Upconversion fluorescent nanoparticles as a potential tool for in-depth imaging

Upconversion nanoparticles (UCNs) are nanoparticles that are excited in the near infrared (NIR) region with emission in the visible or NIR regions. This makes these particles attractive for use in biological imaging as the NIR light can penetrate the tissue better with minimal absorption/scattering. This paper discusses the study of the depth to which cells can be imaged using these nanoparticles. UCNs with NaYF(4) nanocrystals doped with Yb(3+), Er(3+) (visible emission)/Yb(3+), Tm(3+) (NIR emission) were synthesized and modified with silica enabling their dispersion in water and conjugation of biomolecules to their surface. The size of the sample was characterized using transmission electron microscopy and the fluorescence measured using a fluorescence spectrometer at an excitation of 980 nm. Tissue phantoms were prepared by reported methods to mimic skin/muscle tissue and it was observed that the cells could be imaged up to a depth of 3 mm using the NIR emitting UCNs. Further, the depth of detection was evaluated for UCNs targeted to gap junctions formed between cardiac cells.     

Optofluidic fluorescent imaging cytometry on a cell phone

Fluorescent microscopy and flow cytometry are widely used tools in biomedical sciences. Cost-effective translation of these technologies to remote and resource-limited environments could create new opportunities especially for telemedicine applications. Toward this direction, here we demonstrate the integration of imaging cytometry and fluorescent microscopy on a cell phone using a compact, lightweight, and cost-effective optofluidic attachment. In this cell-phone-based optofluidic imaging cytometry platform, fluorescently labeled particles or cells of interest are continuously delivered to our imaging volume through a disposable microfluidic channel that is positioned above the existing camera unit of the cell phone. The same microfluidic device also acts as a multilayered optofluidic waveguide and efficiently guides our excitation light, which is butt-coupled from the side facets of our microfluidic channel using inexpensive light-emitting diodes. Since the excitation of the sample volume occurs through guided waves that propagate perpendicular to the detection path, our cell-phone camera can record fluorescent movies of the specimens as they are flowing through the microchannel. The digital frames of these fluorescent movies are then rapidly processed to quantify the count and the density of the labeled particles/cells within the target solution of interest. We tested the performance of our cell-phone-based imaging cytometer by measuring the density of white blood cells in human blood samples, which provided a decent match to a commercially available hematology analyzer. We further characterized the imaging quality of the same platform to demonstrate a spatial resolution of ~2 μm. This cell-phone-enabled optofluidic imaging flow cytometer could especially be useful for rapid and sensitive imaging of bodily fluids for conducting various cell counts (e.g., toward monitoring of HIV+ patients) or rare cell analysis as well as for screening of water quality in remote and resource-poor settings.     

Surface-modified CdS nanoparticles as a fluorescent probe for the selective detection of cysteine

We present a novel method for the selective detection of cysteine, a sulfur-containing amino acid, which plays a crucial role in many important biological functions such as protein folding. Surface-modified colloidal CdS nanoparticles have been used as a fluorescent probe to selectively detect cysteine in the presence of other amino acids in the micromolar concentration range. Cysteine quenches the emission of CdS in the 0.5-10?μM concentration range, whereas the other amino acids do not affect its emission. Among the other amino acids, histidine is most efficient in quenching the emission of the CdS nanoparticles. The sulfur atom of cysteine plays a crucial role in the quenching process in the 0.5-10?μM concentration range. Cysteine is believed to quench the emission of the CdS nanoparticles by binding to their surface via its negatively charged sulfur atom. This method can potentially be applied for its detection in biological samples.     

Optical imaging fiber-based single live cell arrays: a high-density cell assay platform

A high-density, ordered array containing thousands of microwells is fabricated on an optical imaging fiber. Each individually addressable microwell is used to accommodate a single living cell. A charged coupled device (CCD) detector is employed to monitor and spatially resolve the fluorescence signals obtained from each individual cell, allowing simultaneous monitoring of cellular responses of all the cells in the array using reporter genes (lacZ, EGFP, ECFP, DsRed) or fluorescent indicators. Yeast and bacteria cell arrays were fabricated and used to perform multiplexed cell assays with resolution at the single-cell level. Monitoring gene expression in single yeast cells carrying a two-hybrid system was used to detect in vivo protein-protein interactions. The single-cell array technology provides a new platform for monitoring the unique multiple responses of large populations of individual cells from different strains or cell lines. The rich data acquired by the cell array has the potential to be employed as a new tool for cell biology research as well as to improve cell-based high-throughput screening (HTS) applications, such as the validation of new disease-associated cellular targets and the early-stage evaluation of potential drug candidates.     

Imaging of conformational changes of proteins with a new environment-sensitive fluorescent probe designed for site-specific labeling of recombinant proteins in live cells

We demonstrate herein a new method for imaging conformational changes of proteins in live cells using a new synthetic environment-sensitive fluorescent probe, 9-amino-6,8-bis(1,3,2-dithioarsolan-2-yl)-5H-benzo[a]phenoxazin-5-one. This fluorescent probe can be attached to recombinant proteins containing four cysteine residues at the i, i + 1, i + 4, and i + 5 positions of an alpha-helix. The specific binding of the fluorescent probe to this 4Cys motif enables fluorescent labeling inside cells by its extracellular administration. The high sensitivity of the fluorophore to its environment enables monitoring of the conformational changes of the proteins in live cells as changes in its fluorescence intensity. The present method was applied to calmodulin (CaM), a Ca2+-binding protein that was well-known to expose hydrophobic domains, depending on the Ca2+ concentration. A recombinant CaM fused at its C-terminal with a helical peptide containing a 4Cys motif was labeled with the fluorescent probe inside live cells. The fluorescence intensity changed reversibly depending on the intracellular Ca2+ concentration, which reflected the conformational change of the recombinant CaM in the live cells.     

Bioconjugated quantum dots as fluorescent probes for biomedical imaging

Luminescent semiconductor quantum dots have become an important class of fluorescent labels for biological and biomedical imaging. In comparison with conventional organic dyes and fluorescent proteins, quantum dots have extraordinary fluorescent properties including high brightness, high resistance to photobleaching and tunable wavelengths. In this review, we briefly discuss the properties and modification of quantum dots. We focus on the applications of quantum dots in biomedical imaging, including molecular detection, live cell imaging and in vivo imaging. The toxicity of the quantum dots to cells and animals is also discussed.     

Aqueous self-assembly and surface-functionalized nanodots for live cell imaging and labeling

Nanoparticles have enormous potential for bioimaging and biolabeling applications, in which conventional organically based fluorescent labels degrade and fail to provide long-term tracking. Thus, the development of approaches to make fluorescent probes water soluble and label cells efficient is desirable for most biological applications. Here, we report on the fabrication and charac- terization of self-assembled nanodots （SANDs） from 3-aminopropyltriethoxysilane （APTES） as a probe for protein labeling. We show that fluorescent SAND probes exhibit both bright photoluminescence and biocompatibility in an aqueous environment. Selective in vitro imaging using protein and carbohydrate labeling of hepatoma cell lines are demonstrated using biocompatible SANDs conjugated with avidin and galactose, respectively. Cytotoxicity tests show that conjugated SAND particles have negligible effects on cell proliferation. Unlike other synthetic systems that require multistep treatments to achieve robust surface functionalization and to develop flexible bioconjugation strategies, our results demonstrate the versatility of this one-step SAND fabrication method for creating multicolor fluorescent probes with the tailored functionalities, effident emission, as well as excellent biocompatibility, required for broad biological use.     

Capillary electrophoresis-based noncompetitive immunoassay for the prion protein using fluorescein-labeled protein A as a fluorescent probe

A novel CE-based noncompetitive immunoassay for prion protein (PrP) was established. Fluorescein isothiocyanate (FITC)-labeled protein A (FITC-PrA) was used as a fluorescent probe to tag monoclonal antibody through noncovalent binding of FITC-PrA to the Fc region of the antibody. The FITC-PrA-Ab was incubated with the analyte, prion protein, under optimized condition, forming the immunocomplex FITC-PrA-Ab-PrP. The complex was separated and analyzed by capillary zone electrophoresis. The addition of carboxymethyl-beta-cyclodextrin in the running buffer as dynamical coating reagent improved the reproducibility and the resolution. The complex was isolated in less than 1 min with theoretical plates of 3.8 x 10(4). Relative standard deviations of peak height and migration time for the complex were 3.46 and 1.48%, respectively. A linear relationship was established for the bovine recombinant prion protein (rPrP) concentration in the range from 0.2 to 2.0 mug/mL and the peak height. The correlation factor was r2 = 0.9969. The estimated detection limit for rPrP was approximately 6 ng/mL, which is 3 times the signal-to-noise ratio. The method was successfully applied for testing blood samples from scrapie-infected sheep.     

Reaction-based fluorescent probe for selective discrimination of thiophenols over aliphaticthiols and its application in water samples

The development of highly sensitive and selective detection techniques for the discrimination of relevant toxic benzenethiols and biologically active aliphatic thiols is of considerable importance in the fields of chemical, biological, and environmental sciences. In this article, we describe a new design of reaction-based fluorescent probe for discrimination of thiophenols over aliphaticthiols through intramolecular charge transfer pathways using N-butyl-4-amino-1,8-naphthalimide as a fluorophore, the strongly electron-withdrawing 2,4-dinitrobenzenesulfonamide group as a recognition unit, and 2,3-dihydroimidazo-[1,2-a] pyridine moiety as a linker. This rational design not only affords finely tunable spectroscopic properties by adding 2,3-dihydroimidazo-[1,2-a] pyridine moiety but also provides the chance to regulate the selectivity and sensitivity of the probe due to the formation of a new type of potentially reversible sulfonamide bond through 4-dimethylaminopyridine-like resonance. The developed probe displayed high off/on signal ratios, good selectivity, and sensitivity with a detection limit of 20 nM and a relative standard deviation of 1.7% for 11 replicate detections of 0.33 μM thiophenol and was successfully applied to the determination of thiophenols in water samples with quantitative recovery (from 94% to 97%) demonstrating its application prospect for thiophenols sensing in environmental and biological sciences.     

Double-strand DNA-templated formation of copper nanoparticles as fluorescent probe for label-free aptamer sensor

Double-strand DNA (dsDNA) can act as an efficient template for the formation of copper nanoparticles (Cu NPs) at low concentration of CuSO(4), and the formed Cu NPs have excellent fluorescence, whereas a single-strand DNA (ssDNA) template does not support Cu NPs' formation. This property of dsDNA-Cu NPs makes it suitable for DNA sensing. However, exploration of dsDNA-Cu NPs applied in biological analysis is still at an early stage. In this regard, we report herein for the first time a sensitive, cost-effective, and simple aptamer sensor (aptasensor) using dsDNA-Cu NPs as fluorescent probe. The design consists of a dsDNA with reporter DNA (here, aptamer) as template for the formation of Cu NPs, and the formed dsDNA-Cu NPs show high fluorescence. Using adenosine triphosphate (ATP) as a model analyte, the introduction of ATP triggers the structure switching of reporter DNA to form aptamer-ATP complex, causing the destruction of the double helix and thus no formation of the Cu NPs, resulting in low fluorescence. The preferable linear range (0.05-500 μM), sensitivity (LOD 28 nM), and simplicity for the detection of ATP indicate that dsDNA-Cu NPs may have great prospects in the field of biological analysis. We also use this novel fluorescent probe to determine ATP in 1% human serum and human adenocarcinoma HeLa cells. The dsDNA-Cu NPs probes provide recovery of 104-108% in 1% human serum and a prominent fluorescent signal is obtained in cellular ATP assay, revealing the practicality of using dsDNA-Cu NPs for the determination of ATP in real samples. Besides, this design is simply based on nucleic acid hybridization, so it can be generally applied to other aptamers for label-free detection of a broad range of analytes. Successful detection of cocaine with detection limit of 0.1 μM demonstrates its potential to be a general method.     

Quantum dots with a paramagnetic coating as a bimodal molecular imaging probe

MRI detectable and targeted quantum dots were developed. To that aim, quantum dots were coated with paramagnetic and pegylated lipids, which resulted in a relaxivity, r(1), of nearly 2000 mM(-1)s(-1) per quantum dot. The quantum dots were functionalized by covalently linking alphavbeta3-specific RGD peptides, and the specificity was assessed and confirmed on cultured endothelial cells. The bimodal character, the high relaxivity, and the specificity of this nanoparticulate probe make it an excellent contrast agent for molecular imaging purposes.     

Autofocusing in digital holographic phase contrast microscopy on pure phase objects for live cell imaging

Digital holography enables a multifocus quantitative phase microscopy for the investigation of reflective surfaces and for marker-free live cell imaging. For digital holographic long-term investigations of living cells an automated (subsequent) robust and reliable numerical focus adjustment is of particular importance. Four numerical methods for the determination of the optimal focus position in the numerical reconstruction and propagation of the complex object waves of pure phase objects are characterized, compared, and adapted to the requirements of digital holographic microscopy. Results from investigations of an engineered surface and human pancreas tumor cells demonstrate the applicability of Fourier-weighting- and gradient-operator-based methods for robust and reliable automated subsequent numerical digital holographic focusing.     

Development of biocompatible fluorescent gelatin nanocarriers for cell imaging and anticancer drug targeting

In recent years, fluorescent carbon dots (CDs) have attracted a great deal of attention in imaging and related biomedical applications due to their excellent photoluminescence properties, low cost, high quantum yield and low cytotoxicity in comparison with semiconductor quantum dots based on metallic elements. In this paper, a new and simple design for development of CDs/gelatin nanoparticles (CDs/GNPs) is described which used as a novel methotrexate (MTX) nanocarrier and MCF-7 cell imaging. The obtained fluorescent nanocarriers were characterized using FTIR, SEM, XRD, DLS, PL, TGA, and zeta-potential analysis. Afterward, the performance of developed NPs was investigated through different in vitro tests such as MTT assay, fluorescence microscopy, and flow cytometry analyses. MTX was successfully loaded into the fluorescent NPs at physiological pH (7.4) by ionic interactions between anionic carboxylate groups of MTX and cationic amino groups on the surface of NPs. MTX releasing ability of the obtained nanocarrier was illustrated through the comparison of in vitro drug release at both simulated tumor tissue and physiological environment. The MTT assay revealed that the MTX-loaded nanocarriers have higher cytotoxicity in MCF-7 breast cancer cells than nanocarriers without MTX. Upon the obtained results, our fluorescent nanocarriers hold great potential as drug delivery carriers for the targeted MTX delivery to the cancer cells and biological fluorescent labeling.     

Plasmonic nanosensors for imaging intracellular biomarkers in live cells

Here we present the first intracellular molecular imaging platform using multifunctional gold nanoparticles which incorporate both cytosolic delivery and targeting moieties on the same particle. The utility of these intracellular sensors was demonstrated by monitoring actin rearrangement in live fibroblasts. We observed a strong molecular specific optical signal associated with effective targeting of actin filaments. These multifunctional nanosensors can be adapted to target various intracellular processes especially where transfection or cytotoxic labels are not feasible.     

An engineered virus as a bright fluorescent tag and scaffold for cargo proteins--capture and transport by gliding microtubules

We have demonstrated substantial capture and transport of fluorescently-labeled engineered cowpea mosaic virus (CPMV) using Drosophila kinesin-driven microtubules (MTs). The capture occurred through both NeutrAvidin (NA)-biotin and antibody (IgG)-antigen interactions. The MTs were derivatized with rabbit anti-chicken IgG or biotin, and the virus was conjugated with chicken IgG or NA. The CPMV conjugate was introduced into standard MT motility assays via convective flow at concentrations as high as 1.36 nM, and became bound to the MTs in densities as high as one virus per microm of MT length. When the CPMV conjugate was present at 17 pM, the average speed of the MTs bearing the NA-virus was 0.59 +/- 0.08 microm/sec, and that of those bearing IgG-virus was 0.52 +/- 0.15 microm/sec. These speeds are comparable to those of the unladen MTs (0.61 +/- 0.09 microm/sec), the presence of the virus on the MT causing only a small decrease in MT gliding speeds. The fluorescent CPMV appears to be superior to fluorescent polystyrene spheres of the same size, as both a reporter tag and a scaffold for MT-transported cargo proteins, because of its negligible non-specific adsorption and superior brightness. This work is important for the development of sensors based on nanolocomotion and biological recognition, or new strategies for the nanoassembly of biological structures.     

Graphene transistor as a probe for streaming potential

We explore the dependence of electrical transport in a graphene field effect transistor (GraFET) on the flow of water/sodium chloride electrolyte within the immediate vicinity of that transistor. We find large and reproducible shifts in the charge neutrality point of GraFETs that are dependent on the liquid velocity and the ion concentration. We show that these shifts are consistent with the variation of the local electrochemical potential of the liquid next to graphene that are caused by the fluid flow (streaming potential). Furthermore, we utilize the sensitivity of electrical transport in GraFETs to the parameters of the fluid flow to demonstrate graphene-based mass flow and ionic concentration sensing. We successfully detect a flow as small as ～70 nL/min and detect a change in the ionic concentration as small as ～40 nM.     

A microsphere coupler for a nanowire waveguide plasmonic probe for molecular imaging

Highly forward scattering of light by a silica microsphere is predicted by Mie theory. Finite-difference time-domain simulations are used to examine the use of a silica microsphere for coupling light into a ZnO nanowire terminated with a gold nanoparticle (NP) tip, intended for near-field imaging of single molecules immobilized on a gold substrate or gold nanoparticle-labeled cell membranes. The results show that plasmonic coupling at the Au tip is dependent on the incident angle of the excitation. Pre-conditioning the signal with the microsphere amplifies the coupling while reducing background energy levels, significantly boosting the signal-to-noise ratio.     

Direct Raman imaging techniques for study of the subcellular distribution of a drug

Direct Raman imaging techniques are demonstrated to study the drug distribution in living cells. The advantage of Raman imaging is that no external markers are required, which simplifies the sample preparation and minimally disturbs the drug mechanism during imaging. The major challenge in Raman imaging is the weak Raman signal. In this study, we present a Raman image model to describe the degradation of Raman signals by imaging processes. Using this model, we demonstrate special-purpose image-processing algorithms to restore the Raman images. The processing techniques are then applied to visualize the anticancer agent paclitaxel in living MDA-435 breast cancer cells. Raman images were obtained from a cancer cell before, during, and after drug treatment. The paclitaxel distribution illustrated in these images is explained by means of the binding characteristics of the paclitaxel and its molecular target-the microtubules. This result demonstrates that direct Raman imaging is a promising tool to study the distribution of a drug in living cells.