RESUMO
Hydrogen peroxide (H2O2), a member of small-molecule reactive oxygen species (ROS), plays an important role in physiological and/or pathological process within live systems. Herein, to quantitatively investigate the biological role of H2O2 in subcellular level, we constructed of a novel two-photon (TP) in near-infrared (NIR) out fluorescent probe (TP-NIR-H2O2) for visualization of mitochondria H2O2 in living cells and tissues. Specifically, TP-NIR-H2O2 utilized the oxonium ion as the mitochondrial targeting unit and phenylboric acid as the H2O2 reaction moiety. After the phenylboric acid moiety reaction with H2O2, TP-NIR-H2O2 displayed a ~105-fold fluorescence intensity enhancement in 665â¯nm. Selectivity experiment demonstrated that the probe can detect H2O2 with high selectivity over other ROS. Moreover, TP-NIR-H2O2 could be employed for imaging H2O2 in mice liver tissues with large tissue-image depth (50-170⯵m) under two-photon excitation (800â¯nm).
Assuntos
Corantes Fluorescentes/química , Peróxido de Hidrogênio/análise , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Mitocôndrias/química , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Animais , Células HeLa , Humanos , Peróxido de Hidrogênio/metabolismo , Limite de Detecção , Modelos Lineares , Fígado/citologia , Fígado/diagnóstico por imagem , Camundongos , Camundongos Nus , Mitocôndrias/metabolismoRESUMO
BACKGROUND: The diagnosis of idiopathic atrophoderma of Pasini and Pierini (IAPP) relies on typical clinical features, particularly distinctive pigmented ovular/round depressed plaques. Histologic examination often reveals no obvious changes, but patterns of collagen distribution, using multiphoton imaging and second harmonic generation can help track hidden details of tissue organization contributing to atrophy. OBJECTIVE: To identify histologic features that distinguish IAPP from unaffected skin. METHODS: Eleven patients were included for conventional analyses. Masson trichrome- and Unna-Tanzer orcein-stained sections were evaluated using automated morphometry. Hematoxylin-eosin-stained sections were analyzed by multiphoton imaging using 2-photon excited fluorescence and second harmonic generation. RESULTS: No abnormalities were found under light microscopy or by automated quantification. Multiphoton imaging revealed no difference in optical density of either collagen or elastic fibers in lesioned and unaffected skin; however, horizontal collagen fiber organization in lesion specimens increased toward the lower dermis, whereas elastic fibers featured greater disorganization within the upper dermis. LIMITATIONS: The low number of patients evaluated. CONCLUSION: The atrophic appearance of IAPP lesions reflects changes in organization, but not in collagen and elastic tissue content. Minute organizational differences that are imperceptible to the experienced pathologist and undetectable by automated analyses were revealed by multiphoton analyses, particularly second harmonic generation, in association with texture analyses.
Assuntos
Colágeno/ultraestrutura , Tecido Elástico/ultraestrutura , Esclerodermia Localizada/diagnóstico por imagem , Esclerodermia Localizada/patologia , Adolescente , Adulto , Atrofia/patologia , Biópsia por Agulha , Tecido Elástico/patologia , Feminino , Humanos , Imuno-Histoquímica , Masculino , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Pessoa de Meia-Idade , Valores de Referência , Estudos de Amostragem , Estatísticas não Paramétricas , Adulto JovemRESUMO
Mating of haploid Saccharomyces cerevisiae cells of opposite sex provides a powerful model system to study the cell-cell fusion. However, a rapid and standardized method is much needed for quantitative assessment of fusion efficiency. The gold standard method relies on counting mating pairs in fluorescence microscopy images. This current method is limited by expectancy bias and it is time consuming, restricting the number of both cell-cell fusion events and strains that can be analyzed at once. Automatic approaches present a solution to these limitations. Here, we describe a novel flow cytometric approach that is able to quickly both identify mating pairs within a mixture of gametes and quantify cell fusion efficiency. This method is based on staining the cell wall of yeast populations with different Concanavalin A-fluorophore conjugates. The mating subpopulation is identified as the two-colored events set and fused and unfused mating pairs are subsequently discriminated by green fluorescent protein bimolecular complementation. A series of experiments was conducted to validate a simple and reliable protocol. Mating efficiency in each sample was determined by flow cytometry and compared with the one obtained with the current gold standard technique. The results show that mating pair counts using both methods produce indistinguishable outcomes and that the flow cytometry-based method provides quantitative relevant information in a short time, making possible to quickly analyze many different cell populations. In conclusion, our data show multicolor flow cytometry-based fusion quantitation to be a fast, robust, and reliable method to quantify the cell-cell fusion in yeast.
Assuntos
Parede Celular/química , Parede Celular/metabolismo , Concanavalina A/análise , Citometria de Fluxo/métodos , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Fusão Celular/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodosRESUMO
PURPOSE: To demonstrate lipid-specific imaging of the retina through the use of third harmonic generation (THG), a multiphoton microscopic technique in which tissue contrast is generated from optical inhomogeneities. METHODS: A custom fiber laser and multiphoton microscope was constructed and optimized for simultaneous two-photon autofluorescence (TPAF) and THG retinal imaging. Imaging was performed using fixed-frozen sections of mouse eyes without the use of exogenous fluorescent dyes. In parallel experiments, a fluorescent nuclear stain was used to verify the location of the retinal cell nuclei. RESULTS: Simultaneous THG and TPAF images revealed all retinal layers with subcellular resolution. In BALB/c strains, the THG signal stems from the lipidic organelles of the cellular and nuclear membranes. In the C57BL/6 strain, the THG signal from the RPE cells originates from the pigmented granules. CONCLUSIONS: THG microscopy can be used to image structures of the mouse retina using contrast inherent to the tissue and without the use of a fluorescent dye or exogenously expressed recombinant protein.
Assuntos
Microscopia de Fluorescência por Excitação Multifotônica/métodos , Retina/anatomia & histologia , Retina/metabolismo , Animais , Biomarcadores/metabolismo , Células Cultivadas , Desenho de Equipamento , Humanos , Metabolismo dos Lipídeos , Lipídeos de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Gambás , Imagem Óptica/instrumentação , Imagem Óptica/métodos , Epitélio Pigmentado da Retina/anatomia & histologia , Epitélio Pigmentado da Retina/metabolismoRESUMO
Murine induced colon cancer has been used to demonstrate that Second Harmonic Generation (SHG) microscopy images, combined with Two-Photon Excitation Fluorescence (TPEF) and specific quantization scoring methods allow distinguishing early alterations in colon mucosa. TPEF was used only to identified crypts and submucosa regions, whereas the image analysis was used to get quantitative data (Integrated Intensity and Aspect Ratio scoring) of different cancer stages. The submucosa amount of collagen fibers was significant and their orientation suffering proportional changes with the development of the pathological processes. Both after the fourth and eighth weeks after colon cancer induction, integrated intensity and aspect ratio values have shown significant statistical differences compared with control samples. Thus, SHG microscopy has proved to be a useful quantitative tool to highlight early changes of submucosa and the progression of these through the cancer development.
Assuntos
Algoritmos , Neoplasias Colorretais/patologia , Detecção Precoce de Câncer/métodos , Interpretação de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Imagem Multimodal/métodos , Animais , Aumento da Imagem/métodos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dinâmica não Linear , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: The confirmatory diagnosis of Osteogenesis Imperfecta (OI) requires invasive, commonly bone biopsy, time consuming and destructive methods. This paper proposes an alternative method using a combination of two-photon excitation fluorescence (TPEF) and second-harmonic generation (SHG) microscopies from easily obtained human skin biopsies. We show that this method can distinguish subtypes of human OI. METHODOLOGY/PRINCIPAL FINDINGS: Different aspects of collagen microstructure of skin fresh biopsies and standard H&E-stained sections of normal and OI patients (mild and severe forms) were distinguished by TPEF and SHG images. Moreover, important differences between subtypes of OI were identified using different methods of quantification such as collagen density, ratio between collagen and elastic tissue, and gray-level co-occurrence matrix (GLCM) image-pattern analysis. Collagen density was lower in OI dermis, while the SHG/autofluorescence index of the dermis was significantly higher in OI as compared to that of the normal skin. We also showed that the energy value of GLCM texture analysis is useful to discriminate mild from severe OI and from normal skin. CONCLUSIONS/SIGNIFICANCE: This work demonstrated that nonlinear microscopy techniques in combination with image-analysis approaches represent a powerful tool to investigate the collagen organization in skin dermis in patients with OI and has the potential to distinguish the different types of OI. The procedure outlined in this paper requires a skin biopsy, which is almost painless as compared to the bone biopsy commonly used in conventional methods. The data presented here complement existing clinical diagnostic techniques and can be used as a diagnostic procedure to confirm the disease, evaluate its severity and treatment efficacy.
Assuntos
Colágeno Tipo I/análise , Osteogênese Imperfeita/patologia , Pele/patologia , Adulto , Biópsia , Criança , Colágeno Tipo I/metabolismo , Humanos , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Osteogênese Imperfeita/metabolismo , Patologia/métodosRESUMO
Based on two-photon calcium imaging, Histed et al. (2009) concluded that electrical microstimulation of cortical tissue in mammals activates a sparse and distributed population of neurons. This work has been cited by many as proof that electrical microstimulation is nonfocal, which means it may lack the precision needed for applications in neuroprosthetics. We affirm that the generation of stimulation-evoked behaviour is based primarily on the orthodromic conduction of signals originating mainly from the deepest layers of cortex, while the work of Histed et al. is effectively limited to investigating the antidromic activation of lateral projection neurons of the superficial cortex. The apparent sparse activation is a consequence of the pattern of axonal projections based on activating a volume of axons while imaging cell bodies transecting a single plane through the cortex. This creates the false impression that the distribution of activated neurons is sparse and nonfocal. We recommend how two-photon calcium imaging, which is superb for the study of individual and groups of neurons, might be more effectively used to ascertain how electrical stimulation affects the brains of mammals. This is a timely topic since investigators are using electrical microstimulation in animals to develop prosthetic devices to restore sensory and motor functions in disabled patients.
Assuntos
Cálcio/metabolismo , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Diagnóstico por Imagem/métodos , Neurônios/metabolismo , Animais , Córtex Cerebral/química , Humanos , Imageamento por Ressonância Magnética/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Neurônios/químicaRESUMO
We show that combined multimodal nonlinear optical (NLO) microscopies, including two-photon excitation fluorescence, second-harmonic generation (SHG), third harmonic generation, and fluorescence lifetime imaging microscopy (FLIM) can be used to detect morphological and metabolic changes associated with stroma and epithelial transformation during the progression of cancer and osteogenesis imperfecta (OI) disease. NLO microscopes provide complementary information about tissue microstructure, showing distinctive patterns for different types of human breast cancer, mucinous ovarian tumors, and skin dermis of patients with OI. Using a set of scoring methods (anisotropy, correlation, uniformity, entropy, and lifetime components), we found significant differences in the content, distribution and organization of collagen fibrils in the stroma of breast and ovary as well as in the dermis of skin. We suggest that our results provide a framework for using NLO techniques as a clinical diagnostic tool for human cancer and OI. We further suggest that the SHG and FLIM metrics described could be applied to other connective or epithelial tissue disorders that are characterized by abnormal cells proliferation and collagen assembly.
Assuntos
Neoplasias da Mama/etiologia , Neoplasias da Mama/patologia , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Neoplasias Epiteliais e Glandulares/etiologia , Neoplasias Epiteliais e Glandulares/patologia , Osteogênese Imperfeita/complicações , Osteogênese Imperfeita/patologia , Adulto , Idoso , Feminino , Humanos , Pessoa de Meia-Idade , Dinâmica não Linear , Lesões Pré-Cancerosas/patologia , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
We review the current state of multiphoton microscopy. In particular, the requirements and limitations associated with high-speed multiphoton imaging are considered. A description of the different scanning technologies such as line scan, multifoci approaches, multidepth microscopy, and novel detection techniques is given. The main nonlinear optical contrast mechanisms employed in microscopy are reviewed, namely, multiphoton excitation fluorescence, second harmonic generation, and third harmonic generation. Techniques for optimizing these nonlinear mechanisms through a careful measurement of the spatial and temporal characteristics of the focal volume are discussed, and a brief summary of photobleaching effects is provided. Finally, we consider three new applications of multiphoton microscopy: nonlinear imaging in microfluidics as applied to chemical analysis and the use of two-photon absorption and self-phase modulation as contrast mechanisms applied to imaging problems in the medical sciences.
Assuntos
Microscopia de Fluorescência por Excitação Multifotônica/métodos , Absorção , Algoritmos , Animais , Fluorescência , Humanos , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Modelos Teóricos , Dinâmica não Linear , Óptica e Fotônica/instrumentação , Óptica e Fotônica/métodos , Fotodegradação , Fótons , Fatores de TempoRESUMO
Our view of the structure and function of the interphase nucleus has changed drastically in recent years. It is now widely accepted that the nucleus is a well organized and highly compartmentalized organelle and that this organization is intimately related to nuclear function. In this context, chromatin-initially considered a randomly entangled polymer-has also been shown to be structurally organized in interphase and its organization was found to be very important to gene regulation. Relevant and not completely answered questions are how chromatin organization is achieved and what mechanisms are responsible for changes in the positions of chromatin loci in the nucleus. A significant advance in the field resulted from tagging chromosome sites with bacterial operator sequences, and visualizing these tags using green fluorescent protein fused with the appropriate repressor protein. Simultaneously, fluorescence imaging techniques evolved significantly during recent years, allowing observation of the time evolution of processes in living specimens. In this context, the motion of the tagged locus was observed and analyzed to extract quantitative information regarding its dynamics. This review focuses on recent advances in our understanding of chromatin dynamics in interphase with the emphasis placed on the information obtained from single-particle tracking (SPT) experiments. We introduce the basis of SPT methods and trajectory analysis, and summarize what has been learnt by using this new technology in the context of chromatin dynamics. Finally, we briefly describe a method of SPT in a two-photon excitation microscope that has several advantages over methods based on conventional microscopy and review the information obtained using this novel approach to study chromatin dynamics.