Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 81.815
Filtrar
1.
Food Chem ; 398: 133884, 2023 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-35964575

RESUMO

Development of selective and sensitive methods for the detection of 2, 6-dipicolinic acid (DPA), a biomarker produced by bacterial spores, is of great significance for maintaining public health and food safety. Herein, a ratiometric fluorescence strategy using graphene carbon nitride (g-C3N4) coupled with Eu3+ is designed for the assay of DPA. As the concentration of DPA increases, the emission intensity of g-C3N4 kept constant which acted as a stable internal reference, while the fluorescence of Eu3+ was enhanced obviously due to the antenna effect. The linear calibration ranged from 0.1 to 15 µM with a detection limit of 13 nM was obtained. More Importantly, a paper-based sensor with a smartphone was successfully combined to perform colorimetric and visual detection of DPA in situ. This method has good performance for the detection of DPA, which is expected to broaden the application prospects of preliminary biomarker monitoring.


Assuntos
Antraz , Antraz/diagnóstico , Antraz/microbiologia , Biomarcadores , Európio , Corantes Fluorescentes , Humanos , Nitrilas , Ácidos Picolínicos , Smartphone
2.
Food Chem ; 398: 133935, 2023 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-35986995

RESUMO

Nitrite is a widely used food additive that has been shown to be carcinogenic and can cause health damage when consumed in excess. Therefore, developing a detection method is in demand. Here, we prepared a novel Fe-doped carbon dots (Fe-CDs) using metallic deep eutectic solvent (MDES) which showed high sensitivity and selectivity. Besides, it also showed excellent pH-dependent luminescence characteristics, which proved the feasibility as a pH sensor. Under the optimal conditions, the detection linear of nitrite ranged from 0.2 to 80 µM, and the detection limit was 50 nM. The recovery rate was between 98.8 % and 104.1 % in food and water samples. For pH monitoring, its fluorescence intensity was linearly correlated in the pH range from 2 to 7, accompanying a unique differential solution color change of colorless-yellow-green. Therefore, it can be used as an excellent fluorescent probe for detection of nitrite and pH in food and water environment.


Assuntos
Corantes Fluorescentes , Pontos Quânticos , Carbono , Solventes Eutéticos Profundos , Concentração de Íons de Hidrogênio , Nitritos , Solventes , Água
3.
Food Chem ; 398: 133879, 2023 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-35987002

RESUMO

Accurate and real-time reporting the freshness and quality of meat has far-reaching implications for safeguarding food safety and public health. Owing to the significant indicator role of H2S in meat spoilage, we here designed a ''dual-key-and-lock'' kind of H+-powered H2S-responsive ratiometric fluorescent nanoprobe BODIPY/Cy7Cl@HyNPs for precise and real-time evaluating the meat freshness. After incubation with spoilt meat, BODIPY/Cy7Cl@HyNPs was simultaneously initiated by the abnormal H+ and H2S acted as "dual-keys". As a result, the fluorescence emission of BODIPY/Cy7Cl@HyNPs at âˆ¼ 688 nm was enhanced, and the other emission at âˆ¼ 818 nm was quenched, thereby yielding a typical H+-powered ratiometric fluorescence response toward H2S in meat. In addition, we also successfully exploited BODIPY/Cy7Cl@HyNPs to achieve the real-time monitoring of the meat freshness during storage by measuring the changes of fluorescence signals and solution color.


Assuntos
Corantes , Carne , Fluorescência , Corantes Fluorescentes
4.
Methods Mol Biol ; 2564: 1-45, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36107335

RESUMO

FPbase is a database of fluorescent proteins and their characteristics and a set of online tools that facilitate searching the database and performing experiments with fluorescent probes. This chapter serves as a general reference for using and searching the database and a guide to some of the more commonly used tools including the spectra viewer, custom microscope pages, and FRET calculator. Important caveats when evaluating the data are also discussed.


Assuntos
Corantes Fluorescentes , Proteínas , Bases de Dados de Proteínas
5.
Methods Mol Biol ; 2564: 223-246, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36107345

RESUMO

DNA binding fluorescent proteins are a powerful tool for single-molecule visualization. In this chapter, we discuss a protocol for the synthesis of DNA binding fluorescent proteins and visualization of single DNA molecules. This chapter includes stepwise methods for molecular cloning, reversible staining, two-color staining, sequence-specific staining, and microscopic visualization of single DNA molecules in a microfluidic device. This content will be useful for DNA characterization using DNA binding fluorescent proteins and its visualization at the single-molecule level.


Assuntos
Proteínas de Ligação a DNA , DNA , DNA/química , Proteínas de Ligação a DNA/metabolismo , Corantes Fluorescentes , Microscopia de Fluorescência/métodos , Coloração e Rotulagem
6.
Food Chem ; 399: 133970, 2023 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-35998499

RESUMO

Lateral flow immunoassays (LFIAs) are routine methods for rapid foodborne pollutants screening, with detection limits that are closely associated with the label probes used. The exploitation of high performance and robust probe is highly desirable, and remains a great challenge. Herein, we reported an emerging fluorescent nanobeads i.e. carbon-dots (CD) covalently incorporated mesoporous silicon nanoparticles (CD-MSNs) for LFIAs. CD-MSNs revealed brighter fluorescence, larger particle size and more modification sites in comparison with those of single CD. After bio-functionalisation, CD-MSNs probes were introduced to construct LFIA test strips, and designed for ultrasensitive detection of aflatoxin B1 (AFB1) and Staphylococcus aureus (S. aureus), two representative foodborne pollutants, based on the competitive and sandwich models, respectively. Very competitive quantitative detection limits i.e. 0.05 ng/mL and 102 cfu/mL were correspondingly obtained. Additionally, the test strips were successfully applied to rapidly and accurately screen AFB1 and S. aureus in food samples, highlighting their practicality.


Assuntos
Poluentes Ambientais , Nanopartículas , Aflatoxina B1/análise , Carbono , Corantes Fluorescentes , Imunoensaio/métodos , Limite de Detecção , Silício , Staphylococcus aureus
7.
Food Chem ; 399: 134008, 2023 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-36037693

RESUMO

The Tyndall Effect assay (TEA) has been applied into colorimetric metal ion detection since 2019. However, the TEA-based sensor for pesticide detection has never been reported till now. Herein, a facile fluorescent organic nanoparticle (FON)-based sensor is firstly developed for fluorine-containing pesticide detection through ratiometric fluorescence assay (FLA) and TEA. For FLA, the intensity of the second-order Tyndall scattering peak (STS590nm) and the fluorescence peak of the FON-based sensor would increase and remain unchanged respectively when adding bifenthrin, flufenoxuron, and diflubenzuron. The detection limits were respectively 9.34, 6.91, and 3.60 µg/kg. For TEA, the increased STS590nm intensity displayed a bright and visible light beam. An economical, simple, and portable device was then constructed to visually monitor the analytes. The sensor was successfully used to detect the analytes in teas through FLA and TEA with the recoveries and RSD ranging from 86.27-100.00 %, and 0.00-5.68 %, respectively.


Assuntos
Diflubenzuron , Nanopartículas , Praguicidas , Corantes Fluorescentes , Fluoretos , Flúor , Compostos de Fenilureia , Piretrinas , Chá
8.
Sci Rep ; 12(1): 15098, 2022 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-36065005

RESUMO

Low-molecular synthetic fluorophores are convenient tools in bioimaging applications. Several derivatives of Safirinium dyes as well as their reactive N-hydroxysuccinimide (NHS) esters bearing diverse substituents were synthesized and evaluated experimentally in terms of their lipophilicity by means of reverse-phase and immobilized artificial membrane high-performance liquid chromatography. Subsequently, the selected compounds were employed as novel cellular imaging agents for staining Gram-positive and Gram-negative bacteria, human kidney cell line, as well as human skin tissue. The analyzed dyes allowed for visualization of cellular structures such as mitochondria, endoplasmic reticulum, and cellular nuclei. They proved to be useful in fluorescent staining of stratum corneum, especially in the aspect of xenobiotic exposure and its penetration into the skin. The best results were obtained with the use of moderately lipophilic NHS esters of Safirinium Q. The development of Safirinium dyes is a promising alternative for commercially available dyes since the reported molecules have low molecular masses and exhibit efficient staining and remarkable water solubility. Moreover, they are relatively simple and low-cost in synthesis.


Assuntos
Antibacterianos , Bactérias Gram-Negativas , Compostos Azo , Epiderme , Ésteres , Corantes Fluorescentes/química , Bactérias Gram-Positivas , Humanos , Rim , Coloração e Rotulagem
9.
Sci Rep ; 12(1): 15091, 2022 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-36065053

RESUMO

The orientation factor of fluorescence resonance energy transfer (FRET) between photosynthetic light-harvesting 2 complex (LH2) and artificial fluorophore (Alexa Fluor 647: A647) was theoretically investigated. The orientation factor of 2/3, i.e., the isotropic mean, is widely used to predict the donor-acceptor distance from FRET measurements. However, this approximation seems inappropriate because the movement of A647 is possibly restricted by the bifunctional linker binding to LH2. In this study, we performed molecular dynamics (MD) simulations and electronic coupling calculations on the LH2-A647 conjugate to analyze its orientation factor. The MD results showed that A647 keeps a position approximately 26 Å away from the bacteriochlorophyll (BChl) assembly in LH2. The effective orientation factor was extracted from the electronic coupling calculated using the transition charge from electrostatic potential (TrESP) method. With MD snapshots, an averaged orientation factor was predicted to be 1.55, significantly different from the isotropic mean value. The analysis also suggested that the value of the refractive index employed in the previous studies is not suitable for this system. Furthermore, optimal orientations of A647 with larger orientation factors to improve FRET efficiency were searched using Euler angles. The present approach is useful for extending the applicability of FRET analysis.


Assuntos
Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes , Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/química , Ionóforos , Complexos de Proteínas Captadores de Luz/metabolismo , Simulação de Dinâmica Molecular , Fotossíntese
10.
Curr Protoc ; 2(9): e531, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36066206

RESUMO

Mitochondria are fundamental for human spermatozoa motility and fertilizing ability. Mitochondria participate not only in ATP production, but also in reactive oxygen species production, redox equilibrium, and calcium regulation, all of which are central for human spermatozoa motility, capacitation, acrosome reaction, and ultimately, oocyte fertilization. Mitochondrial membrane potential is a key indicator of mitochondrial health and activity. Most commonly used methods for the study of mitochondrial membrane potential, however, cannot be applied to human spermatozoa due to their unique characteristics, including high motility and time-dependent decay of quality, limiting the study of this important parameter in these cells. Here, we describe an easy, fast, and cheap protocol for the quantitative evaluation of human spermatozoa mitochondrial membrane potential, using the fluorescent cationic dye 5,5,6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimi-dazoylcarbocyanine iodide (JC-1). JC-1 is a sensitive marker for mitochondrial membrane potential, exhibiting a potential-dependent accumulation in the mitochondria. At high mitochondrial membrane potential, JC-1 forms J-aggregates, which emit red fluorescence, whereas at low mitochondrial membrane potential, JC-1 remains at its monomer state, which emits green fluorescence. We first describe how to evaluate human spermatozoa mitochondrial membrane potential using JC-1 and a fluorescence plate reader, for high-throughput studies. The calculation of the JC-1 ratio (indicative of the J-aggregates/monomers ratio) is then used to quantitatively evaluate mitochondrial health and activity. In addition, we describe an imaging protocol for the qualitative evaluation of human spermatozoa mitochondrial membrane potential using a fluorescence microscope. This allows for a visual analysis of the results that can complement the quantitative data. These protocols can be used to study the effects of spermatozoa exposure to compounds of interest, and alterations due to diseases or different conditions. While these protocols are illustrated with human spermatozoa, they can be adapted and used on spermatozoa of different species. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Quantitative evaluation of human spermatozoa mitochondrial membrane potential using the JC-1 dye and a fluorescence plate reader Basic Protocol 2: Qualitative evaluation of human spermatozoa mitochondrial membrane potential using the JC-1 dye and fluorescence microscopy Support Protocol: Preparation of the JC-1 working solution.


Assuntos
Benzimidazóis , Espermatozoides , Benzimidazóis/metabolismo , Carbocianinas/metabolismo , Corantes Fluorescentes/metabolismo , Humanos , Masculino , Potencial da Membrana Mitocondrial , Espermatozoides/metabolismo
11.
Anal Chim Acta ; 1226: 340153, 2022 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-36068047

RESUMO

A convenient and intuitive method for detecting trace heavy metal ions is vital for food safety and quality monitoring. Herein, a dual-emission ratiometric fluorescence probe based on Zr-based metal-organic frameworks (Zr-MOF) and silver nanoclusters (AgNCs) was assembled to sensitive and visual detect mercury ions (Hg2+). The sensor exhibits good sensitivity from 0.010 to 0.5 µg mL-1 with a low detection limit of 1.8 µg L-1 (corresponding to 0.72 µg kg-1 by weight). The proposed method was finally applied to determine Hg2+ in the Porphyra matrix with satisfactory outcomes, the analytical recoveries were in the range of 94.74%-101.1%, indicating the practicability of the developed method. Meanwhile, the smartphone-based colorimetric method was established by capturing the changes in fluorescence colors of the probes exposed to different Hg2+ concentrations, with a quick sample-to-answer monitoring time of 10 min. The fluorescence probe, with these merits of simplicity, rapid response, and high sensitivity, offered a promising means for evaluating the safety of food polluted with Hg2+.


Assuntos
Mercúrio , Porphyra , Corantes Fluorescentes , Íons , Limite de Detecção , Smartphone
12.
Anal Chim Acta ; 1226: 340192, 2022 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-36068051

RESUMO

Viscosity is an essential microenvironmental parameter, which is related to various diseases such as acute inflammation. So it is necessary to develop a probe to monitor viscosity changes during the inflammatory progression in vivo. Herein, a HPQ (2-(2'-hydroxyphenyl)-4(3H)-quinazolinone)-based fluorescent probe named HPQ-BI-V is prepared for detecting viscosity in biological systems. The introduction of benzindole groups extends the π conjugation of HPQ, resulting in far-red emission wavelength at 610 nm. When the viscosity raises from 3.11 cP to 567.1 cP, the fluorescence signal increases 711 times, indicating the high sensitivity of the probe. Furthermore, this probe displays excellent selectivity for viscosity in comparison with other interfering analytes. Furthermore, the probe has excellent photostability and outstanding response capability in the physiological pH range. Given these advantages, HPQ-BI-V can be applied for detecting viscosity changes in HepG2 cells and zebrafish. In particular, the probe can successfully visualize viscosity changes in acute inflammatory mice induced by LPS and the assessment of anti-inflammatory drug.


Assuntos
Corantes Fluorescentes , Peixe-Zebra , Animais , Modelos Animais de Doenças , Células HeLa , Humanos , Inflamação/induzido quimicamente , Camundongos , Mitocôndrias , Viscosidade
13.
Anal Chim Acta ; 1226: 340262, 2022 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-36068061

RESUMO

The efficient and selective detection of isomers is an attractive but challenging area. In this study, a supramolecular fluorescent probe based on cucurbit[8]uril (Q[8]) and a pyrene-based derivative (G) was prepared, which effectively recognized and removed o-nitrophenol (o-NP) from a mixture of nitrophenol isomers. The newly designed probe G@Q[8] was characterized by NMR spectroscopy, fluorescence emission and UV-Vis spectroscopy, and its host-guest properties in aqueous solution were investigated. The results revealed that the system forms a stable inclusion complex with a stoichiometric ratio of 1:1, which was accompanied by a distinct fluorescence enhancement of G. Moreover, it was employed for the rapid detection of nitrophenol isomers where o-NP showed a dramatical quenching efficiency with a detection limit of 1.53 × 10-7 mol·L-1. This highly efficient supramolecular fluorescent probe offers a new strategy for the convenient detection and removal of o-NP from mixtures in aqueous medium.


Assuntos
Corantes Fluorescentes , Nitrofenóis , Hidrocarbonetos Aromáticos com Pontes/química , Corantes Fluorescentes/química , Compostos Heterocíclicos com 2 Anéis , Imidazolidinas , Compostos Macrocíclicos , Espectroscopia de Ressonância Magnética , Água/química
14.
Anal Chim Acta ; 1226: 340288, 2022 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-36068069

RESUMO

As a member of reactive sulfur molecules, hydrogen polysulfide (H2Sn) plays a vital role in cell protection, anti-oxidative stress and regulation of redox signaling. The highly selective and sensitive detection of H2Sn was still challenging due to its special nucleophilic and electrophilic reactivity. By incorporating phenyl 2-(benzoylthio) benzoate into semi-naphthofluorescein, we developed a novel red emissive fluorescent probe SNAFL-H2Sn for the detection of a representative H2Sn (e. g. H2S2). The addition of H2S2 would rapidly trigger SNAFL-H2Sn to produce significant turn-on fluorescence signal changes at 626 nm with a linear response over a range of 2-30 µM and a detection limit of 16 nM. SNAFL-H2Sn was capable of mapping exogenous and endogenous H2S2 in living cells and zebrafish. Moreover, SNAFL-H2Sn was applied to detect endogenous H2S2 under atorvastatin stimulation. The present study demonstrated that SNAFL-H2Sn potentially served as a promising tool for interrogating H2Sn functions in biological systems.


Assuntos
Corantes Fluorescentes , Sulfeto de Hidrogênio , Animais , Dissulfetos , Hidrogênio , Sulfeto de Hidrogênio/metabolismo , Sulfetos/metabolismo , Regulação para Cima , Peixe-Zebra
15.
Nihon Yakurigaku Zasshi ; 157(5): 371-375, 2022.
Artigo em Japonês | MEDLINE | ID: mdl-36047156

RESUMO

Visualization and measurement of drugs themselves as well as biological responses to those drugs are crucial in pharmacological research. To this end, various fluorescent dyes and proteins have been developed. Despite such progresses, there still remains technical difficulties to overcome in bioimaging that keep many pharmacological targets and phenomena invisible. Outside the fields of biology where fluorescence and luminescence prevail, variety of other optical phenomena are well known and utilized. These optical phenomena can shed unique lights on biological phenomena based on their specific physical and chemical properties. Although applications of these optical phenomena to biology are yet to be explored, they have high potentials in realizing visualization and measurement of currently invisible targets and phenomena, and thereby bringing new insights into pharmacological research. Thus, here I will introduce Raman scattering microscopy that visualize vibration of functional groups as an alternative imaging platform to fluorescence and luminescence. Special focus will be put on two recent technical advancements; namely, nonlinear Raman scattering microscopy that utilizes multi-photon effect of highly tissue penetrating near-infrared lights, and Raman-tag that realizes tagging of targets that could not have been labeled, combination of which is expected to pave a way toward imaging previously invisible targets in pharmacology.


Assuntos
Microscopia , Análise Espectral Raman , Corantes Fluorescentes/química , Microscopia/métodos , Proteínas , Análise Espectral Raman/métodos , Vibração
16.
Opt Lett ; 47(17): 4455-4458, 2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-36048677

RESUMO

Imaging of molecular-specific photophysical parameters such as fluorescence intensity, emission band shape, or fluorescence decay is widely used in biophysics. Here we propose a method for quantitative mapping of another molecular-specific parameter in living cells, two-photon absorption cross section, based on the fluorescence saturation effect. Using model dye solutions and cell culture, we show that the analysis of the fluorescence signal dependencies on the intensity of two-photon excitation within the range typical for routine two-photon microscopy experiments allows one to reconstruct two-photon absorption cross section maps across the sample. We believe that the absorption cross section contrast visualized by the proposed fluorescence saturation imaging microscopy could be a new tool for studying processes in living cells and tissues.


Assuntos
Corantes Fluorescentes , Fótons , Corantes Fluorescentes/farmacologia , Microscopia de Fluorescência/métodos , Imagem Óptica , Espectrometria de Fluorescência
17.
Commun Biol ; 5(1): 895, 2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-36050388

RESUMO

Glioblastoma (GBM) is the most aggressive primary brain tumour for which both effective treatments and efficient tools for an early-stage diagnosis are lacking. Herein, we present curcumin-based fluorescent probes that are able to bind to aldehyde dehydrogenase 1A3 (ALDH1A3), an enzyme overexpressed in glioma stem cells (GSCs) and associated with stemness and invasiveness of GBM. Two compounds are selective versus ALDH1A3, without showing any appreciable interaction with other ALDH1A isoenzymes. Indeed, their fluorescent signal is detectable only in our positive controls in vitro and absent in cells that lack ALDH1A3. Remarkably, in vivo, our Probe selectively accumulate in glioblastoma cells, allowing the identification of the growing tumour mass. The significant specificity of our compounds is the necessary premise for their further development into glioblastoma cells detecting probes to be possibly used during neurosurgical operations.


Assuntos
Neoplasias Encefálicas , Curcumina , Glioblastoma , Aldeído Desidrogenase/metabolismo , Aldeído Oxirredutases/metabolismo , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/cirurgia , Curcumina/metabolismo , Curcumina/farmacologia , Diagnóstico Precoce , Corantes Fluorescentes/metabolismo , Glioblastoma/diagnóstico , Glioblastoma/metabolismo , Glioblastoma/cirurgia , Humanos , Células-Tronco Neoplásicas/metabolismo
18.
Biomed Pharmacother ; 153: 113408, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36076535

RESUMO

G-protein coupled receptors (GPCRs) have been largely targeted in a wide range of diseases, but few therapies have been directed against GPCRs in the field of cancer, partly because of the lack of effective target identification strategies. Here, using colorectal cancer (CRC) as a model, we explored the gene expression of a panel of GPCRs in tumor and stromal cells, identifying specific gene sets defining each cellular compartment. We selected the adenosine receptor 2B (A2BAR), specifically expressed in cancer cell lines compared with stromal cells, to explore the use of fluorescent ligands that can be used for target visualization. Fluorescent probes allowed semi-quantitative receptor mapping in living cells and validated the specific expression of A2BAR in CRC cell lines. As well, fluorescent ligands were effective at monitoring real-time A2BAR receptor labeling using live-imaging modalities, and displayed high efficiency when used to label complex 3D cellular systems such as tumor spheroids. Finally, we validated A2BAR as a potential pharmacological tool in CRC, using selective antagonists, finding a reduction in tumor cell proliferation. This proof-of-concept study suggests the use of fluorescent ligands for GPCR characterization through imaging, and as possible new tools used for target validation in drug screening methodologies.


Assuntos
Neoplasias Colorretais , Receptores Acoplados a Proteínas G , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Corantes Fluorescentes , Humanos , Ligantes , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais
19.
Open Biol ; 12(9): 220175, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36099931

RESUMO

The plasma membrane, as a highly complex cell organelle, serves as a crucial platform for a multitude of cellular processes. Its collective biophysical properties are largely determined by the structural diversity of the different lipid species it accommodates. Therefore, a detailed investigation of biophysical properties of the plasma membrane is of utmost importance for a comprehensive understanding of biological processes occurring therein. During the past two decades, several environment-sensitive probes have been developed and become popular tools to investigate membrane properties. Although these probes are assumed to report on membrane order in similar ways, their individual mechanisms remain to be elucidated. In this study, using model membrane systems, we characterized the probes Pro12A, NR12S and NR12A in depth and examined their sensitivity to parameters with potential biological implications, such as the degree of lipid saturation, double bond position and configuration (cis versus trans), phospholipid headgroup and cholesterol content. Applying spectral imaging together with atomistic molecular dynamics simulations and time-dependent fluorescent shift analyses, we unravelled individual sensitivities of these probes to different biophysical properties, their distinct localizations and specific relaxation processes in membranes. Overall, Pro12A, NR12S and NR12A serve together as a toolbox with a wide range of applications allowing to select the most appropriate probe for each specific research question.


Assuntos
Corantes Fluorescentes , Simulação de Dinâmica Molecular , Membrana Celular/química , Colesterol , Corantes Fluorescentes/análise , Corantes Fluorescentes/química
20.
Methods Mol Biol ; 2478: 141-240, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36063322

RESUMO

We present an instrument that combines high-resolution optical tweezers and multicolor confocal fluorescence spectroscopy. Biological macromolecules exhibit complex conformation and stoichiometry changes in coordination with their motion and activity. To further our understanding of the complex machinery of life, we need methods that can simultaneously probe more than one degree of freedom of single molecules and complexes. Fluorescence optical tweezers, or "fleezers," combine the capabilities of optical tweezers and single-molecule fluorescence microscopy into a single instrument. Here we present the latest generation of a high-resolution fleezers instrument integrated with multicolor fluorescence spectroscopy. The tweezers portion of the instrument can manipulate biological macromolecules with pN scale forces while measuring subnanometer distances. Simultaneous with tweezers measurements, the multicolor fluorescence capability allows the direct observation of multiple molecules or multiple degrees of freedom which allows, for example, the observation of multiple proteins simultaneously within a complex. The instrument incorporates three fluorescence excitation lasers, all sourced from a single-mode optical fiber allowing a reliable alignment scheme, that allows, for example, three independent fluorescent probes or fluorescence resonance energy transfer (FRET) measurements and also increases flexibility in the choice of fluorescent probes. To avoid photobleaching and improve tweezers stability, the instrument implements a timesharing (using a single trap laser to produce a pair of traps via rapid switching between two locations) and interlacing (turning the trapping beam off when the fluorescence excitation beams are on and vice versa) scheme using acousto-optic modulators (AOM) to rapidly and precisely modulate lasers. Our latest "random phase" trap AOM control method obliterates previous residual trap positioning and bead position measurement errors. Here we present the general design principles and detailed construction and testing protocols for the instrument.


Assuntos
Pinças Ópticas , Imagem Individual de Molécula , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes , Nanotecnologia/métodos , Imagem Individual de Molécula/métodos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...