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Second-harmonic generation (SHG) is a noninvasive imaging technique that enables the exploration of physiological structures without the use of an exogenous label. However, traditional SHG imaging is limited by optical diffraction, which restricts the spatial resolution. To break this limitation, we developed a novel approach called multifocal structured illumination microscopy-SHG (MSIM-SHG). By combination of SHG with MSIM, SHG-based super-resolution imaging of material molecules can be achieved, and this SHG super-resolution imaging has a wide range of applications for biological tissues and cells. MSIM-SHG achieved a lateral full width at half-maximum (fwhm) of 147 ± 13 nm and an axial fwhm of 493 ± 47 nm by imaging zinc oxide (ZnO) particles. Furthermore, MSIM-SHG was utilized to quantify collagen fiber alignment in various tissues such as the ovary, muscle, heart, kidney, and cartilage, demonstrating its feasibility for identifying collagen characteristics. MSIM-SHG has potential as a powerful tool for clinical diagnosis and biological research.
Assuntos
Microscopia , Microscopia de Geração do Segundo Harmônico , Feminino , Humanos , Iluminação , Matriz Extracelular , CoraçãoRESUMO
In biological research, rapid wide-field fluorescence lifetime imaging has become an important imaging tool. However, the biological samples with weak fluorescence signals and lower sensitivity often suffer from very low precision in lifetime determinations which restricts its widespread utilization in many bioimaging applications. To address this issue, a method is presented in this paper to substantially enhance the precision of rapid lifetime determination (RLD). It expedites the discrimination of fluorescence lifetimes, even for the weak signals coming from the cells, stained with long-lived biocompatible AIS/ZnS QDs. The proposed method works in two phases. The first phase deals with the systematic noise analysis based on the signal and contrast of the images in a time-gated imaging system, wherein acquiring the high-quality imaging data through optimization of hardware parameters improves the overall system performance. In the second phase, the chosen images are treated using total variation denoising method combined with the Max/Min filtering method for extracting the region of interest to reconstruct the intensity images for RLD. We performed several experiments on live cells to demonstrate the improvements in imaging performance by the systematic optimizations and data treatment. Obtained results demonstrated a great enhancement in signal-to-noise and contrast-to-noise ratios beside witnessing an obvious improvement in RLD for weak signals. This approach can be used not only to improve the quality of time-gated imaging data but also for efficient fluorescence lifetime imaging of live biological samples without compromising imaging speed and light exposure.
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Imagem Óptica , Microscopia de Fluorescência/métodos , Imagem Óptica/métodosRESUMO
Fluorescence lifetime imaging microendoscopy (FLIME) has been reported to investigate the physicochemical microenvironment in biological tissue. In this work, we designed a two-photon (TP) FLIME system based on a fiber-bundle glued with an achromatic mini-objective with 1.4-mm diameter, which was coupled to a standard TP microscope containing a dispersion precompensation module in the laser source. With 840â nm excitation, the field of view and lateral resolution of our system are 390â µm and 1.55â µm, respectively. To examine the capability of imaging in vivo, we obtained Z-stack (0-130â µm) TP-FLIME images from the intestine's surface of a mouse injected with squaraine dye. Further, we utilized the TP-FLIME system to image the kidney, liver, and xenografted tumor at 100-µm depth in vivo with cellular resolution, which features the distribution of cells and tissue structures with better contrast than intensity images. These results demonstrated that the proposed system is capable of measuring fluorescence lifetime in situ and provides a powerful tool to research the deep tissue microenvironment naturally.
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Rim , Fótons , Animais , Lasers , Camundongos , Imagem ÓpticaRESUMO
In this paper, we present a method to distinguish neoplastic tissues from non-neoplastic ones using calibration-free laser-induced breakdown spectroscopy (CF-LIBS). For this propose, plasma emission was collected from neoplastic and non-neoplastic tissues taken from the ovarian cancer mice models. Results were obtained by utilizing the characteristic plasma emission lines of different elements that have been confirmed in the investigated samples. From the temporal evolution of plasma emission, the optimum temporal-observation-windows are identified for LIBS investigation. The concentrations of the detected elements in tissues were measured by a calibration-free approach based on data process of plasma parameters at the local thermodynamic equilibrium. The neoplastic specimens provided more energetic plasma than non-neoplastic ones that resulting in higher peaks intensities, electron density and electron temperature especially in the early windows (between 0.1 µs to 0.8 µs). Results demonstrated higher concentrations of major and trace elements such as Mg, Fe, Ca, Na, and K in the neoplastic tissues. Finally, the results using CF-LIBS method were found to be in good agreement with that of Inductive coupled plasma-optical emission spectroscopy (ICP-OES).
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Neoplasias Ovarianas/química , Análise Espectral/métodos , Oligoelementos/análise , Animais , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Interaction between the alteration/deficiency in activation-2b (ADA2b) and histone H3/switch-3B (SWI3B) proteins was evaluated in arabidopsis mesophyll protoplasts by quantitative fluorescence resonance energy transfer (FRET) analysis. Microscopic image showed that ADA2b, SWI3B and H3 proteins colocalized in nucleus, and quantitative FRET measurements showed 0.31 of FRET efficiency (E) for the protoplasts coexpressing ECFP-ADA2b and EYFP-SWI3B, and 0.285 of E for the protoplasts coexpressing ECFP-H3 and EYFP-ADA2b, demonstrating the direct interaction of ADA2b with SWI3B/H3 protein. Collectively, SWI3B and H3 proteins are the inherent components of the ADA2b complex in which ADA2b directly interacts with SWI3B/H3 protein.
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Arabidopsis , Transferência Ressonante de Energia de Fluorescência , Histonas , Proteínas Luminescentes , ProtoplastosRESUMO
As an important cellular signal transduction messenger, Ca2+ has the capability to regulate cell function and control many biochemical processes, including metabolism, gene expression, and cell survival and death. Here, we introduce an accessible method for the photoactivation of Ca2+ channels mediated by squaraine (SQ) to rapidly induce cellular Ca2+ release and activate signal transduction. With a short preparation time, the maximum Ca2+ concentration increase could reach approximately 450% in 30 s, resulting from marked Ca2+ release channel opening in the endoplasmic reticulum (ER). This release was enhanced by another target location of SQ, that is, the outer mitochondrial-associated membrane where Ca2+ channels accumulate, and by the consequent large amounts of reactive oxygen species resulting from the respiratory chain activity stimulated by Ca2+ load. We used this method to investigate cellular signal transduction in different cancer cells and revealed rapid intracellular Ca2+ flow, unidirectional intercellular signaling processes, and neuronal signaling activity, which demonstrated the potential and convenience of the method for routine Ca2+ research.
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Canais de Cálcio/metabolismo , Ciclobutanos/metabolismo , Fenóis/metabolismo , Animais , Canais de Cálcio/química , Sinalização do Cálcio , Ciclobutanos/análise , Retículo Endoplasmático/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Fenóis/análise , Processos Fotoquímicos , Espécies Reativas de Oxigênio/análise , Espécies Reativas de Oxigênio/metabolismo , Células Tumorais CultivadasRESUMO
A versatile twisted-intramolecular-charge-transfer (TICT)-based near-infrared (NIR) fluorescent probe (L) has been judiciously designed and synthesized that could be utilized for potential cancer diagnosis and to track lymph node(s) in mice through distinct emission signals. Essentially, the probe rendered the capability to preferentially recognize the cancer cells over the noncancer cells by polarity-guided lipid droplet specific differential bioimaging (in green emission channel) studies. The probe also exhibited selective turn-on fluorescence response toward HSA/BSA in physiological media (aqueous PBS buffer; pH 7.4) at far-red/NIR regions, because of the 1:1 chelation between the probe and HSA/BSA. Therefore, the fluorescent probe was then maneuvered to track the draining lymphatic system and sentinel lymph node in tumor mice model by fluorescence imaging (NIR/deep-red channel), wherein the accumulated albumin protein in the draining tumor lymphatic system facilitated the in situ formation of the fluorescent albumin-L complex.
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Neoplasias da Mama/diagnóstico por imagem , Corantes Fluorescentes/química , Linfonodos/diagnóstico por imagem , Sistema Linfático/diagnóstico por imagem , Imagem Óptica , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Corantes Fluorescentes/síntese química , Camundongos , Estrutura MolecularRESUMO
Here we integrate multiple Gaussian-functions analysis into fluorescence resonance energy transfer (FRET) two-hybrid assays (Gaussian FRET two-hybrid assay) to determine the stoichiometric ratios of intracellular hetero-oligomers in single living cells. This method adopts in multiple Gaussian-functions to fit the E-count histograms of both donor- and acceptor-centric FRET efficiency (ED and EA) images of a single cell for obtaining the peak values (EDi and EAi), thus yielding the corresponding stoichiometric ratios (EDi/EAi) of intracellular hetero-oligomers. We performed Gaussian FRET two-hybrid assay for living Hela cells coexpressing different FRET tandem plasmids, and obtained consistent results with the expected values. Gaussian FRET two-hybrid assay for cells coexpressing Bad-CFP and Bcl-XL-YFP reveals that Bcl-XL binds with Bad to form a hetero-oligomeric complex with a stoichiometry of 2:1 on mitochondria.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Análise de Célula Única/métodos , Técnicas do Sistema de Duplo-Híbrido , Células HeLa , Humanos , Multimerização Proteica , Transdução de Sinais , Proteína de Morte Celular Associada a bcl/metabolismo , Proteína bcl-X/metabolismoRESUMO
Two-photon excited fluorescence (TPEF) plays an important role in bioimaging, the longer excitation wavelength improves its imaging depths, which gives us deeper biological information. Here, we reported the two-photon absorption of a small squaraine dye (SD), and we found that the TPEF of the small SD can be enhanced significantly using albumin, the TPEF of SD in water was enhanced 17.7 times by adding bull serum albumin (BSA) in the solution. Meanwhile, the cell imaging results indicated that the SD can enter cell effectively in less than 30 min and emit bright TPEF. Furthermore, the SD showed excellent stability against photobleaching in near-infrared II (1200 nm). The cytotoxicity experiment showed that the cytotoxicity of SD is relatively low. Our work demonstrates the excellent two-photon effect of SD in cells, potential application value of SD in two-photon bioimaging, protein detection and near infrared sensing.
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Laser-induced breakdown spectroscopy (LIBS) is a powerful tool in the soil monitoring field, but the poor spectral quality limits its application. To improve the spectral quality of major elements in soil samples, a method based on controlling the ambient pressure and time sequence was introduced. Spectral qualities that include signal-to-background ratio (SBR), spectral stability, and spectral profile were all studied in different ambient pressures and delay times. The results show that the SBRs of Na and K elements increased from 20 to about 300, when the air pressure and delay time were controlled. Meanwhile, the relative standard deviations were improved from more than 30% to less than 5% due to the release of the self-absorption effect. This work proved that the spectral qualities of LIBS can be improved a lot by controlling the ambient pressure in the field of detecting major elements in soil.
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Acceptor-sensitized quantitative Förster resonance energy transfer (FRET) measurement (E-FRET) is mainly impeded by donor emission crosstalk and acceptor direct excitation crosstalk. In this paper, we develop a novel E-FRET approach (Lux-E-FRET) based on linear unmixing (Lux) of the fluorescence intensity ratio between two detection channels with each excitation of two different wavelengths. The two detection channels need not to selectively collect the emission of donor or acceptor, and the excitation wavelengths need not to selectively excite donor or acceptor. For a tandem FRET sensor, Lux-E-FRET only needs single excitation wavelength. We performed Lux-E-FRET measurements on our dual-channel wide-field fluorescence microscope for FRET constructs in living cells, and obtained consistent FRET efficiencies with those measured by other methods. Collectively, Lux-E-FRET completely overcomes all spectral crosstalks and thus is applicable to the donor-acceptor pair with larger spectral overlapping.
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In this work, a biosensor based on Fano resonance metasurface is proposed for the specific detection of CA242 which is a typical marker of pancreatic cancer. The biosensor consists of a chiral symmetric plasma double "N" structure, which utilises coherent coupling of bright and dark modes to generate Fano resonance, achieving suppression of radiation loss, concentrating and storing energy more efficiently in the structure, and contributing to increased sensitivity to changes in ambient refractive index, resulting in a sensitivity of the sensor of up to 842.8 nm /RIU. After a series of antibody functionalization modifications, the metasurface has become an immune biosensor that can specifically detect the tumor marker CA242 of pancreatic cancer. The detection of mixed and single antigen solutions with different concentrations has verified the high sensitivity, high specificity, and high linear relationship of the biosensor to CA242, and the detection limit is as low as 0.0692 ng/mL. It is superior to other common methods and breaks the traditional disadvantages of lower detection accuracy and greater damage in tumour detection methods. The detection of the wavelength shift of localized surface plasmon resonance in plasma metasurface has been successfully applied to the highly sensitive detection of tumor markers. This study demonstrates the sensitivity and maneuverability of the chiral symmetric double "N" plasmonic metasurface biosensor, suggesting the potential application of metamaterials in biosensing based on environmental refractive index changes.
Assuntos
Técnicas Biossensoriais , Neoplasias Pancreáticas , Humanos , Ressonância de Plasmônio de Superfície/métodos , Anticorpos , Sensibilidade e Especificidade , Biomarcadores TumoraisRESUMO
Immunotherapy and its evaluation have shown great promise for cancer treatment. Here, a mouse subcutaneous transplantable tumor model was applied to testing therapeutic strategies. The mouse model was treated by regulating anti-PD-L1, anti-CTLA-4, cisplatin and their combined therapy. Biochemistry experiments have found that after immunotherapy, mice have more immune responses, which were manifested by an increase in the content of growth factors and the activation of T cells. Meanwhile, multimodal nonlinear optical microscopy imaging combined with algorithms was used to evaluate the treatment's effectiveness. By detecting the metabolism rate and microstructure in tissue, it was proved that combined therapies including immune checkpoint inhibitors do have a better effect on ovarian tumors. Our discovery of valid treatments for mice with ovarian tumor and provides an evaluation tool via nonlinear optics combined with algorithms offers new insights into therapeutic effect.
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Fluxapyroxad is a broad-spectrum and high-efficiency succinate dehydrogenase inhibitor fungicide that can control plant fungal pathogens on many crops. However, fluxapyroxad can enter the aquatic environment when applied in the field, which has an impact on the aquatic environment. The potential threat and toxicological mechanisms of fluxapyroxad in aquatic organisms remain poorly understood. In this study, zebrafish embryos were exposed to fluxapyroxad to investigate the toxic effects and potential mechanisms of fluxapyroxad. In the acute toxicity test, the lethal sensitivity rank of the zebrafish during the three stages was larvae (0.699 mg/L) > adult fish (0.913 mg/L) > embryo (1.388 mg/L). Fluxapyroxad induced abnormal spontaneous movement, malformations and decreased heartbeat, hatching percentage, and body length of the embryos. In the sublethal toxicity test, succinate dehydrogenase activity was significantly increased in all treatment groups, while the activities of the electron transport chain complex II and ATPase were markedly inhibited in 0.347 and 0.694 mg/L fluxapyroxad groups compared to that of the control group. Exposure to fluxapyroxad resulted in significant increases in MDA production, and GPx activity was significantly reduced at 0.694 mg/L. Moreover, caspase-3 activity was significantly increased in the 0.694 mg/L group, and the expression of the genes related to growth (bmp4 and lox) was inhibited after fluxapyroxad exposure. These results indicated that oxidative stress, cell apoptosis and mitochondrial damage might be the potential mechanism underlying the toxic effects of fluxapyroxad on zebrafish embryos.
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Poluentes Químicos da Água , Peixe-Zebra , Amidas , Animais , Embrião não Mamífero/metabolismo , Larva , Estresse Oxidativo , Poluentes Químicos da Água/metabolismo , Poluentes Químicos da Água/toxicidadeRESUMO
Precise and efficient cell-to-cell communication is critical to the growth and differentiation of organisms, the formation of various organism, the maintenance of tissue function and the coordination of their various physiological activities, especially to the growth and invasion of cancer cells. Tunneling nanotubes (TNTs) were discovered as a new method of cell-to-cell communication in many cell lines. In this paper, we investigated TNTs-like structures in ovarian cancer cells and proved their elements by fluorescent staining, which showed that TNTs are comprised of natural lipid bilayers with microtubules as the skeleton that can transmit ions and organelles between adjacent cells. We then used fluorescence resonance energy transfer (FRET) based on two-photon excitation fluorescence lifetime imaging microscopy (FLIM) (TP-FLIM-FRET) to detect material transport in TNTs. The experimental results showed that the number of TNTs have an impact on the drug treatment of cancer cells, which provided a new perspective for TNTs involvement in cancer treatment. Our results also showed that TP-FLIM-FRET would potentially become a new optical method for TNTs study.
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The mortality of high-grade serous ovarian cancer (HGSOC) accounts for 70% to 80% of all ovarian cancer deaths and overall mortality rate has not declined in the last decade. Recently, many studies have demonstrated that HGSOC originates from the fallopian tubes. The extracellular matrix (ECM) is present in all tissues, its remodeling and interaction with cells are crucial for regulating cell proliferation, migration, and differentiation. In this paper, we used label-free nonlinear optical microscopy to image tissues of the fallopian tube and ovary. Combining a set of image processing algorithms, we monitored the remodeling of ECM in the fallopian tube and ovary during the invasion of primary serous fallopian tube tumor into the ovary in microscopic dimension. With this approach, we can obtain physiological information of HGSOC at the early stage, which provided useful data for auxiliary clinical diagnosis.
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Neoplasias das Tubas Uterinas , Neoplasias Ovarianas , Matriz Extracelular , Feminino , Humanos , Microscopia Óptica não Linear , Neoplasias Ovarianas/diagnóstico por imagemRESUMO
Ovarian cancer (OVC), the most lethal form of all gynecological cancers, is a big threat to women's health. Late diagnosis at the advanced stages is one of the major reasons for the ovarian cancer-related deaths. Conventionally, the up-regulated proteins CA125 (cancer antigen 125) and HE4 (human epididymis protein 4) are used as biomarkers to diagnose the OVC malignancies. The lack of sensitivity/specificity and the false-positive results create complexity in the diagnostic process. With specificity over 90 %, HE4 is suitable for diagnosing ovarian cancer. Herein, we have developed an ultrasensitive all-graphene quantum dot (GQD) Förster resonance energy transfer (FRET) probe for the ratiometric detection of HE4 biomarker. A set of two GQD samples were solvothermally prepared and then analyzed by the morphological, structural, and photophysical characterization. One GQD sample exhibited a strong green emission, peaked at around 515 nm, while the other GQD sample displayed a strong red emission with maximum at around 615 nm. The good spectral overlap between the emission and excitation spectra of the green and red GQDs, respectively, all allowed us to consider them for the design of FRET-based probe. The green and red-emitting GQDs were conjugated with HE4 antibody and used as donor and acceptor, respectively for the ratiometric sensing of HE4 ovarian cancer biomarker. The all GQD FRET probe was able to detect as low as 4.8 pM, along with a large dynamic detection range up to 300 nM. The selectivity and interference effect of the developed FRET probe was also investigated against different protein combinations.
Assuntos
Grafite , Neoplasias Ovarianas , Pontos Quânticos , Biomarcadores Tumorais , Feminino , Transferência Ressonante de Energia de Fluorescência , Humanos , Neoplasias Ovarianas/diagnósticoRESUMO
Human serum albumin (HSA) is a depot and carrier for many endogenous and exogenous molecules in blood. Many studies have demonstrated that the transport of HSA in tumor microenvironments contributes to tumor development and progression. In this report, we set up a multimodal nonlinear optical microscope system, combining two-photon excitation fluorescence, second harmonic generation, and two-photon fluorescence lifetime imaging microscopy. The fluorescence lifetime of a small squaraine dye (SD) is used to evaluate HSA concentrations in tumor tissue based on specific binding between SD and HSA. We used SD to stain the cryosections from serous ovarian cancer patients in high-grade (HGSOC) and low-grade (LGSOC), respectively, and found a gradient descent of HSA concentration from normal connective tissue to extracellular matrix to tumor masses from 13 to 2 µM for LGSOC patients and from 36 to 12 µM for HGSOC patients. We demonstrated that multimodal nonlinear optical microscopy can obtain similar results as those from traditional histologic staining, thus it is expected to move to clinical applications.
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Bovine serum albumin (BSA) has a wide range of physiological functions involving the binding, transportation, and delivery of fatty acids, porphyrins, bilirubin, steroids, etc. In the present study, we prepared a small squaraine dye (SD), which can selectively detect BSA using fluorescence lifetime imaging microscopy (FLIM), to monitor the endocytosis of BSA in live cultured cells in real time. This approach revealed that BSA uptake is concentration-dependent in living cells. Furthermore, we used paclitaxel (PTX), a chemotherapeutic drug, to influence the endocytosis of BSA in living cells. The results demonstrated that the endocytic rate was clearly reduced after pretreatment with 0.4 µM PTX for 2â h. The present study demonstrates the potential value of using the fluorescence lifetime of SD to detect BSA concentration and study the physiological mechanism of chemotherapeutic drugs.
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The spontaneous excitation-emission (ExEm) spectrum is introduced to the quantitative mExEm-spFRET methodology we recently developed as a spectral unmixing component for quantitative fluorescence resonance energy transfer measurement, named as SPEES-FRET method. The spectral fingerprints of both donor and acceptor were measured in HepG2 cells with low autofluorescence separately expressing donor and acceptor, and the spontaneous spectral fingerprint of HEK293 cells with strong autofluoresence was measured from blank cells. SPEES-FRET was performed on improved spectrometer-microscope system to measure the FRET efficiency (E) and concentration ratio (R C ) of acceptor to donor vales of FRET tandem plasmids in HEK293 cells, and obtained stable and consistent results with the expected values. Moreover, SPEES-FRET always obtained stable results for the bright and dim cells coexpressing Cerulean and Venus or Cyan Fluorescent Protein (CFP)-Bax and Yellow fluorescent protein (YFP)-Bax, and the E values between CFP-Bax and YFP-Bax were 0.02 for healthy cells and 0.14 for the staurosporine (STS)-treated apoptotic cells. Collectively, SPEES-FRET has very strong robustness against cellular autofluorescence, and thus is applicable to quantitative evaluation on the protein-protein interaction in living cells with strong autofluoresence.