. METHODOLOGY/PRINCIPAL FINDINGS: Using a Vivascope®-1500 Multilaser microscope, we found that the fluorophore Indocyanine-Green (ICG) is well suited as a fluorescent marker for skin imaging in vivo after intradermal injection. ICG is one of few fluorescent dyes approved for use in humans. Its fluorescence properties are compatible with the application of a near-infrared laser, which penetrates deeper into the tissue than the standard 488 nm laser for fluorescein. ICG-fluorescence turned out to be much more stable than fluorescein in vivo, persisting for more than 48 hours without significant photobleaching whereas fluorescein fades within 2 hours. The well-defined intercellular staining pattern of ICG allows automated cell-recognition algorithms, which we accomplished with the free software CellProfiler, providing the possibility of quantitative high-content imaging. Furthermore, we demonstrate the superiority of ICG-based fluorescence microscopy for selected skin pathologies, including dermal nevi, irritant contact dermatitis and necrotic skin. CONCLUSIONS/SIGNIFICANCE: Our results introduce a novel in vivo skin imaging technique using ICG, which delivers a stable intercellular fluorescence signal ideal for morphological assessment down to sub-cellular detail. The application of ICG in combination with the near infrared laser opens new ways for minimal-invasive diagnosis and monitoring of skin disorders.
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Project description:The pH in the aqueous pores of poly(lactide-co-glycolide) (PLGA) matrix, also referred to as microclimate pH (?pH), is often uncontrolled, ranging from highly acidic to neutral pH range. The ?pH distribution inside protein-encapsulated PLGA microspheres was quantitatively evaluated using confocal laser scanning microscopy. The fluorescent response of Lysosensor yellow/blue dextran used to map ?pH in PLGA was influenced by the presence of encapsulated protein. The nonprotonated form of pyridyl group on the fluorescence probe at neutral pH was responsible for the interference, which was dependent on the type and concentration of protein. A method for correction of this interference based on estimating protein concentration inside the microspheres was established and validated. After correction of the influence, the ?pH distribution kinetics inside microspheres was evaluated for different PLGA 50/50 microsphere formulations under physiological conditions for 4 weeks. Generally, the ?pH acidity increased with the progression of incubation time. The coincorporation of poorly soluble base, magnesium carbonate, in the microspheres prolonged the appearance of detectable acidity for up to 3 weeks. Co-addition of an acetate buffer was able to control the ?pH over a slightly acidic range (around pH 4.7) after two week incubation. Microspheres prepared from a lower polymer concentration exhibited a higher ?pH, likely owing to reduced diffusional resistance to acidic degradation products. The stability of protein was enhanced by addition of MgCO(3), acetate buffer, or by reduced polymer concentration in the preparation, as evidenced by more soluble protein recovered after incubation. Hence, the ?pH imaging technique developed can be employed in the future for optimization of formulation strategies for controlling ?pH and stabilizing encapsulated proteins.
| S-EPMC3817562 | biostudies-literature