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Vibrational spectroscopic imaging and live cell video microscopy for studying differentiation of primary human alveolar epithelial cells
摘要： Alveolar type II(ATII) cells in the peripheral human lung spontaneously differentiate towards alveolar type I(ATI) cells, thus enabling air-blood barrier formation. Here, linear Raman and coherent anti-Stokes Raman scattering (CARS) microscopy are applied to study cell differentiation of freshly isolated ATII cells. The Raman spectra can successfully be correlated to gradual morphological and molecular changes during cell differentiation. Alveolar surfactant rich vesicles in ATII cells are identified based on phospholipid vibrations, while ATI-like cells are characterized by the absence of vesicular structures. Complementary, CARS microscopy allows for 3D visualization of lipid vesicles within ATII cells and their secretion, while hyperspectral CARS enables the distinction between cellular proteins and lipids according to their vibrational signatures. This study paves the path for further label-free investigations of lung cells and the role of the pulmonary surfactant, thus also providing a basis for rational development of future lung therapeutics.
关键词： confocal laser scanning microscopy,confocal Raman microscopy,pneumocyte type II differentiation,CARS microscopy,cell imaging
更新于2025-09-23 15:23:52
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Tracking host infection and reproduction of <i>Peronospora salviaea??officinalis</i> using an improved method for confocal laser scanning microscopy
摘要： Peronospora salviae-officinalis, the causal agent of downy mildew on common sage, is an obligate biotrophic pathogen. It grows in the intercellular spaces of the leaf tissue of sage and forms intracellular haustoria to interface with host cells. Although P. salviae-officinalis was described as a species of its own 10 years ago, the infection process remains obscure. To address this, a histological study of various infection events, from the adhesion of conidia on the leaf surface to de novo sporulation is presented here. As histological studies of oomycetes are challenging due to the lack of chitin in their cell wall, we also present an improved method for staining downy mildews for confocal laser scanning microscopy as well as evaluating the potential of autofluorescence of fixed nonstained samples. For staining, a 1:1 mixture of aniline blue and trypan blue was found most suitable and was used for staining of oomycete and plant structures, allowing discrimination between them as well as the visualization of plant immune responses. The method was also used to examine samples of Peronospora lamii on Lamium purpureum and Peronospora belbahrii on Ocimum basilicum, demonstrating the potential of the presented histological method for studying the infection processes of downy mildews in general.
关键词： aniline blue,Peronospora belbahrii,confocal laser scanning microscopy,Peronospora salviae-officinalis,trypan blue,Peronospora lamii
更新于2025-09-23 15:21:01
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Fluorescence spectroscopy of ancient sedimentary organic matter via confocal laser scanning microscopy (CLSM)
摘要： Fluorescence spectroscopy via confocal laser scanning microscopy (CLSM) was used to analyze ancient sedimentary organic matter, including Tasmanites microfossils in Devonian shale and Gloeocapsomorpha prisca (G. prisca) in Ordovician kukersite from North American basins. We examined fluorescence emission as a function of excitation laser wavelength, sample orientation, and with respect to location within individual organic entities and in transects across bedded organic matter. Results from spectral scans of the same field of view in Tasmanites with different laser lines showed progressive red-shift in emission maxima with longer excitation wavelengths. This result indicates steady-state Tasmanites fluorescence emission is an overlapping combination of emission from multiple fluorophore functions. Stokes shift decreased with increasing excitation wavelength, further suggesting the presence of multiple fluorophore functions with different S1 → S0 transition energies. This observation also indicates that at longer excitation wavelengths, less absorbed light energy is dissipated via collisional transfer than at shorter excitation wavelengths and may suggest fewer polar functions are preferentially absorbing. Confirming earlier results, emission spectra observed from high fluorescence intensity regions (fold apices) in individual Tasmanites are blue-shifted relative to emission from other locations in the same microfossil. We suggest high intensity emission is from photoselective alignment of polarized excitation with the fluorophore absorption and emission transition moment. The blue shift observed in regions of high intensity emission may be due to relative absence or realignment of polar species, e.g., bridging ether or ester functions, although variations in O abundance could not be confirmed with preliminary time-of-flight secondary ion mass spectrometry (TOF-SIMS) analysis. Tasmanites occurring in consolidated sediments are flattened from original spherical morphology and, in optical microscopy, this burial deformation results in generally parallel extinction (strain-influenced) and positive elongation. The deformation also induces fluorescence anisotropy observed as variations in emission wavelength when individual Tasmanites are measured from their long axis parallel to bedding, whereas this effect is absent in bedding-normal view. Transects from G. prisca-rich source layers into adjacent reservoir layers show decrease in fluorescence intensity and spectral red-shift (increase in full-width half-maximum with increasing red portion of the half-width). These results may suggest an increase in fluorescence quenching across the source-to-reservoir transition zone, consistent with an increase in aromaticity following petroleum expulsion and migration. These observations are supported by increasing reflectance values measured across similar micro-scale transects. Our results highlight the applicability of CLSM as a broad and under-utilized approach for the characterization of sedimentary organic matter and are discussed with perspective toward petroleum processes and thermal indices research.
关键词： Gloeocapsomorpha prisca,Confocal laser scanning microscopy,Tasmanites,Petroleum processes,Fluorescence spectroscopy,Sedimentary organic matter,Thermal indices
更新于2025-09-23 15:21:01
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Editorial: Advances in Label Free Tissue Imaging With Laser Scanning Microscopy Techniques
摘要： Significant efforts are being spent at the time being for transferring various laser scanning microscopy (LSM) techniques to the realm of tissue characterization, because of their potential to circumvent some of the most important disadvantages of traditional histopathology approaches based on excisional biopsy and tissue staining. Although conventional histopathology is currently regarded as a golden standard for the diagnosing pathologies that reflect in tissular modification (e.g., cancers), limitations such as long diagnosis time, invasiveness, artifacts, sampling error, time consumption, high costs, and interpretive variability make such approaches to be impractical in many scenarios, while also placing considerable pressure on the sustainability of healthcare systems around the world. The potential of LSM techniques to contribute to overcoming these aspects derives from their “non-invasive” character. They can exploit various endogenous optical signals generated by tissues upon interaction with a laser beam and are able to provide optical sections (virtual biopsies) that reflect the tissular architecture at controlled depths. Many studies reported to date showed that LSM techniques can provide label-free information of similar pathologic relevance to the information collected for characterization/confirmation purposes with traditional histopathology approaches. These techniques are thus capable of probing optical properties of tissues with deep implications for resolving important anatomical and physiological aspects which represent hallmarks for disease predisposition and progression. To date techniques such as Confocal Laser Scanning Microscopy (CLSM) [1], Fluorescence Lifetime Imaging (FLIM) [2], Two-Photon Excited Fluorescence Microscopy (TPEF) [2–6], Second Harmonic Generation Microscopy (SHG) [5, 6], Third Harmonic Generation Microscopy (THG) [4], Coherent Anti-Stokes Raman Scattering Microscopy (CARS) [3, 7], as well as other LSM variants such as the Brillouin Microscopy [8] have already been demonstrated to be powerful tools for investigating tissue morphology, functionality, and biochemical composition with high spatial and temporal resolution. In the opinion of many, these techniques, together with investigations approaches based on their combined use, will soon become the central element of the default tissue characterization frameworks for both ex vivo and in vivo assays. Furthermore, emerging LSM techniques exploiting various ingenious strategies to achieve superresolved images in a label-free manner [9–12] are also likely to be transferred soon toward applications addressing tissue imaging.
关键词： tissue imaging,two photon excitation fluorescence microscopy,Brillouin microspectroscopy,laser scanning microscopy,second harmonic generation microscopy
更新于2025-09-23 15:19:57
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Characterization of Nanoencapsulated Food Ingredients || Confocal laser scanning microscopy (CLSM) of nanoencapsulated food ingredients
摘要： Encapsulation is a process of entrapping bioactive compounds (e.g., antioxidants, antimicrobials, essential fatty acids, probiotics, vitamins, etc.) within a protecting wall material such as lipid-based or biopolymeric carriers. Encapsulation can help to enhance the stability and bioaccessibility as well as controlled release of bioactive materials and mask unwanted properties of encapsulated ingredients. Nanoparticles have a diameter range from 0.1 nm to 1 μm and show great potential to develop new products and applications in food and packaging industries. The performance of nanocarriers formed by encapsulation processes fundamentally depends on various technical aspects such as carrier wall thickness, morphology, and quality. Therefore, measuring and monitoring of coating thickness and morphology of the nanocarriers is an essential process to control the nanoencapsulation procedure. An excessively thick coating layer would lead to delayed release of bioactive materials, increase of coating time, and consequently, encapsulation costs, while an excessively thin capsule would cause interruption in the release and stability of nanoparticles. Spectroscopic and microscopic techniques are important tools for analyzing the morphology, structure, and coating thickness of nanoparticles. Different electron microscopies have been introduced to investigate nano- and micro-carriers. Scanning electronic microscopy (SEM), transmission electron microscopy (TEM), atomic force microscopy (AFM), near-IR spectra, laser-induced breakdown spectroscopy (LIBS), and confocal laser scanning microscopy (CLSM) are some of these light and microscopic methods.
关键词： Encapsulation,Nanoencapsulated food ingredients,Confocal laser scanning microscopy,Bioactive compounds,Nanoparticles
更新于2025-09-23 15:19:57
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Determination of parvovirus retention profiles in virus filter membranes using laser scanning microscopy
摘要： Virus filtration is a highly effective method in the downstream processing of biotherapeutic products to provide effective removal of potential infectious agents based on a size exclusion mechanism. The direct visualization of viruses retained inside the filter membrane represents a valuable tool to get a deeper understanding of the filtration process and to explain observations of virus breakthrough under particular operating conditions. Parvoviruses, which are used as worst-case models in validation studies, were purified and labeled with fluorescent dyes to detect their retention pattern inside the filter membrane using laser scanning microscopy. Critical factors influencing the reproducibility and accuracy of the approach were identified and optimized. The retention profiles revealed detectable differences between viruses, suggesting that the use of bacteriophages or nanoparticles as surrogates is limited in their applicability to accurately predict the behavior of parvoviruses in filter membranes. The established method enables a direct and quantitative analysis of the virus retention profile, adding a valuable tool to the conventional measurement of the viral load reduction to better understand the mechanism underlying the removal of viruses during nanofiltration of biotherapeutic products.
关键词： Parvovirus retention profile,Virus filtration,Nanofiltration,Laser scanning microscopy,Filter membrane
更新于2025-09-23 15:19:57
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Visualization of materials using the confocal laser scanning microscopy technique
摘要： The development of materials science always benefits from advanced characterizations. Currently, imaging techniques are of great technological importance in both fundamental and applied research on materials. In comparison to conventional visualization methods, confocal laser scanning microscopy (CLSM) is non-invasive, with macroscale and high-contrast scanning, a simple and fast sample preparation procedure as well as easy operation. In addition, CLSM allows rapid acquisition of longitudinal and cross-sectional images at any position in a material. Therefore, the CLSM-based visualization technique could provide direct and model-independent insight into material characterizations. This review summarizes the recent applications of CLSM in materials science. The current CLSM approaches for the visualization of surface structures, internal structures, spatial structures and reaction processes are discussed in detail. Finally, we provide our thoughts and predictions on the future development of CLSM in materials science. The purpose of this review is to guide researchers to build a suitable CLSM approach for material characterizations, and to open viable opportunities and inspirations for the development of new strategies aiming at the preparation of advanced materials. We hope that this review will be useful for a wide range of research communities of materials science, chemistry, and engineering.
关键词： confocal laser scanning microscopy,reaction processes,internal structures,materials science,surface structures,spatial structures,visualization
更新于2025-09-19 17:13:59
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Evaluation of the Bactericidal Activity of a Hyaluronic Acid-Vehicled Clarithromycin Antibiotic Mixture by Confocal Laser Scanning Microscopy
摘要： Confocal laser scanning microscopy (CLSM) was used to evaluate the antibacterial effect and depth of action of a novel clarithromycin-containing triple antibiotic mixture, which was proposed for root canal disinfection in dental pulp regeneration. A previous study reported that this mixture had no tooth discoloration effects in vitro. After infection with Enterococcus faecalis for 3 weeks, the dentinal tubules in the cylindrical root specimens were exposed to different antibiotic mixtures: ciprofloxacin, metronidazole and minocycline (3-MIX); ciprofloxacin, metronidazole and clarithromycin (3-MIXC) and ciprofloxacin and metronidazole (2-MIX). Each antibiotic formulation was mixed with macrogol (MG) or hyaluronic acid (HA) vehicles. CLSM and viability staining were used to quantitatively analyze the mean depth of the antibacterial effect and the proportions of dead and live bacteria inside the dentinal tubules. The 3-MIX and 3-MIXC demonstrated a similar depth of action. The mean proportion of dead bacteria was similar in the 3-MIX and 3-MIXC groups, and both were statistically higher than that of 2-MIX (p = 0.014). Each antibiotic mixture showed a higher bactericidal efficacy if conveyed with HA, compared to MG (3-MIX, p = 0.019; 3-MIXC, p = 0.013 and 2-MIX, p = 0.0125). The depth of action and the antibacterial efficacy of 3-MIXC seemed comparable with 3-MIX.
关键词： triple antibiotic paste,confocal laser scanning microscopy,double antibiotic paste,regenerative endodontic procedures,hyaluronic acid
更新于2025-09-19 17:13:59
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Microscopy and Analysis || Automatic Interpretation of Melanocytic Images in Confocal Laser Scanning Microscopy
摘要： The frequency of melanoma doubles every 20 years. The early detection of malignant changes augments the therapy success. Confocal laser scanning microscopy (CLSM) enables the noninvasive examination of skin tissue. To diminish the need for training and to improve diagnostic accuracy, computer-aided diagnostic systems are required. Two approaches are presented: a multiresolution analysis and an approach based on deep layer convolutional neural networks. For the diagnosis of the CLSM views, architectural structures such as micro-anatomic structures and cell nests are used as guidelines by the dermatologists. Features based on the wavelet transform enable an exploration of architectural structures at different spatial scales. The subjective diagnostic criteria are objectively reproduced. A tree-based machine-learning algorithm captures the decision structure explicitly and the decision steps are used as diagnostic rules. Deep layer neural networks require no a priori domain knowledge. They are capable of learning their own discriminatory features through the direct analysis of image data. However, deep layer neural networks require large amounts of processing power to learn. Therefore, modern neural network training is performed using graphics cards, which typically possess many hundreds of small, modestly powerful cores that calculate massively in parallel. Readers will learn how to apply multiresolution analysis and modern deep learning neural network techniques to medical image analysis problems.
关键词： convolutional neural networks,skin lesions,multiresolution image analysis,computer-aided diagnosis,confocal laser scanning microscopy,machine learning
更新于2025-09-19 17:13:59
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Nonlinear absorption and scattering of a single plasmonic nanostructure characterized by <i>x</i> -scan technique
摘要： 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.
关键词： nonlinear absorption,absorption cross section,nonlinear scattering,single gold nanostructures,laser scanning microscopy,nanoplasmonics
更新于2025-09-16 10:30:52