In this study, we employed stimulated Raman scattering (SRS) microscopy, augmented with sum frequency generation, to characterize complex solid-state mixtures, containing many solid-state forms of the same compound, for the first time. Five crystalline forms and one amorphous form of lactose were characterized and resolved, including two more recently defined anhydrous solid-state forms. Additionally, the complex solid-state character of several commercially available pharmaceutical tableting and inhalation grades of lactose was profiled. The advanced multimodal label-free microscopy method enabled visualization of the distribution of the solid-state forms with submicron spatial resolution, including the detection of trace levels. In addition, quantitative solid-state compositions of the lactose products were estimated. Overall SRS microscopy allows sensitive and specific spatially resolved solid-state characterization of complex mixtures, beyond what is possible with established (nonspatially resolved) characterization methods.

• MeSH
• Lactose * chemistry   analysis   MeSH
• Pharmaceutical Preparations chemistry   MeSH
• Microscopy MeSH
• Nonlinear Optical Microscopy * methods   MeSH
• Spectrum Analysis, Raman * methods   MeSH
• Tablets chemistry   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Lactose * MeSH
• Pharmaceutical Preparations MeSH
• Tablets MeSH

AIM: Clubfoot is a congenital deformity affecting the musculoskeletal system, resulting in contracted and stiff tissue in the medial part of the foot. Minoxidil (MXD) has an inhibitory effect on lysyl hydroxylase, which influences the quality of extracellular matrix crosslinking, and could therefore be used to reduce the stiffness and to improve the flexibility of the tissue. We assessed the in vitro antifibrotic effects of minoxidil on clubfoot-derived cells. METHODS: Cell viability and proliferation were quantified by xCELLigence, MTS, and LIVE/DEAD assays. The amount of collagen I deposited into the extracellular matrix was quantified using immunofluorescence with subsequent image segmentation analysis, hydroxyproline assay, and Second Harmonic Generation imaging. Extracellular matrix contraction was studied in a 3D model of cell-populated collagen gel lattices. RESULTS: MXD concentrations of 0.25, 0.5, and 0.75 mM inhibited the cell proliferation in a concentration-dependent manner without causing a cytotoxic effect. Exposure to ≥0.5 mM MXD resulted in a decrease in collagen type I accumulation after 8 and 21 days in culture. Changes in collagen fiber assembly were observed by immunofluorescence microscopy and nonlinear optical microscopy (second harmonic generation). MXD also inhibited the contraction of cell-populated collagen lattices (0.5 mM by 22%; 0.75 mM by 28%). CONCLUSIONS: Minoxidil exerts an in vitro inhibitory effect on the cell proliferation, collagen accumulation, and extracellular matrix contraction processes that are associated with clubfoot fibrosis. This study provides important preliminary results demonstrating the potential relevance of MXD for adjuvant pharmacological therapy in standard treatment of relapsed clubfoot.

• Keywords
• CTEV, Relapsed clubfoot, collagen type I, congenital idiopathic talipes equinovarus, fibrosis, minoxidil,
• MeSH
• Collagen Type I MeSH
• Collagen MeSH
• Conservative Treatment MeSH
• Humans MeSH
• Minoxidil pharmacology   MeSH
• Clubfoot * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Collagen Type I MeSH
• Collagen MeSH
• Minoxidil MeSH

Nitrogen (N) is an essential macronutrient for microalgae, influencing their productivity, composition, and growth dynamics. Despite the dramatic consequences of N starvation, many free-living and endosymbiotic microalgae thrive in N-poor and N-fluctuating environments, giving rise to questions about the existence and nature of their long-term N reserves. Our understanding of these processes requires a unequivocal identification of the N reserves in microalgal cells as well as their turnover kinetics and subcellular localization. Herein, we identified crystalline guanine as the enigmatic large-capacity and rapid-turnover N reserve of microalgae. The identification was unambiguously supported by confocal Raman, fluorescence, and analytical transmission electron microscopies as well as stable isotope labeling. We discovered that the storing capacity for crystalline guanine by the marine dinoflagellate Amphidiniumcarterae was sufficient to support N requirements for several new generations. We determined that N reserves were rapidly accumulated from guanine available in the environment as well as biosynthesized from various N-containing nutrients. Storage of exogenic N in the form of crystalline guanine was found broadly distributed across taxonomically distant groups of microalgae from diverse habitats, from freshwater and marine free-living forms to endosymbiotic microalgae of reef-building corals (Acropora millepora, Euphyllia paraancora). We propose that crystalline guanine is the elusive N depot that mitigates the negative consequences of episodic N shortage. Guanine (C5H5N5O) may act similarly to cyanophycin (C10H19N5O5) granules in cyanobacteria. Considering the phytoplankton nitrogen pool size and dynamics, guanine is proposed to be an important storage form participating in the global N cycle.

• Keywords
• coral, guanine, nitrogen cycle, nutrient storage, phytoplankton,
• MeSH
• Dinoflagellida chemistry   metabolism   MeSH
• Nitrogen metabolism   MeSH
• Ecosystem MeSH
• Guanine chemistry   metabolism   MeSH
• Kinetics MeSH
• Anthozoa MeSH
• Crystallization MeSH
• Microalgae chemistry   metabolism   MeSH
• Nonlinear Optical Microscopy methods   MeSH
• Symbiosis MeSH
• Microscopy, Electron, Transmission MeSH
• Tropical Climate MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Video-Audio Media MeSH
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Arctic Regions MeSH
• Names of Substances
• Nitrogen MeSH
• Guanine MeSH

The synthesis and characterization of two thiophenol-modified fluorographene derivatives, namely methoxythiophenol-and dimethylaminothiophenol-modified fluorographenes, are reported, while their third-order nonlinear optical response were thoroughly investigated under both visible (532 nm) and infrared (1064 nm) with 35 ps and 4 ns laser pulses. The graphene derivatives were obtained by partial nucleophilic substitution/reduction of fluorographene by the corresponding organic thiophenols, and were fully characterized by techniques including infrared/Raman spectroscopy, X-ray photoelectron spectroscopy, atomic force spectroscopy, and high-resolution transmission microscopy. This type of modification resulted in graphenic structures where the attached thiol groups, sp2 domains, and the residual fluorine groups act as donors, π bridges, and acceptors, respectively. Both derivatives exhibited large nonlinear optical response compared to fluorographene, and have potential applications in optical limiting as an alternative to fullerenes.

• Keywords
• fluorographene, nonlinear optical properties, optical limiting, surface modification, thiophenol derivatives,
• Publication type
• Journal Article MeSH

INTRODUCTION: Remodeling of human placental membranes (amniochorionic or fetalmembrane) throughout gestation, a necessity to accommodate increasing uterine volume, involves continuous alterations (replacement of cells and remodeling of extracellular matrix). Methodologic limitations have obscured microscopic determination of cellular and layer-level alterations. This study used a combination of advanced imaging by multiphoton autofluorescence microscopy (MPAM) and second harmonic generation (SHG) microscopy along with tissue optical clearing to characterize the 3Dimensional multilayer organization of placental membranes. METHODS: Placental membranes biopsies (6 mm) collected from term, not-in-labor cesarean deliveries (n = 7) were fixed in 10% formalin (native) or treated with 2,2'-thiodiethanol to render them transparent for deeper imaging. Native and cleared tissues were imaged using MPAM (cellular autofluorescence) and SHG (fibrillar collagen). Depth z-stacks captured the amnion epithelium, underlying matrix layers, and in the cleared biopsies, the decidua layer. RESULTS: MPAM and SHG revealed fetal membrane epithelial topography and collagen organization in multiple matrix layers. Term amnion layers showed epithelial shedding and gaps. Optical clearing provided full-depth imaging with improved visualization of collagen structure, mesenchymal cells in extracellular matrix layers, and decidua morphology. Layer thicknesses measured by imaging corroborated with histology. Mosaic tiling of MPAM/SHG image stacks allowed large area visualization of entire biopsies. CONCLUSION: MPAM-SHG microscopy allowed for study of this multi-layered tissue and revealed shedding, gap formation, and other structural changes. This approach could be used to study structural changes associated with membranes as well as other uterine tissues to better understand events in normal and abnormal parturition.

• Keywords
• Amniochorion, Epithelial shedding, Membrane rupture, Multiphoton microscopy, Nonlinear optical microscopy, Optical clearing, Placenta, Pregnancy and parturition, Second harmonic generation microscopy,
• MeSH
• Extracellular Matrix MeSH
• Extraembryonic Membranes anatomy & histology   diagnostic imaging   MeSH
• Histocytological Preparation Techniques MeSH
• Humans MeSH
• Microscopy, Fluorescence, Multiphoton * MeSH
• Second Harmonic Generation Microscopy * MeSH
• Placenta anatomy & histology   diagnostic imaging   MeSH
• Pregnancy MeSH
• Imaging, Three-Dimensional MeSH
• Check Tag
• Humans MeSH
• Pregnancy MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH

Several methods based on single- and two-photon fluorescence detected linear dichroism have recently been used to determine the orientational distributions of fluorescent dyes in lipid membranes. However, these determinations relied on simplified descriptions of nonlinear anisotropic properties of the dye molecules, using a transition dipole-moment-like vector instead of an absorptivity tensor. To investigate the validity of the vector approximation, we have now carried out a combination of computer simulations and polarization microscopy experiments on two representative fluorescent dyes (DiI and F2N12S) embedded in aqueous phosphatidylcholine bilayers. Our results indicate that a simplified vector-like treatment of the two-photon transition tensor is applicable for molecular geometries sampled in the membrane at ambient conditions. Furthermore, our results allow evaluation of several distinct polarization microscopy techniques. In combination, our results point to a robust and accurate experimental and computational treatment of orientational distributions of DiI, F2N12S, and related dyes (including Cy3, Cy5, and others), with implications to monitoring physiologically relevant processes in cellular membranes in a novel way.

• MeSH
• Cell Membrane chemistry   MeSH
• Fluorescent Dyes chemistry   MeSH
• Phospholipids * MeSH
• Molecular Conformation MeSH
• Nonlinear Dynamics * MeSH
• Optical Phenomena * MeSH
• Molecular Dynamics Simulation MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Fluorescent Dyes MeSH
• Phospholipids * MeSH

This paper highlights some of the key technologies of using two innovative molecular imaging modalites, magnetic resonance imaging (MRI) and nonlinear optical microscopy, for imaging intravenously injected ultra small paramagnetic iron oxide nanoparticles cross linked with antibodies (CLUSPIO) in the amyotrophic lateral sclerosis (ALS) experimental model in vivo or ex vivo, respectively. Intensive efforts have been made in investigating the causes of abnormalities in lipid metabolism, monitored in some neurodegenerative disorders systems. It has been shown that an abnormal accumulation of some common lipids in motor nerve cells may play a critical role in the development of amyotrophic lateral sclerosis. The presented experiments were performed on brain specimens from the transgenic rat model expressing multiple copies of mutated (G93A) human SOD-1 gene, after CD4+ lymphocytes were magnetically labeled with i.v.i. CLUSPIO antibodies. In vivo MRI revealed marked signal intensity enhancements in specific pathological regions of the ALS rat brain as compared to the wild type. Surface-enhanced coherent anti-Stokes Raman scattering (SECARS) microscopy indicated cellular interactions based on lipids association to anti-CD4 CLUSPIO.

• MeSH
• Amyotrophic Lateral Sclerosis genetics   immunology   metabolism   pathology   MeSH
• CD4-Positive T-Lymphocytes immunology   metabolism   pathology   MeSH
• Dextrans MeSH
• Contrast Media MeSH
• Rats MeSH
• Humans MeSH
• Magnetic Resonance Imaging * MeSH
• Magnetite Nanoparticles MeSH
• Lipid Metabolism * MeSH
• Microscopy * MeSH
• Disease Models, Animal MeSH
• Molecular Imaging methods   MeSH
• Brain immunology   metabolism   pathology   MeSH
• Mutation MeSH
• Lipid Peroxidation MeSH
• Rats, Sprague-Dawley MeSH
• Rats, Transgenic MeSH
• Antibodies MeSH
• Spectrum Analysis, Raman * MeSH
• Superoxide Dismutase-1 MeSH
• Superoxide Dismutase genetics   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Dextrans MeSH
• ferumoxtran-10 MeSH Browser
• Contrast Media MeSH
• Magnetite Nanoparticles MeSH
• Antibodies MeSH
• SOD1 protein, human MeSH Browser
• Sod1 protein, rat MeSH Browser
• Superoxide Dismutase-1 MeSH
• Superoxide Dismutase MeSH

Fluorescence microscopy using single molecule imaging and localization (PALM, STORM, and similar approaches) has quickly been adopted as a convenient method for obtaining multicolor, 3D superresolution images of biological samples. Using an approach based on extensive Monte Carlo simulations, we examined the performance of various noise reducing filters required for the detection of candidate molecules. We determined a suitable noise reduction method and derived an optimal, nonlinear threshold which minimizes detection errors introduced by conventional algorithms. We also present a new technique for visualization of single molecule localization microscopy data based on adaptively jittered 2D histograms. We have used our new methods to image both Atto565-phalloidin labeled actin in fibroblast cells, and mCitrine-erbB3 expressed in A431 cells. The enhanced methods developed here were crucial in processing the data we obtained from these samples, as the overall signal to noise ratio was quite low.

• MeSH
• Actins metabolism   MeSH
• Algorithms * MeSH
• Phalloidine metabolism   MeSH
• Humans MeSH
• Monte Carlo Method MeSH
• Microscopy methods   MeSH
• Cell Line, Tumor MeSH
• Nonlinear Dynamics MeSH
• Computer Simulation MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Actins MeSH
• Phalloidine MeSH

We investigated possibilities of the combination of the one- and two-photon excitation microscopy for examination of the experimental melanoma tissue in vivo, in mice under general anesthesia, and ex vivo on freshly harvested specimens. Our aim was to obtain sufficiently informative images of unstained tumor tissues and their modifications after hyperthermia treatment. The mouse experimental melanoma structure was studied and compared with normal tissue from the same animal by using confocal and nonlinear microscopy techniques based on (i) one-photon excitation (1PE) fluorescence, (ii) 1PE reflectance, (iii) second harmonic generation imaging, and (iv) two-photon excitation autofluorescence. We checked different spectral conditions and other settings of image acquisition, as well as combinations of the above imaging modalities, to fully exploit the potential of these techniques in the evaluation of treated and untreated cancer tissue morphology. Our approach enabled to reveal the collagen fiber network in relation with the other tissues, and to identify invasive tumor cells. It also proved to be useful for the examination of interrelationships between functional and morphological aspects based on optical properties of the tissues, especially in studies of changes between the tumor and control tissue, as well as changes induced by physical treatments, e.g., delivery of microwave hyperthermia treatment. These differences were also evaluated quantitatively, when we found out that the maximum Euler-Poincaré characteristic reflects well the melanoma morphological structure. The results showed that the proposed investigative approach could be suitable also for a direct evaluation of tissue modifications induced by clinical interventions.

• MeSH
• Hyperthermia, Induced * MeSH
• Melanoma, Experimental pathology   radiotherapy   MeSH
• Microscopy * MeSH
• Mice MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH