Progress in understanding the cell biology and diseases depends on advanced imaging and labeling techniques. Here, we address this demand by exploring novel multilayered nanocomposites (MNCs) with plasmonic nanoparticles and absorbing dyes in thin nonabsorbing shells as supercontrast multimodal photoacoustic (PA) and fluorescent agents in the near‐infrared range. The proof of concept was performed with gold nanorods (GNRs) and indocyanine green (ICG) dispersed in a matrix of biodegradable polymers. We demonstrated synergetic PA effects in MNCs with the gold‐ICG interface that could not be achieved with ICG and GNRs alone. We also observed ultrasharp PA and emission peaks that could be associated with nonlinear PA and spaser effects, respectively. Low‐toxicity multimodal MNCs with unique plasmonic, thermal and acoustic properties have the potential to make a breakthrough in PA flow cytometry and near‐infrared spasers in vivo by using the synergetic interaction of plasmonic modes with a nearby absorbing medium. 相似文献
Reversibly switchable fluorescent proteins (RSFPs) are GFP-like proteins that may be repeatedly switched by irradiation with light from a fluorescent to a nonfluorescent state, and vice versa. They can be utilized as genetically encodable probes and bear large potential for a wide array of applications, in particular for new protein tracking schemes and subdiffraction resolution microscopy. However, the currently described monomeric RSFPs emit only blue-green or green fluorescence; the spectral window for their use is thus rather limited. Using a semirational engineering approach based on the crystal structure of the monomeric nonswitchable red fluorescent protein mCherry, we generated rsCherry and rsCherryRev. These two novel red fluorescent RSFPs exhibit fluorescence emission maxima at ∼610 nm. They display antagonistic switching modes, i.e., in rsCherry irradiation with yellow light induces the off-to-on transition and blue light the on-to-off transition, whereas in rsCherryRev the effects of the switching wavelengths are reversed. We demonstrate time-lapse live-cell subdiffraction microscopy by imaging rsCherryRev targeted to the endoplasmic reticulum utilizing the switching and localization of single molecules. 相似文献
We present a dual‐modality technique based on wide‐field photothermal (PT) interferometric phase imaging and simultaneous PT ablation to selectively deplete specific cell populations labelled by plasmonic nanoparticles. This combined technique utilizes the plasmonic reaction of gold nanoparticles under optical excitation to produce PT imaging contrast by inducing local phase changes when the excitation power is weak, or ablation of selected cells when increasing the excitation power. Controlling the entire process is carried out by dynamic quantitative phase imaging of all cells (labelled and unlabelled). We demonstrate our ability to detect and specifically ablate in vitro cancer cells over‐expressing epidermal growth factor receptors (EGFRs), labelled with plasmonic nanoparticles, in the presence of either EGFR under‐expressing cancer cells or white blood cells. The latter demonstration establishes an initial model for depletion of circulating tumour cells in blood. The proposed system is able to image in wide field the label‐free quantitative phase profile together with the PT phase profile of the sample, and provides the ability of both detection and selective cell ablation in a controlled environment.
Quantitative phase imaging with molecular specificity and specific cell depletion. ( a ) Label‐free quantitative phase profiles of mixed population of EGFR+/EGFR– cancer cells. ( b ) When weak modulated PT excitation is applied, selective phase contrast is generated in the modulation frequency only for the EGFR+ cancer cells labelled with plasmonic nanoparticles. ( c ) When stronger modulated PT excitation is applied, selective ablation of the EGFR+ cancer cells labelled with plasmonic nanoparticles occurs. White scalebars represent 10 µm upon sample. 相似文献
Bacterial infections are a primary cause of morbidity and mortality worldwide. Bacteremia is a particular concern owing to the possibility of septic shock and the development of metastatic infections. Treatment of bacteremia is increasingly compromised by the emergence of antibiotic resistant strains, creating an urgent need for alternative therapy. Here, we introduce a method for in vivo photoacoustic (PA) detection and photothermal (PT) eradication of Staphylococcus aureus in tissue and blood. We show that this method could be applicable for label-free diagnosis and treatment of in the bloodstream using intrinsic near-infrared absorption of endogenous carotenoids with nonlinear PA and PT contrast enhancement. To improve sensitivity and specificity for detection of circulating bacteria cells (CBCs), two-color gold and multilayer magnetic nanoparticles with giant amplifications of PA and PT contrasts were functionalized with an antibody cocktail for molecular targeting of S. aureus surface-associated markers such as protein A and lipoprotein. With a murine model, the utility of this approach was demonstrated for ultrasensitive detection of CBCs with threshold sensitivity as low as 0.5 CBCs/mL, in vivo magnetic enrichment of CBCs, PT eradication of CBCs, and real-time monitoring of therapeutic efficacy by CBC counting. Our PA-PT nano-theranostic platform, which integrates in vivo multiplex targeting, magnetic enrichment, signal amplification, multicolor recognition, and feedback control, could be used as a biological tool to gain insights on dissemination pathways of CBCs, infection progression by bacteria re-seeding, and sepsis development and treatment, and could potentially be feasible in humans, especially using bypass schematic. 相似文献
Confocal fluorescence microscopy is a powerful biological tool providing high-resolution, three-dimensional (3D) imaging of fluorescent molecules. Many cellular components are weakly fluorescent, however, and thus their imaging requires additional labeling. As an alternative, label-free imaging can be performed by photothermal (PT) microscopy (PTM), based on nonradiative relaxation of absorbed energy into heat. Previously, little progress has been made in PT spectral identification of cellular chromophores at the 3D microscopic scale. Here, we introduce PTM integrating confocal thermal-lens scanning schematic, time-resolved detection, PT spectral identification, and nonlinear nanobubble-induced signal amplification with a tunable pulsed nanosecond laser. The capabilities of this confocal PTM were demonstrated for high-resolution 3D imaging and spectral identification of up to four chromophores and fluorophores in live cells and Caenorhabditis elegans. Examples include cytochrome c, green fluorescent protein, Mito-Tracker Red, Alexa-488, and natural drug-enhanced or genetically engineered melanin as a PT contrast agent. PTM was able to guide spectral burning of strong absorption background, which masked weakly absorbing chromophores (e.g., cytochromes in the melanin background). PTM provided label-free monitoring of stress-related changes to cytochrome c distribution, in C. elegans at the single-cell level. In nonlinear mode ultrasharp PT spectra from cyt c and the lateral resolution of 120 nm during calibration with 10-nm gold film were observed, suggesting a potential of PTM to break through the spectral and diffraction limits, respectively. Confocal PT spectromicroscopy could provide a valuable alternative or supplement to fluorescence microscopy for imaging of nonfluorescent chromophores and certain fluorophores. 相似文献
The fluorescent labeling of target proteins is useful for analyzing their functions and localization in cells, and several fluorescent probes have been developed. However, the fusion of tags such as green fluorescent protein (GFP) to target proteins occasionally affects their functions and/or localization in living cells. Therefore, an imaging method that uses short peptide tags such as hexa-histidine (the His tag) has been attracting increasing attention. Few studies have investigated ON/OFF switchable fluorescent probes for intracellular His-tagged proteins. We herein developed a novel ON/OFF switchable probe for imaging targeted intracellular proteins fused with a CH6 tag, which is composed of one cysteine residue and six histidine residues. 相似文献
Here we demonstrate multiplex and simultaneous detection of four different rare RNA species from plant, Arabidopsis thaliana, using surface‐enhanced Raman spectroscopy (SERS) and gold nanoprobes at single‐cell resolution. We show the applicability of nanoparticle‐based Raman spectroscopic sensor to study intracellular RNA copies. First, we demonstrate that gold‐nanoparticles decorated with Raman probes and carrying specific nucleic acid probe sequences can be uptaken by the protoplasts. We confirm the internalization of gold nanoprobes by transmission electron microscopy, inductively‐coupled plasma‐mass spectrometry and fluorescence imaging. Second, we show the utility of a SERS platform to monitor individual alternatively spliced (AS) variants and miRNA copies within single cells. Finally, the distinctive spectral features of Raman‐active dyes were exploited for multiplex analysis of AtPTB2, AtDCL2, miR156a and miR172a. Furthermore, single‐cell studies were validated by in vitro quantification and evaluation of nanotoxicity of gold probes. Raman tag functionalized gold nanosensors yielded an approach for the tracking of rare RNAs within the protoplasts. The SERS‐based approach for quantification of RNAs has the capability to be a highly sensitive, accurate and discerning method for single‐cell studies including AS variants quantification and rare miRNA detection in specific plant species. 相似文献
Light can manipulate molecular biological processes with high spatial and temporal precision and optical manipulation has become increasingly popular during the last years. In combination with absorbing dyes or gold nanoparticles light is a valuable tool for cell and protein inactivation with high precision. Here we show distinct differences in the underlying mechanisms whether gold nanoparticles or fluorescent dyes are used for the inactivation of the Ki‐67 protein. The proliferation‐associated protein Ki‐67 was addressed by the antibody MIB‐1. In vitro studies showed a fragmentation of the Ki‐67 protein after laser irradiation of 15 nm gold nanoparticle antibody conjugates with nanosecond pulsed laser, while continuous wave (cw) irradiation of fluorescein isothiocyanate (FITC)‐ and Alexa 488‐labeled antibodies led to specific crosslinking of Ki‐67. The irradiation energy for the gold nanoparticles was above cavitation bubble formation threshold. We observed a fragmentation of the target protein and also of the gold particles. The understanding of the underlying inactivation mechanisms is important for the application and further development of these two techniques, which can harness nanotechnology to introduce molecular selectivity to biological systems. 相似文献
Gold nanoparticles with a 12-nm diameter were used as probes for the determination of proteins by resonance Rayleigh-scattering techniques. In weak acidic solution, large amounts of citrate anions will self-assemble on the surface of positively charged gold nanoparticles to form supermolecular compounds with negative charges. Below the isoelectric point, proteins with positive charges such as human serum albumin (HSA), bovine serum albumin (BSA), and ovalbumin (Ova) can bind gold nanoparticles to form larger volume products (the diameter of the binding product of gold nanoparticles with HSA is 23 nm.) through electrostatic force, hydrogen bonds, and hydrophobic effects, which can result in a red shift of the maximum absorption wavelength, the remarkable enhancement of the resonance Rayleigh-scattering intensity (RRS), and the appearance of the RRS spectra. At the same time, the second-order-scattering (SOS) and frequency-doubling-scattering (FDS) intensities are also enhanced. The binding products of gold nanoparticles with different proteins have similar spectral characteristics and the maximum wavelengths are located near 303 nm for RRS, 540 nm for SOS, and 390 for FDS, respectively. The scattering enhancement (DeltaI) is directly proportional to the concentration of proteins. Among them, the RRS method has the highest sensitivity and the detection limits are 0.38 ng/ml for HSA, 0.45 ng/ml for BSA, and 0.56 ng/ml for Ova, separately. The methods have good selectivity. A new RRS method for the determination of trace proteins using a gold nanoparticle probe has been developed. Because gold nanoparticle probes do not need to be modified chemically in advance, the method is very simple and fast. 相似文献
In this study, a novel electroconductive interface was prepared based on Fe3O4 magnetic nanoparticle and cysteamine functionalized gold nanoparticle. The engineered interface was used as signal amplification substrate in the electrochemical analysis of antibody‐antigen binding. For this purpose, biotinilated‐anti‐prostate‐specific antigen (PSA) antibody was bioconjugated with iron oxide magnetic nanoparticles (Fe3O4) and drop‐casted on the surface of glassy carbon electrode (GCE). Also, secondary antibody (HRP‐Ab2) encapsulated on gold nanoparticles caped by cysteamine was immobilized on the surface of GCE modified electrode. A transmission electron microscopy images shows that a sandwich immunoreaction was done and binding of Ab1 and Ab2 performed successfully. Various parameters of immunoassay, including the loading of magnetic nanoparticles, the amount of gold nanoparticle conjugate, and the immunoreaction time, were optimized. The detection limit of 0.001 μg. L?1 of PSA was obtained under optimum experimental conditions. It is found that such magneto‐bioassay could be readily used for simultaneous parallel detection of multiple proteins by using multiple inorganic metal nanoparticle tracers and are expected to open new opportunities for early stage diagnosis of cancer in near future. 相似文献
Conventional flow cytometry using scattering and fluorescent detection methods has been a fundamental tool of biological discoveries for many years. Invasive extraction of cells from a living organism, however, may lead to changes in cell properties and prevents the long-term study of cells in their native environment. Here, we summarize recent advances of new generation flow cytometry for in vivo noninvasive label-free or targeted detection of cells in blood, lymph, bone, cerebral and plant vasculatures using photoacoustic (PA) detection techniques, multispectral high-pulse-repetition-rate lasers, tunable ultrasharp (up to 0.8nm) rainbow plasmonic nanoprobes, positive and negative PA contrasts, in vivo magnetic enrichment, time-of-flight cell velocity measurement, PA spectral analysis, and integration of PA, photothermal (PT), fluorescent, and Raman methods. Unique applications of this tool are reviewed with a focus on ultrasensitive detection of normal blood cells at different functional states (e.g., apoptotic and necrotic) and rare abnormal cells including circulating tumor cells (CTCs), cancer stem cells, pathogens, clots, sickle cells as well as pharmokinetics of nanoparticles, dyes, microbubbles and drug nanocarriers. Using this tool we discovered that palpation, biopsy, or surgery can enhance CTC release from primary tumors, increasing the risk of metastasis. The novel fluctuation flow cytometry provided the opportunity for the dynamic study of blood rheology including red blood cell aggregation and clot formation in different medical conditions (e.g., blood disorders, cancer, or surgery). Theranostics, as a combination of PA diagnosis and PT nanobubble-amplified multiplex therapy, was used for eradication of CTCs, purging of infected blood, and thrombolysis of clots using PA guidance to control therapy efficiency. In vivo flow cytometry using a portable fiber-based devices can provide a breakthrough platform for early diagnosis of cancer, infection and cardiovascular disorders with a potential to inhibit, if not prevent, metastasis, sepsis, and strokes or heart attack by well-timed personalized therapy. 相似文献
Fluorescent proteins that can be reversibly photoswitched between a fluorescent and a nonfluorescent state are important for innovative microscopy schemes, such as protein tracking, fluorescence resonance energy transfer imaging, sub-diffraction resolution microscopy and others. However, all available monomeric reversibly switchable fluorescent proteins (RSFPs) have similar properties and switching characteristics, thereby limiting their use. Here, we introduce two bright green fluorescent RSFPs, bsDronpa and Padron, generated by extensive mutagenesis of the RSFP Dronpa, with unique absorption and switching characteristics. Whereas bsDronpa features a broad absorption spectrum extending into the UV, Padron displays a switching behavior that is reversed to that of all green fluorescent RSFPs known to date. These two RSFPs enable live-cell fluorescence microscopy with multiple labels using a single detection color, because they can be distinguished by photoswitching. Furthermore, we demonstrate dual-color fluorescence microscopy with sub-diffraction resolution using bsDronpa and Dronpa whose emission maxima are separated by <20 nm. 相似文献
Structural DNA nanotechnology, in which Watson-Crick base pairing drives the formation of self-assembling nanostructures, has rapidly expanded in complexity and functionality since its inception in 1981. DNA nanostructures can now be made in arbitrary three-dimensional shapes and used to scaffold many other functional molecules such as proteins, metallic nanoparticles, polymers, fluorescent dyes and small molecules. In parallel, the field of dynamic DNA nanotechnology has built DNA circuits, motors and switches. More recently, these two areas have begun to merge—to produce switchable DNA nanostructures, which change state in response to their environment. In this review, we summarise switchable DNA nanostructures into two major classes based on response type: molecular actuation triggered by local chemical changes such as pH or concentration and external actuation driven by light, electric or magnetic fields. While molecular actuation has been well explored, external actuation of DNA nanostructures is a relatively new area that allows for the remote control of nanoscale devices. We discuss recent applications for DNA nanostructures where switching is used to perform specific functions—such as opening a capsule to deliver a molecular payload to a target cell. We then discuss challenges and future directions towards achieving synthetic nanomachines with complexity on the level of the protein machinery in living cells. 相似文献
Proteomic data from embryos are essential for the completion of whole proteome catalog due to embryo‐specific expression of certain proteins. In this study, using reverse phase LC‐MS/MS combined with 1‐D SDS‐PAGE, we identified 1625 mammalian and 735 Sus scrofa proteins from porcine zygotes that included both cytosolic and membranous proteins. We also found that the global protein profiles of parthenogenetically activated (PA) and in vitro fertilized (IVF) zygotes were similar but differences in expression of individual proteins were also evident. These differences were not due to culture conditions, polyspermy or non‐activation of oocytes, as the same culture method was used in both groups, the frequency of polyspermy was 24.3±3.0% and the rates of oocyte activation did not differ (p>0.05) between PA and IVF embryos. Consistent with proteomic data, fluorescent Hoechst 33 342 staining and terminal deoxynucleotidyl transferase dUTP nick end labeling assay also revealed that PA embryos were of poor quality as they contained less cells per blastocyst and were more predisposed to apoptosis (p<0.05), although their in vitro development rates were similar. To our knowledge, this is the first report on global peptide sequencing and quantification of protein in PA and IVF embryos by LC‐MS/MS that may be useful as a reference map for future studies. 相似文献
Photoactivatable and photoconvertible fluorescent proteins capable of pronounced light‐induced spectral changes are a powerful addition to the fluorescent protein toolbox of the cell biologist. They permit specific tracking of one subcellular structure (organelle or cell subdomain) within a differentially labelled population. They also enable pulse–chase analysis of protein traffic. The Kaede gene codes for a tetrameric protein found in the stony coral Trachyphyllia geoffroyi, which emits green fluorescence that irreversibly shifts to red following radiation with UV or violet light. We report here the use of Kaede to explore the plant secretory pathway. Kaede versions of the Golgi marker sialyl‐transferase (ST‐Kaede) and of the vacuolar pathway marker cardosin A (cardA‐Kaede) were engineered. Several optical devices enabling photoconversion and observation of Kaede using these two constructs were assessed to optimize Kaede‐based imaging protocols. Photoconverted ST‐Kaede red‐labelled organelles can be followed within neighbouring populations of non‐converted green Golgi stacks, by their gradual development of orange/yellow coloration from de novo synthesis of Golgi proteins (green). Results highlight some aspects on the dynamics of the plant Golgi. For plant bio‐imaging, the photoconvertible Kaede offers a powerful tool to track the dynamic behaviour of designated subpopulations of Golgi within living cells, while visualizing the de novo formation of proteins and structures, such as a Golgi stack. 相似文献