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1.
J Agric Food Chem ; 67(35): 9934-9941, 2019 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-31402655

RESUMO

A fluorescence microscopic method for characterizing size, quantity, and oxidation of lipid droplets (LDs) in HepG2 cells was developed. LDs were induced by palmitic (PA), oleic (OA), or linoleic acids (LA) and stained with two fluorescent probes for neutral lipids and lipid peroxides. Each fatty acid increased the number of LDs and oxidized LDs (oxLDs) and the degree of LD oxidation time dependently, as well as increased intracellular triglyceride hydroperoxides. LDs induced by LA without 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH) showed the most significant oxidation degree over PA and OA, especially in large LDs (area ≥ 3 µm2, oxLD/LD = 52.3 ± 21.7%). Under this condition, two food-derived antioxidants were evaluated, and both of them significantly improved the LD characteristics. Moreover, chlorogenic acid reduced the quantity of large LDs by 74.0-87.6% in a dose-dependent manner. The proposed method provides a new approach to evaluate the effect of dietary antioxidants on LD characteristics.


Assuntos
Antioxidantes/metabolismo , Hepatócitos/química , Hepatócitos/metabolismo , Gotículas Lipídicas/metabolismo , Microscopia de Fluorescência/métodos , Antioxidantes/química , Ácido Clorogênico/química , Ácido Clorogênico/metabolismo , Fluorescência , Células Hep G2 , Humanos , Gotículas Lipídicas/química , Oxirredução
2.
Chem Commun (Camb) ; 55(59): 8583-8586, 2019 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-31274135

RESUMO

We present a near-infrared (NIR) fluorescent probe, NR-HNO, which was successfully applied to visualizing H2S/NO "crosstalk" by the fluorescence detection of nitroxyl with a fast response time (5 min) and a large Stokes shift (131 nm) in living cells and tissue; it was also used to image nitroxyl in live mice.


Assuntos
Compostos de Benzilideno/química , Corantes Fluorescentes/química , Sulfeto de Hidrogênio/metabolismo , Óxido Nítrico/metabolismo , Óxidos de Nitrogênio/análise , Animais , Compostos de Benzilideno/efeitos da radiação , Linhagem Celular Tumoral , Corantes Fluorescentes/efeitos da radiação , Humanos , Rim/metabolismo , Luz , Limite de Detecção , Fígado/metabolismo , Camundongos , Microscopia de Fluorescência/métodos , Nitritos/química , Óxidos de Nitrogênio/metabolismo , Espectrometria de Fluorescência/métodos
3.
Chem Commun (Camb) ; 55(65): 9681-9684, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31347618

RESUMO

Here, we report a convenient, fast labeling strategy for the imaging of cell surface sialic acids (SAs, nine-carbon monosaccharides located at the terminals of cell surface sugar chains). This strategy is based on the synthesis of sticky, furry and fluorescent "wool-balls", which are wound into nanoclusters from p-benzoquinone/ethylenediamine polymer "wires". With abundant amino groups at the surface, the wool-balls can easily stick to the C-7 aldehyde group generated at the ends of periodate treated SAs in less than 30 min.


Assuntos
Benzoquinonas/química , Etilenodiaminas/química , Corantes Fluorescentes/química , Nanopartículas/química , Polímeros/química , Ácidos Siálicos/análise , Animais , Benzoquinonas/síntese química , Linhagem Celular Tumoral , Etilenodiaminas/síntese química , Fluorescência , Humanos , Camundongos , Microscopia de Fluorescência/métodos , Neuraminidase/química , Tamanho da Partícula , Polímeros/síntese química , Células RAW 264.7 , Bases de Schiff/síntese química , Bases de Schiff/química , Ácidos Siálicos/química
5.
Chem Commun (Camb) ; 55(65): 9629-9632, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31353368

RESUMO

Excessive accumulation of reducing agents in the ER leads to a constitutively high UPR. And the co-function of GSH, Cys and HOCl in biological processes is not well understood. To address this, a TP probe, NPCC, was developed for monitoring reductive stress in the ER. It can also distinguish cancer cells from normal cells.


Assuntos
Cumarínicos/química , Estresse do Retículo Endoplasmático/fisiologia , Retículo Endoplasmático/metabolismo , Corantes Fluorescentes/química , Pirazóis/química , Animais , Cumarínicos/síntese química , Cisteína/química , Cisteína/metabolismo , Corantes Fluorescentes/síntese química , Glutationa/química , Glutationa/metabolismo , Cabras , Células HeLa , Humanos , Ácido Hipocloroso/química , Ácido Hipocloroso/metabolismo , Camundongos , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Oxirredução , Pirazóis/síntese química , Peixe-Zebra
6.
Nat Commun ; 10(1): 2781, 2019 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-31273194

RESUMO

Recent advances in optical clearing and light-sheet microscopy have provided unprecedented access to structural and molecular information from intact tissues. However, current light-sheet microscopes have imposed constraints on the size, shape, number of specimens, and compatibility with various clearing protocols. Here we present a multi-immersion open-top light-sheet microscope that enables simple mounting of multiple specimens processed with a variety of clearing protocols, which will facilitate wide adoption by preclinical researchers and clinical laboratories. In particular, the open-top geometry provides unsurpassed versatility to interface with a wide range of accessory technologies in the future.


Assuntos
Microscopia de Fluorescência/métodos , Animais , Encéfalo/diagnóstico por imagem , Desenho de Equipamento , Humanos , Imagem Tridimensional/instrumentação , Imagem Tridimensional/métodos , Pulmão/diagnóstico por imagem , Linfonodos/diagnóstico por imagem , Masculino , Camundongos , Microscopia de Fluorescência/instrumentação , Próstata/diagnóstico por imagem
7.
Anal Chim Acta ; 1074: 123-130, 2019 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-31159932

RESUMO

Abnormal levels of Cys, Hcy and GSH are associated with various diseases, thus monitoring biothiols is of great significance. In this work, a dual-emission responsive near-infrared fluorescent probe NIR-NBD for detecting Hcy and Cys/GSH was developed based on the conjugation of a dicyanoisophorone based fluorophore (NIR-OH) and 7-nitrobenzofurazan (NBD). To our surprise, the addition of Hcy induced significant fluorescence enhancement at both 549 and 697 nm; while Cys/GSH resulted in major fluorescence emission at 697 nm. The detection limit was determined to be 33.2 nM for Cys, 33.5 nM for Hcy, and 34.4 nM for GSH. Therefore, the probe can be used for discriminative detection of Hcy and Cys/GSH. Moreover, fluorescence imaging of HeLa cells indicated that the probe was cell membrane permeable and could be used for visualizing Hcy and Cys/GSH in living cells.


Assuntos
4-Cloro-7-nitrobenzofurazano/análogos & derivados , Aminofenóis/química , Cisteína/análise , Corantes Fluorescentes/química , Glutationa/análise , Homocisteína/análise , 4-Cloro-7-nitrobenzofurazano/síntese química , 4-Cloro-7-nitrobenzofurazano/toxicidade , Aminofenóis/síntese química , Aminofenóis/toxicidade , Fluorescência , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/toxicidade , Células HeLa , Humanos , Limite de Detecção , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos
8.
Anal Chim Acta ; 1074: 43-53, 2019 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-31159938

RESUMO

This work evaluates the possibility of placement of high-resolution imaging and single-cell analysis via laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) within precision medicine by assessing the suitability of LA-ICP-MS as a micro-analytical technique for the localization and quantification of membranous receptors in heterogeneous cell samples that express both the membrane-bound receptors C-X-C chemokine receptor type 4 (CXCR4) and epidermal growth factor receptor (EGFR). Staining of the breast cancer cell lines MDA-MB-231 X4 and MDA-MB-468 was achieved using receptor-specific hybrid tracers, containing both a fluorophore and a DTPA single-lanthanide chelate. Prior to LA-ICP-MS imaging, fluorescence confocal microscopy (FCM) imaging was performed to localize the receptors, hereby enabling direct comparison. Based on the different expression levels of CXCR4 and EGFR, a distinction could be made between the cell lines using both imaging modalities. Furthermore, FCM and LA-ICP-MS demonstrated complementary characteristics, as a more distinct discrimination could be made between both cell lines based on the EGFR-targeting hybrid tracer via LA-ICP-MS, due to the intrinsic CXCR4-related green fluorescent protein (GFP) signal present in the MDA-MB-231 X4 cells. Employing state-of-the-art LA-ICP-MS instrumentation in bidirectional area scanning mode for sub-cellular imaging of MDA-MB-231 X4 cells enabled the specific binding of the CXCR4-targeting hybrid tracer to the cell membrane to be clearly demonstrated. The stretching of cells over the glass substrate led to a considerably higher signal response for pixels at the cell edges, relative to the more central pixels. The determination of the expression levels of CXCR4 and EGFR for the MDA-MB-468 cell line was performed using LA-ICP-MS single-cell analysis (sc-LA-ICP-MS) and external calibration, based on the quantitative ablation of Ho-spiked dried gelatin droplet standards. Additionally, a second calibration approach was applied based on spot ablation of highly homogeneous dried gelatin gels in combination with the determination of the ablated volume using atomic force microscopy (AFM) and yielded results which were in good agreement with the expression levels determined via flow cytometry (FC) and mass cytometry (MC). Hybrid tracers enable a direct comparison between (i) FCM and LA-ICP-MS imaging for the evaluation of the microscopic binding pattern and between (ii) FC, MC and sc-LA-ICP-MS for the quantification of receptor expression levels in single cells.


Assuntos
Corantes Fluorescentes/química , Receptores CXCR4/análise , Calibragem , Linhagem Celular Tumoral , Cetuximab/química , Quelantes/química , Receptores ErbB/análise , Citometria de Fluxo , Fluoresceínas/química , Fluorescência , Humanos , Elementos da Série dos Lantanídeos/química , Terapia a Laser , Limite de Detecção , Espectrometria de Massas/métodos , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Ácido Pentético/análogos & derivados , Peptídeos Cíclicos/química , Análise de Célula Única/métodos
9.
Histochem Cell Biol ; 152(2): 133-143, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31154480

RESUMO

Actin fulfills important cytoplasmic but also nuclear functions in eukaryotic cells. In the nucleus, actin modulates gene expression and chromatin remodeling. Monomeric (G-actin) and polymerized actin (F-actin) have been analyzed by fluorescence microscopy in the nucleus; however, the resolution at the ultrastructural level has not been investigated in great detail. We provide a first documentation of nuclear actin in mouse fibroblasts by electron microscopy (EM). For this, we employed correlative light and electron microscopy on the same section using actin-directed nanobodies recognizing endogenous monomeric and polymeric actin proteins (so-called nuclear Actin-chromobody-GFP; nAC-GFP). Indeed, using this strategy, we could identify actin proteins present in the nucleus. Here, immunogold-labeled actin proteins were spread throughout the entire nucleoplasm. Of note, nuclear actin was complementarily localized to DAPI-positive areas, the latter marking preferentially transcriptionally inactive heterochromatin. Since actin aggregates in rod structures upon cell stress including neurodegeneration, we analyzed nuclear actin at the ultrastructural level after DMSO or UV-mediated cell damage. In those cells the ratio between cytoplasmic and nuclear gold-labeled actin proteins was altered compared to untreated control cells. In summary, this EM analysis (i) confirmed the presence of endogenous nuclear actin at ultrastructural resolution, (ii) revealed the actin abundance in less chromatin-dense regions potentially reflecting more transcriptionally active euchromatin rather than transcriptionally inactive heterochromatin and (iii) showed an altered abundance of actin-associated gold particles upon cell stress.


Assuntos
Actinas/análise , Núcleo Celular/química , Microscopia Eletrônica/métodos , Microscopia de Fluorescência/métodos , Actinas/metabolismo , Animais , Núcleo Celular/metabolismo , Células Cultivadas , Fibroblastos/química , Fibroblastos/citologia , Camundongos , Células NIH 3T3 , Tamanho da Partícula , Conformação Proteica
10.
Chem Commun (Camb) ; 55(54): 7776-7779, 2019 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-31210218

RESUMO

An unusual class, compact in size, of fluorescent probes based on pyridazino-1,3a,6a-triazapentalene scaffolds exhibits promising fluorescent properties (quantum yield values up to 73%, large Stokes shifts, emission wavelengths located in the green-yellow range, excellent solubility) with good photostability suitable for optical imaging applications.


Assuntos
Corantes Fluorescentes/química , Compostos Heterocíclicos com 3 Anéis/química , Fluorescência , Corantes Fluorescentes/síntese química , Células HeLa , Compostos Heterocíclicos com 3 Anéis/síntese química , Humanos , Microscopia de Fluorescência/métodos , Imagem Óptica/métodos , Fotodegradação
11.
Inorg Chem ; 58(13): 8369-8378, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31247863

RESUMO

This article reports the effect of Gd(III) doping on the structure, microstructure, and optical properties of boehmite nanoparticles. The bright-blue fluorescence along with a long lifetime makes our material an efficient candidate for optical applications. Our material particularly targets and eliminates hexavalent chromium ions (Cr(VI)) from aqueous media, which turns it into a multifunctional fluorescent nanosensor (MFNS). The development of an efficient hexavalent chromium ion (Cr(VI)) sensor to detect and quantify Cr(VI) ions is still a serious issue worldwide. Thus, this work will be very beneficial for various environmental applications. No such work has been reported so far which includes cost-effective and biocompatible boehmite nanoparticles in this field. Detailed synthesis and characterization procedures for the MFNS have been incorporated here. The biocompatibility of the MFNS has also been studied rigorously by performing cell survivability assay (MTT) and cellular morphology assessments. Our extensive research confirmed that the "turn-off" sensing mechanism of this sensor material is based on a collisional quenching model which initiates the photoinduced electron transfer (PET) process. High selectivity and sensitivity (∼1.05 × 10-5 M) of the MFNS toward hexavalent chromium ions even in real life wastewater samples have been confirmed, which makes this fluorescent probe a potential candidate for new age imaging and sensing technologies.


Assuntos
Hidróxido de Alumínio/química , Óxido de Alumínio/química , Cromo/análise , Corantes Fluorescentes/química , Nanopartículas/química , Águas Residuárias/análise , Adsorção , Hidróxido de Alumínio/síntese química , Óxido de Alumínio/síntese química , Linhagem Celular Tumoral , Fluorescência , Corantes Fluorescentes/síntese química , Gadolínio/química , Humanos , Limite de Detecção , Microscopia de Fluorescência/métodos , Porosidade , Espectrometria de Fluorescência/métodos
12.
Chem Commun (Camb) ; 55(60): 8860-8863, 2019 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-31219109

RESUMO

We report the first dual-responsive 19F MRI and fluorescence imaging probe for cellular hypoxia. The Cu2+-based probe exhibits no 19F MR signal and reduced fluorescence signal due to paramagnetic quenching; however, the probe turns-on in both modes following reduction to Cu+. This bimodal agent can differentiate hypoxic and normoxic cells in both modalities.


Assuntos
Hipóxia Celular/fisiologia , Complexos de Coordenação/química , Fluoresceínas/química , Corantes Fluorescentes/química , Complexos de Coordenação/síntese química , Complexos de Coordenação/toxicidade , Cobre/química , Fluoresceínas/síntese química , Fluoresceínas/toxicidade , Fluorescência , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/toxicidade , Flúor , Imagem por Ressonância Magnética de Flúor-19/métodos , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Microscopia de Fluorescência/métodos
13.
Biochemistry (Mosc) ; 84(Suppl 1): S19-S31, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31213193

RESUMO

Super-resolution fluorescence microscopy (nanoscopy) enables imaging with a spatial resolution much higher than the diffraction limit of optical microscopy. However, the methods of fluorescence nanoscopy are still poorly suitable for studying living cells. In this review, we describe some of methods for nanoscopy and specific fluorescent labeling aimed to decrease the damaging effects of light illumination on live samples.


Assuntos
Células/ultraestrutura , Microscopia de Fluorescência/métodos , Nanotecnologia/métodos
14.
Biochemistry (Mosc) ; 84(Suppl 1): S51-S68, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31213195

RESUMO

Classic time-correlated single photon counting (TCSPC) technique involves detection of single photons of a periodic optical signal, registration of the photon arrival time in respect to the reference pulse, and construction of photon distribution with regard to the detection times. This technique achieves extremely high time resolution and near-ideal detection efficiency. Modern TCSPC is multi-dimensional, i.e., in addition to the photon arrival time relative to the excitation pulse, spatial coordinates within the image area, wavelength, time from the start of the experiment, and many other parameters are determined for each photon. Hence, the multi-dimensional TCSPC allows generation of photon distributions over these parameters. This review describes both classic and multi-dimensional types of TCSPC microscopy and their application for fluorescence lifetime imaging in different areas of biological studies.


Assuntos
Microscopia de Fluorescência/métodos , Imagem Óptica/métodos , Fluorescência , Fótons , Fatores de Tempo
15.
Nat Commun ; 10(1): 2637, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31201332

RESUMO

The brain stores and recalls memories through a set of neurons, termed engram cells. However, it is unclear how these cells are organized to constitute a corresponding memory trace. We established a unique imaging system that combines Ca2+ imaging and engram identification to extract the characteristics of engram activity by visualizing and discriminating between engram and non-engram cells. Here, we show that engram cells detected in the hippocampus display higher repetitive activity than non-engram cells during novel context learning. The total activity pattern of the engram cells during learning is stable across post-learning memory processing. Within a single engram population, we detected several sub-ensembles composed of neurons collectively activated during learning. Some sub-ensembles preferentially reappear during post-learning sleep, and these replayed sub-ensembles are more likely to be reactivated during retrieval. These results indicate that sub-ensembles represent distinct pieces of information, which are then orchestrated to constitute an entire memory.


Assuntos
Hipocampo/fisiologia , Memória/fisiologia , Neurônios/fisiologia , Animais , Mapeamento Encefálico/métodos , Feminino , Hipocampo/citologia , Microscopia Intravital/métodos , Proteínas Luminescentes/química , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Microscopia de Fluorescência/métodos , Modelos Animais , Imagem Óptica/métodos , Optogenética/métodos , Sono/fisiologia
16.
Nat Commun ; 10(1): 2704, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31221964

RESUMO

Attachment of lipid tails to oligonucleotides has emerged as a powerful technology in constructing cell membrane-anchorable nucleic acid-based probes. In practice, however, conventional lipid-conjugated oligonucleotides fail to distinguish among different cell membranes. Herein, a phosphorylated lipid-conjugated oligonucleotide (DNA-lipid-P) is reported for alkaline phosphatase (ALP)-dependent cell membrane adhesion. In the absence of ALP, DNA-lipid-P with its poor hydrophobicity shows only weak interaction with cell membrane. However, in the presence of the highly expressed plasma membrane-associated ALP, DNA-lipid-P is converted to lipid-conjugated oligonucleotide (DNA-lipid) by enzymatic dephosphorylation. As a result of such conversion, the generated DNA-lipid has greater hydrophobicity than DNA-lipid-P and is thus able to insert into cell membranes in situ. Accordingly, DNA-lipid-P enables selective anchoring on cell membranes with elevated ALP level. Since elevated ALP level is a critical index of some diseases and even cancers, DNA-lipid-P holds promise for cell membrane engineering and disease diagnostics at the molecular level.


Assuntos
Fosfatase Alcalina/metabolismo , Membrana Celular/metabolismo , Sondas Moleculares/metabolismo , Oligonucleotídeos/metabolismo , Linhagem Celular Tumoral , Humanos , Interações Hidrofóbicas e Hidrofílicas , Lipídeos/química , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Imagem Molecular/métodos , Sondas Moleculares/química , Oligonucleotídeos/química , Compostos Organofosforados/química , Fosforilação
17.
Nat Commun ; 10(1): 2745, 2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-31227705

RESUMO

Small molecule probes are indispensable tools to explore diverse cellular events. However, finding a specific probe of a target remains a high challenge. Here we report the discovery of Fast-TRFS, a specific and superfast fluorogenic probe of mammalian thioredoxin reductase, a ubiquitous enzyme involved in regulation of diverse cellular redox signaling pathways. By systematically examining the processes of fluorophore release and reduction of cyclic disulfides/diselenides by the enzyme, structural factors that determine the response rate and specificity of the probe are disclosed. Mechanistic studies reveal that the fluorescence signal is switched on by a simple reduction of the disulfide bond within the probe, which is in stark contrast to the sensing mechanism of published probes. The favorable properties of Fast-TRFS enable development of a high-throughput screening assay to discover inhibitors of thioredoxin reductase by using crude tissue extracts as a source of the enzyme.


Assuntos
Descoberta de Drogas/métodos , Corantes Fluorescentes/química , Imagem Molecular/métodos , Sondas Moleculares/química , Tiorredoxina Redutase 1/metabolismo , Animais , Produtos Biológicos/farmacologia , Misturas Complexas , Dissulfetos/química , Corantes Fluorescentes/metabolismo , Células HeLa , Ensaios de Triagem em Larga Escala/métodos , Humanos , Microscopia Intravital/métodos , Microscopia de Fluorescência/métodos , Sondas Moleculares/metabolismo , Oxirredução , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Tiorredoxina Redutase 1/antagonistas & inibidores , Tiorredoxina Redutase 1/genética
18.
Methods Mol Biol ; 1966: 137-149, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31041744

RESUMO

Increases in levels of protoporphyrin IX (PPIX; a heme precursor) may be driven by xenobiotic induction of aminolevulinic acid synthase 1 (ALAS1) expression. ALAS1 is the rate-limiting enzyme of heme biosynthesis and may be upregulated to satisfy the increased need for heme in CYP450 enzymes. Therefore, a high-throughput fluorescence spectroscopy method that detects PPIX would enable the screening of drugs that increase ALAS1 through nuclear hormone receptor-mediated induction of transcription that may cause toxicity or even provide utility in the diagnosis or treatment of cancers that have elevated cellular PPIX levels. This chapter describes a high-throughput plate-based imaging technique for determining cellular protoporphyrin levels by using the GE Healthcare InCell 6000 confocal imaging system to detect the presence and location of PPIX in each cell and may be adapted for use with other imaging systems. Laser excitation and a scientific-grade complementary metal oxide semiconductor (CMOS) camera generate short exposure times, decreasing photobleaching in the target cells that may result in inaccurate measurements of PPIX and increasing screening throughput. Nuclear staining was detected by using a laser with 405-nm excitation and 455-nm emission wavelengths, and the presence of PPIX was measured using 405-nm excitation and 706-nm emission wavelengths. Image analysis involving top-hat segmentation on both nuclear and PPIX staining was performed by using the InCell Analyzer Workstation software. This assay may be adapted to screen for PPIX formation, degradation, and transportation effectors. Indeed, the inclusion of PPIX transport inhibition would be expected to further widen the linear range of fluorescence and improve the method.


Assuntos
Ensaios de Triagem em Larga Escala/métodos , Microscopia Confocal/métodos , Protoporfirinas/análise , Espectrometria de Fluorescência/métodos , Células Hep G2 , Humanos , Interpretação de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos
19.
Int J Mol Sci ; 20(9)2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-31067698

RESUMO

We review the contribution of bioimaging in building a coherent understanding of Ca 2 + signalling during legume-bacteria symbiosis. Currently, two different calcium signals are believed to control key steps of the symbiosis: a Ca 2 + gradient at the tip of the legume root hair is involved in the development of an infection thread, while nuclear Ca 2 + oscillations, the hallmark signal of this symbiosis, control the formation of the root nodule, where bacteria fix nitrogen. Additionally, different Ca 2 + spiking signatures have been associated with specific infection stages. Bioimaging is intrinsically a cross-disciplinary area that requires integration of image recording, processing and analysis. We used experimental examples to critically evaluate previously-established conclusions and draw attention to challenges caused by the varying nature of the signal-to-noise ratio in live imaging. We hypothesise that nuclear Ca 2 + spiking is a wide-range signal involving the entire root hair and that the Ca 2 + signature may be related to cytoplasmic streaming.


Assuntos
Sinalização do Cálcio , Fabaceae/metabolismo , Simbiose , Fabaceae/microbiologia , Microscopia de Fluorescência/métodos , Rhizobium/metabolismo , Rhizobium/patogenicidade
20.
Analyst ; 144(11): 3546-3551, 2019 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-31041945

RESUMO

A turn-on two-photon fluorescent probe HCA-Green for hypochlorous acid (HOCl) was synthesized using 4-methylamino-1,8-naphthalimide (MNA) as a two-photon fluorophore and p-hydroxyaniline as a leaving-recognition domain. Both the probe and the fluorophore were investigated under one- and two-photon excitation modes. The fluorescence intensity of the probe was enhanced by ∼229-fold and ∼193-fold under one-photon and two-photon excitation, respectively, after reacting with HOCl. A maximal two-photon action cross-section of 50 GM was obtained under excitation at 810 nm. The probe exhibited high sensitivity with a detection limit of 42.3 nM, as well as high selectivity, low cytotoxicity, and good photostability. Two-photon microscopy (TPM) was conducted to visualize HOCl levels in living cells and tissues. The production of endogenous HOCl induced by lipopolysaccharide-mediated inflammation was successfully monitored with this probe.


Assuntos
Corantes Fluorescentes/química , Ácido Hipocloroso/análise , Inflamação/metabolismo , Animais , Fluorescência , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/efeitos da radiação , Corantes Fluorescentes/toxicidade , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Inflamação/induzido quimicamente , Luz , Limite de Detecção , Lipopolissacarídeos , Camundongos Nus , Microscopia de Fluorescência/métodos , Ratos
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