Morphology, fluorescence properties, and their photothermal changes of poly(substituted thiophene) films revealed by near-field fluorescence microspectroscopy

The morphology and fluorescence spectrum of poly{3-[2-(N-dodecylcarbamoyloxy)ethyl]thiophene-2,5-diyl} film were examined with spatial resolution of 100 nm using near-field fluorescence microspectroscopy. Fluorescence spectra observed at protruding domains were blue-shifted compared with flat areas, and further blue-shift was observed there more appreciably by long-time irradiation via a near-field scanning optical microscope probe. It is considered that the polymer chains at the protruding domains take disordered conformations, in which conjugated lengths are shorter and further disordering can be induced more easily by irradiation compared with those in the flat areas.     

Near-field nano-ellipsometer for ultrathin film characterization

We describe a near‐field ellipsometer for accurate characterization of ultrathin dielectric films. Optical tunnelling mimics the absorption in metallic films, enabling accurate measurement of the refractive index of ultrathin dielectric film. A regression model shows that a refractive index resolution of 0.001 for films as thin as 1 nm is possible. A solid‐immersion nano‐ellipsometer that incorporates this near‐field ellipsometric technique with a solid‐immersion lens is constructed to demonstrate the viability of this technique. Such a nano‐ellipsometer can accurately characterize thin films ranging in thickness from subnanometre to micrometres with potential transverse resolution of the order of 100 nm.     

透射式光热偏转技术实验装置的建立及应用

应用透射式的光热偏转光谱技术 ,建立了一套实验装置。应用这套实验装置 ,测量了 5 2 0 nm～ 5 5 5 nm(Ti O2 +Zr O2 ) /Si O2 多层高反膜的激光损伤情况。实验结果表明透射式的光热偏转技术是一种非常灵敏的热波探测技术 ,即使在小功率的泵浦能量条件下 ,它也能获得比较大的光热偏转信号     

Infra-red microspectroscopy of hydrated biological systems: design and construction of a new cell with atmospheric control for the study of plant cell walls

A new hydration cell has been constructed that allows wet biological samples, or samples of controlled moisture content, to be analysed in situ using infra-red microspectroscopy. The cell has been used to show that there are minor spectral changes associated with the hydration of pectin and tomato pericarp cell walls and slightly more significant changes in onion and carrot epidermal walls. The cell was also used to show that molecular orientations of polymers, previously observed in dry cell walls, were also to be seen in hydrated walls. For cell walls of onion and carrot epidermis, it was shown that the orientations of cell wall polymers are not affected by hydration. Furthermore, the polymer orientations in cell walls of fully elongated onion epidermal cells are different from those of elongating carrot epidermis. By using the hydration cell, it is now possible to investigate both fresh samples and wet systems routinely. The applications of this to the study of biological materials with infra-red microspectroscopy are discussed.     

统计最优近场声全息中正则化方法的仿真与实验研究

基于统计最优近场声全息的声场重建是典型的反问题,测量误差的存在使得该反问题的求解具有"不适定"特性.为此,通过两种不同的正则化方法解决上述问题:第一种方法采用标准的Tikhonov正则算子,并结合需要测量信号先验知识的Morozov偏差原则;第二种方法采用标准的Tikhonov正则算子,并结合不需要测量信号先验知识的Engl误差最小化原则.统计最优近场声全息在空间域直接实现声场重建的过程可以转化为矩阵的特征值求解问题,这为在重建过程中应用上述正则化方法提供了可能.数值仿真和实验结果表明,两种正则化方法可以有效地提高声场的重建精度.     

Comparative characterization of human fetal neural stem cells and induced neural stem cells from peripheral blood mononuclear cells

Human-induced neural stem cells (iNSCs) transplantation is a potential treatment of neurodegenerationdiseases. However, whether the reprogrammed cells have the same characterizations as human fetal neural stem cellsneeds further exploration. Here we isolated human fetal neural stem cells from aborted 12-week fetal brains andcompared with iNSCs reprogrammed from human peripheral blood mononuclear cells in gene expression, proliferationability, differentiation capacity, and the responses to tumor necrosis factor-α. We found that iNSCs and NSCs bothexpressed neural stem cell markers Nestin, SOX1, and SOX2. However, only iNSCs can be patterned into dopaminergicneurons and motor neurons. Furthermore, both iNSCs and NSCs can differentiate into oligodendrocyte progenitorcells. In addition, a low dose of tumor necrosis factor-α did not inhibit the proliferation and differentiation of iNSCsand NSCs. In conclusion, iNSCs have properties similar to, and even better than, fetal neural stem cells and may besuitable for disease modeling and transplantation.     

Brief Note: Isolation,culture, characterization and optimization of human corneal stem cells

The effects of human versus mouse EGF on cell growth and culture duration were studied to optimize a human limbal stem cells culture method for therapeutical autologous transplantation. Limbal cells were obtained by trypsin digestion and transferred to a culture medium. The time needed to reach full confluence in culture was determined. Specific antibodies to corneal stem cell marker (P63) versus corneal epithelial differentiation marker (K3) were used for histochemical determinations. A high proportion of P63 positive cells (85± 4.6%), and a correspondingly low proportion K3 positive cells (15 ± 3.8%) indicated that most cultured cells remained undifferentiated and were considered as stem cells (mean ± SE, n=10). Cultures reached full confluency after 17.3 ± 1.2 days when the medium was supplemented with human EGF, while 21.7 ± 1.5 days were needed when the medium was supplemented with mouse EGF. The results showed that limbal stem cells proliferate more easily and reach to full confluency in a shorter time if the medium is supplemented with hEGF rather than with mEGF.     

Phenotypic,morphological, and metabolic characterization of vascular-spheres from human vascular mesenchymal stem cells

The ability to form spheroids under non-adherent conditions is a well-known property of human mesenchymal stem cells (hMSCs), in addition to stemness and multilineage differentiation features. In the present study, we tested the ability of hMSCs isolated from the vascular wall (hVW-MSCs) to grow as spheres, and provide a characterization of this 3D model. hVW-MSCs were isolated from femoral arteries through enzymatic digestion. Spheres were obtained using ultra-low attachment and hanging drop methods. Immunophenotype and pluripotent genes (SOX-2, OCT-4, NANOG) were analyzed by immunocytochemistry and real-time PCR, respectively. Spheres histological and ultrastructural architecture were examined. Cell viability and proliferative capacity were measured using LIVE/DEATH assay and ki-67 proliferation marker. Metabolomic profile was obtained with liquid chromatography–mass spectrometry. In 2D, hVW-MSCs were spindle-shaped, expressed mesenchymal antigens, and displayed mesengenic potential. 3D cultures of hVW-MSCs were CD44⁺, CD105^low, CD90^low, exhibited a low propensity to enter the cell cycle as indicated by low percentage of ki-67 expression and accumulated intermediate metabolites pointing to slowed metabolism. The 3D model of hVW-MSCs exhibits stemness, dormancy and slow metabolism, typically observed in stem cell niches. This culture strategy can represent an accurate model to investigate hMSCs features for future clinical applications in the vascular field.     

Femtosecond two-photon high-resolution 3D imaging, spatial-volume rendering and microspectral characterization of immunolocalized MHC-II and mLangerin/CD207 antigens in the mouse epidermis

Langerhans cells (LCs) play a sentinel role by initiating both adaptive and innate immune responses to antigens pertinent to the skin. With the discovery of various LCs markers including antibodies to major histocompatibility complex class II (MHC-II) molecules and CD1a, intracellular presence of racket-shaped "Birbeck granules," and very recently Langerin/CD207, LCs can be readily distinguished from other subsets of dendritic cells. Femtosecond two-photon laser scanning microscopy (TPLSM) in recent years has emerged as an alternative to the single photon-excitation based confocal laser scanning microscope (CLSM), particularly for minimally-invasive deep-tissue 3D and 4D vital as well as nonvital biomedical imaging. We have recently combined high resolution two-photon immunofluorescence (using anti MHC-II and Langerin/CD207 antibodies) imaging with microspectroscopy and advanced image-processing/volume-rendering modalities. In this work, we demonstrate the use of this novel state-of-the-art combinational approach to characterize the steady state 3D organization and spectral features of the mouse epidermis, particularly to identify the spatial distribution of LCs. Our findings provide unequivocal direct evidence that, in the mouse epidermis, the MHC-II and mLangerin/CD207 antigens do indeed manifest a high degree of colocalization around the nucleus of the LCs, while in the distal dendritic processes, mLangerin/CD207 antigens are rather sparsely distributed as punctuate structures. This unique possibility to simultaneously visualize high resolution 3D-resolved spatial distributions of two different immuno-reactive antigens, namely MHC-II and mLangerin/CD207, along with the nuclei of LCs and the adjacent epidermal cells can find interesting applications. These could involve aspects associated with pragmatic analysis of the kinetics of LCs migration as a function of immuno-dermatological responses during (1) human Immunodeficiency virus disease progression, (2) vaccination and targeted gene therapy, (3) skin transplantation/plastic surgery, (4) ultraviolet and other radiation exposure, (5) tissue-engineering of 3D skin constructs, as well as in (6) cosmetic industry, to unravel the influence of cosmeceuticals.     

Immunophenotypic,immunocytochemistry, ultrastructural,and cytogenetic characterization of mesenchymal stem cells from equine bone marrow

The aim of this study was to isolate, culture, and characterize mesenchymal stem cells (MSCs) from horse bone marrow (BM) using the techniques of flow cytometry, immunocytochemistry, cytogenetics, and electron microscopy. Immunophenotypic analysis revealed the presence of MSCs with high expression of the CD90 marker, lower expression of the CD44 marker, and absent expression of the CD34 marker. In assays of differentiation, the positive response to osteogenic (OST), chondrogenic (CDG), and adipogenic (ADP) differentiation signals was observed and characterized by deposition of calcium‐rich extracellular matrix (OST), proteoglycans and collagen II (CDG) and intracellular deposition of fat drops (ADP). In immunocytochemical characterization, MSCs were immunopositive for CD44, vimentin, and PCNA, and they were negative for CD13. In the ultrastructural analysis of MSCs, the most outstanding characteristic was the presence of rough endoplasmic reticulum with very dilated cisterns filled with a low electrodensity material. Additionally, MSCs had normal karyotypes (2n = 64) as evidenced by cytogenetic analysis, and aneuploidy in metaphase was not observed. The protocols for isolating, culturing, and characterizing equine MSCs used in this study were shown to be appropriate for the production of a cell population with a good potential for differentiation and without aneuploidy that can be used to study future cellular therapies. Microsc. Res. Tech. 76:618–624, 2013. © 2013 Wiley Periodicals, Inc.     

Mesenchymal stem cells and cell-free preparations for treating atopic dermatitis

Atopic dermatitis (AD) is a chronic cutaneous inflammatory disease caused by an interaction between genetic, immune and epidermal barrier factors. Several treatments can be used to treat this disease but there are patients that do not respond to actual drugs. So, there is a need to develop effective therapies for AD. Mesenchymal stem cells (MSCs) are non-hematopoietic multipotent adult progenitor cells with immunomodulatory power and self-regenerating capacity to repair tissue damage, so they could be a potential effective treatment for AD. MSCs-Conditioned Medium (CM) and MSCs-exosomes are cell-free preparation with molecules secreted by stem cells that could be also beneficial for AD. This viewpoint reviews the actual development of MSCs, MSCs-CM and MSCs-exosomes for treating patients with AD.     

CD34⁺ CD38^- subpopulation without CD123 and CD44 is responsible for LSC and correlated with imbalance of immune cell subsets in AML

Acute myeloid leukemia (AML) is regarded as a stem cell disease. However, no one unique marker is expressedon leukemia stem cells (LSC) but not on leukemic blasts nor normal hematopoietic stem cells (HSC). CD34⁺CD38- withor without CD123 or CD44 subpopulations are immunophenotypically defined as putative LSC fractions in AML.Nevertheless, markers that can be effectively and simply held responsible for the intrinsical heterogeneity of LSC isstill unclear. In the present study, we examined the frequency of three different LSC subtypes (CD34⁺CD38^-,CD34⁺CD38^-CD123⁺, CD34⁺CD38^-CD44⁺) in AML at diagnosis. We then validated their prognostic significance onthe relevance of spectral features for diagnostic stratification, immune status, induction therapy response, treatmenteffect maintenance, and long-term survival. In our findings, high proportions of the above three different LSCsubtypes were all significantly characterized with low complete remission (CR) rate, high relapse/refractory rate, pooroverall survival (OS), frequent FLT3-ITD mutation, the high level of regulatory T cells (Treg) and monocyticmyeloid-derived suppressor cells (M-MDSC). However, there was no significant statistical difference in all kinds ofother clinical performance among the three different LSC groups. It was demonstrated that CD34⁺CD38^-subpopulation without CD123 and CD44 might be held responsible for LSC and correlated with an imbalance ofimmune cell subsets in AML.     

Evaluation of fracture planes and cell morphology in complementary fractures of cultured cells in the frozen-hydrated state by field-emission secondary electron microscopy: feasibility for ion localization and fluorescence imaging studies

We have employed field-emission secondary electron microscopy (FESEM) for morphological evaluation of freeze-fractured frozen-hydrated renal epithelial LLC-PK₁ cells prepared with our simple cryogenic sandwich-fracture method that does not require any high-vacuum freeze-fracture instrumentation (Chandra et al. (1986) J. Microsc. 144 , 15–37). The cells fractured on the substrate side of the sandwich were matched one-to-one with their corresponding complementary fractured faces on the other side of the sandwich. The FESEM analysis of the frozen-hydrated cells revealed three types of fracture: (i) apical membrane fracture that produces groups of cells together on the substrate fractured at the ectoplasmic face of the plasma membrane; (ii) basal membrane fracture that produces basal plasma membrane-halves on the substrate; and (iii) cross-fracture that passes randomly through the cells. The ectoplasmic face (E-face) and protoplasmic face (P-face) of the membrane were recognized based on the density of intramembranous particles. Feasibility of fractured cells was shown for intracellular ion localization with ion microscopy, and fluorescence imaging with laser scanning confocal microscopy. Ion microscopy imaging of freeze-dried cells fractured at the apical membrane revealed well-preserved intracellular ionic composition of even the most diffusible ions (total concentrations of K⁺, Na⁺ and Ca⁺). Structurally damaged cells revealed lower K⁺ and higher Na⁺ and Ca⁺ contents than in well-preserved cells. Frozen-freeze-dried cells also allowed imaging of fluorescently labelled mitochondria with a laser scanning confocal microscope. Since these cells are prepared without washing away the nutrient medium or using any chemical pretreatment to affect their native chemical and structural makeup, the characterization of fracture faces introduces ideal sample types for chemical and morphological studies with ion and electron microscopes and other techniques such as laser scanning confocal microscopy, atomic force microscopy and near-field scanning optical microscopy.