Bioimaging Studies Research Articles

Sodium Cholate-Templated Blue Light-Emitting Ag Subnanoclusters: In Vivo Toxicity and Imaging in Zebrafish Embryos

We report a novel green chemical approach for the synthesis of blue light-emitting and water-soluble Ag subnanoclusters, using sodium cholate (NaC) as a template at a concentration higher than the critical micelle concentration (CMC) at room temperature. However, under photochemical irradiation, small anisotropic and spherically shaped Ag nanoparticles (3-11 nm) were obtained upon changing the concentration of NaC from below to above the CMC. The matrix-assisted laser desorption ionization time-of-flight and electrospray ionization mass spectra showed that the cluster sample was composed of Ag4 and Ag6. The optical properties of the clusters were studied by UV-visible and luminescence spectroscopy. The lifetime of the synthesized fluorescent Ag nanoclusters (AgNCs) was measured using a time-correlated single-photon counting technique. High-resolution transmission electron microscopy was used to assess the size of clusters and nanoparticles. A protocol for transferring nanoclusters to organic solvents is also described. Toxicity and bioimaging studies of NaC templated AgNCs were conducted using developmental stage zebrafish embryos. From the survival and hatching experiment, no significant toxic effect was observed at AgNC concentrations of up to 200 μL/mL, and the NC-stained embryos exhibited blue fluorescence with high intensity for a long period of time, which shows that AgNCs are more stable in living system.

Exploitation of a new Schiff-base ligand for boric acid fluorescent sensing in aqueous medium with bio-imaging studies in a living plant system

A new Schiff-base ligand owing to 1 : 1 chelation with boric acid exhibits selective boric acid fluorosensor at physiological condition along with bio-imaging facilities.

Targeted In Vivo Imaging of Mouse Hindlimb Ischemia Using Fluorescent Gelatin Nanoparticles

Critical limb ischemia is one of the most advanced forms of peripheral artery disease, which seriously threat the human health and even cause amputation. In this study, we developed a fluorescent gelatin nanoparticle (FGNP) by covalent conjugation of the nanoparticles with two fluorogens, Cy7 and rhodamine B. The FGNPs have a volume average hydrodynamic diameter of about 168 nm, which also show low cytotoxicity against NIH/3T3 normal cells. The in vivo ischemia bioimaging studies in live mice and in ischemic limb slices demonstrate that the FGNPs can be preferentially accumulated to the ischemic site, which can thus serve as a safe and effective probe for targeted visualization of ischemia in the limb.

Selective fluorescence swing from cysteine to glutathione by switchover from solid to in situ probe in 100% water and bio-imaging studies for living species

Two novel non-toxic Schiff-base-Cu2+ complexes, acting as fluorescent probes, synthesized in solid (1) and generated in situ (2), exhibit unprecedented selectivity and rapid sensitivity for sensing cysteine (Cys) and gluthathione (GSH), respectively, in 100% aqueous medium in off–on mode, exploiting the “displacement approach”. Reason behind remarkable swing from Cys to GSH for switchover from 1 to 2 is well established by experimental as well as theoretical evidences. The solid complex (probe 1), reluctant to interact with bulky tri-peptide GSH owing to large steric congestion around Cu(II) centers, selectively detects small amino acid Cys by exploiting prompt decomplexation followed by Schiff-base ligand (L1) hydrolysis and subsequent fluorogenic 4-methyl-2,6-diformyl phenol (DFC) generation. On the contrary, for sterically less congested in situ complex (probe 2), the generation of the formyl moiety by means of partial hydrolysis of L1 during complexation, fails to sense Cys owing to formation of stable non-fluorescent cyclic thiazolidine derivative, but reacts with GSH to produce DFC with almost same selectivity and sensitivity similar to probe 1. Ratiometric responses at physiological detection level and bio-imaging studies to monitor Cys and GSH individually are analytically tuned for the first time in multi-cellular domain using the nematode, Caenorhabditis elegans.

Pyrene Schiff base: photophysics, aggregation induced emission, and antimicrobial properties.

Pyrene containing Schiff base molecule, namely 4-[(pyren-1-ylmethylene)amino]phenol (KB-1), was successfully synthesized and well characterized by using (1)H, (13)C NMR, FT-IR, and EI-MS spectrometry. UV-visible absorption, steady-state fluorescence, time-resolved fluorescence, and transient absorption spectroscopic techniques have been employed to elucidate the photophysical processes of KB-1. It has been demonstrated that the absorption characteristics of KB-1 have been bathochromatically tuned to the visible region by extending the π-conjugation. The extended π-conjugation is evidently confirmed by DFT calculations and reveals that π→π* transition is the major factor responsible for electronic absorption of KB-1. The photophysical property of KB-1 was carefully examined in different organic solvents at different concentrations and the results show that the fluorescence of this molecule is completely quenched due to photoinduced electron transfer. Intriguingly, the fluorescence intensity of KB-1 increases enormously by the gradual addition of water up to 90% with concomitant increase in fluorescence lifetime. This clearly signifies that this molecule has aggregation-induced emission (AIE) property. The mechanism of AIE of this molecule is suppression of photoinduced electron transfer (PET) due to hydrogen bonding interaction of imine donor with water. A direct evidence of PET process has been presented by using nanosecond transient absorption measurements. Further, KB-1 was successfully used for antimicrobial and bioimaging studies. The antimicrobial studies were carried out through disc diffusion method. KB-1 is used against both Gram-positive (Rhodococcus rhodochrous and Staphylococcus aureus) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacterial species and also fungal species (Candida albicans). The result shows KB-1 can act as an excellent antimicrobial agent and as a photolabeling agent. S. aureus, P. aeruginosa, and C. albicans were found to be the most susceptible microorganisms at 1 mM concentration among the bacteria used in the present investigation.

Colorimetric and fluorometric dual-modal probes for cyanide detection based on the doubly activated Michael acceptor and their bioimaging applications

In this study, we synthesized CTB and CB probes based on doubly activated Michael acceptors to selectively detect cyanide (CN−) anions through a one-step condensation reaction of coumarinyl acrylaldehyde with the corresponding derivatives of malonyl urea (thiourea). Through the conjugated addition of CN− to the β-site of the Michael acceptor, both probes displayed colorimetric and fluorometric dual-modal responses that were highly reactive and selective. CTB generates an active fluorescent response, whereas CB displays a ratiometric fluorescent response. The fluorescent signal of the probes reached its maximum given only 1 CN− equivalent and the signal change was linearly proportional to CN− concentrations ranging from 0 to 5μM with the detection limits 18 and 23nM, respectively. The reaction rate of the probes is highly dependent on the methylene acidity of malonyl urea derivatives. Thus, the response rate of CTB to CN− is 1.2-fold faster than that of CB, and the response rate of CB to CN− is 1.2-fold faster than that of the previously examined CM. We then verified the highly reactive nature of the β-site of the probes through density functional reactivity theory calculations. In addition, according to proof-of-concept experiments, these probes may be applied to analyze CN− contaminated water and biomimetic samples. Finally, cell cytotoxicity and bioimaging studies revealed that the probes were cell-permeable and could be used to detect CN− with low cytotoxicity.

A ratiometric two-photon fluorescent probe for cysteine and homocysteine in living cells

A ratiometric two-photon fluorescent probe (MQ) was designed and synthesized to detect cysteine (Cys) and homocysteine (Hcy) with high selectivity. MQ exhibited a large two-photon absorption cross-section at 710nm (265GM). Significant blue-shifts of both fluorescent and UV–vis spectra of MQ were found upon the addition of Cys or Hcy. MQ responded to Cys/Hcy much faster than previous reported two-photon fluorescent probes for Cys/Hcy. The response mechanism was proved by 1H NMR, Maldi-TOF MS and theoretical calculation. Cell cytotoxicity and bio-imaging studies revealed that MQ was cell-permeable and could be used to detect Cys with low cytotoxicity under two-photon excitation.

In vitro toxicity and bioimaging studies of gold nanorods formulations coated with biofunctional thiol-PEG molecules and Pluronic block copolymers.

In this work, we investigated the cytotoxicity, colloidal stability and optical property of gold nanorods before and after functionalizing them with thiolated PEG and Pluronic triblock copolymer (PEO–PPO–PEO) molecules. The morphology of functionalized gold nanorods was characterized by UV–visible absorption spectroscopy, transmission electron microscopy, and dynamic light scattering. Solution phase synthesis of gold nanorods has remained the method of choice for obtaining varying shapes and aspect ratios of rod nanoparticles. This method typically involves the use of cetyltrimethylammonium bromide (CTAB) surfactants as directing agents to grow gold nanorods in the solution phase. The as-synthesized gold nanorods surfaces are terminated with CTAB molecules and this formulation gives rise to adverse toxicity in vitro and in vivo. To employ the gold nanorods for biological studies, it is important to eliminate or minimize the exposure of CTAB molecules from the gold nanorods surface to the local environment such as cells or tissues. Complete removal of CTAB molecules from the gold nanorods surface is unfeasible as this will render the gold nanorods structurally unstable, causing the aggregation of particles. Here, we investigate the individual use of thiolated PEG and PEO–PPO–PEO as capping agents to reduce the cytotoxicity of gold nanorods formulation, while maintaining the optical, colloidal, and structural properties of gold nanorods. We found that encapsulating gold nanorods with the thiolated PEG or PEO–PPO–PEO molecules guarantees the stability and biocompatibility of the nanoformulation. However, excessive use of these molecules during the passivation process leads to a reduction in the overall cell viability. We also demonstrate the use of the functionalized gold nanorods as scattering probes for dark-field imaging of cancer cells thereby demonstrating their biocompatibility. Our results offer a unique solution for the future development of safe scattering color probes for clinical applications such as the long term imaging of cells and tissues.

FRET ratiometric probes reveal the chiral-sensitive cysteine-dependent H2S production and regulation in living cells

Hydrogen sulfide (H2S) is an endogenously produced gaseous signalling molecule with multiple biological functions. In order to visualize and quantify the endogenous in situ production of H2S in living cells, here we developed two new sulphide ratiometric probes (SR400 and SR550) based on fluorescence resonance energy transfer (FRET) strategy for live capture of H2S. The FRET-based probes show excellent selectivity toward H2S in a high thiol background under physiological buffer. The probe can be used to in situ visualize cysteine-dependent H2S production in a chiral-sensitive manner in living cells. The ratiometric imaging studies indicated that D-Cys induces more H2S production than that of L-Cys in mitochondria of human embryonic kidney 293 cells (HEK293). The cysteine mimics propargylglycine (PPG) has also been found to inhibit the cysteine-dependent endogenous H2S production in a chiral-sensitive manner in living cells. D-PPG inhibited D-Cys-dependent H2S production more efficiently than L-PPG, while, L-PPG inhibited L-Cys-dependent H2S production more efficiently than D-PPG. Our bioimaging studies support Kimura's discovery of H2S production from D-cysteine in mammalian cells and further highlight the potential of D-cysteine and its derivatives as an alternative strategy for classical H2S-releasing drugs.

Facile synthesis, cytotoxicity and bioimaging of Fe3+ selective fluorescent chemosensor

The designing and development of fluorescent chemosensors have recently been intensively explored for sensitive and specific detection of environmentally and biologically relevant metal ions in aqueous solution and living cells. Herein, we report the photophysical results of alanine substituted rhodamine B derivative 3 having specific binding affinity toward Fe3+ with micro molar concentration level. Through fluorescence titration at 599nm, we were confirmed that ligand 3 exhibited ratiometric fluorescence response with remarkable enhancement in emission intensity by complexation between 3 and Fe3+ while it appeared no emission in case of the competitive ions (Sc3+, Yb3+, In3+, Ce3+, Sm3+, Cr3+, Sn2+, Pb2+, Ni2+, Co2+, Cu2+, Ba2+, Ca2+, Mg2+, Ag+, Cs+, Cu+, K+) in aqueous/methanol (60:40, v/v) at neutral pH. However, the fluorescence as well as colorimetric response of ligand–iron complex solution was quenched by addition of KCN which snatches the Fe3+ from complex and turn off the sensor confirming the recognition process was reversible. Furthermore, bioimaging studies against L-929 cells (mouse fibroblast cells) and BHK-21 (hamster kidney fibroblast), through confocal fluorescence microscopic experiment indicated that ligand showed good permeability and minimum toxicity against the tested cell lines.

A water-soluble fluorescent probe for Fe(III): Improved selectivity over Cr(III)

A water-soluble rhodamine-based fluorescent probe (RD2) was designed and synthesized. Compared with the precursor RD1, RD2 shows excellent selectivity for Fe3+ over Cr3+ during the detection. The enhanced selectivity was achieved by incorporating another coordination moiety to the chelating moiety of the precursor RD1. Furthermore, bioimaging studies indicated that RD2 was cell permeable and suitable for the real-time imaging of Fe3+ in the living cells by confocal microscopy.

Phosphorescent proteins for bio-imaging and site selective bio-conjugation of peptides and proteins with luminescent cyclometalated iridium(iii) complexes

A new bio-conjugation reaction for site selective modification of proteins and peptides with phosphorescent iridium(III) complexes has been developed; the Ir(III)-modified proteins and peptides display long emission lifetimes and large Stoke shifts that can be used for bio-imaging studies.

A novel Zn2+ complex as the ratiometric two-photon fluorescent probe for biological Cd2+ detection

A novel Zn2+ complex as the ratiometric two-photon fluorescent probe (HQZn) for Cd2+ detection was synthesized. Fluorescence emission spectra of the probe showed a large red-shift (75 nm) and obvious enhancement of fluorescent intensity upon the addition of Cd2+. HQZn shows high selectivity for Cd2+ over other metal ions, and can eliminate the interference of Zn2+ during Cd2+ detection. Maldi-TOF MS spectra indicated that the response of HQZn to Cd2+ was caused by central metal displacement. Cell cytotoxicity and bio-imaging studies revealed that HQZn was cell-permeable and it could be used to detect intracellular Cd2+ with low cytotoxicity under two-photon excitation.

Bovine Serum Albumin Nanoparticles with Fluorogenic Near-IR-Emitting Squaraine Dyes

Two squaraine (SQ) dyes, N-propanesulfonate-benzothiazolium squaraine (SQ-1) and N-propanesulfonate-benzoindolium squaraine (SQ-2), were synthesized with sulfonate groups to increase water solubility. Both dyes are almost nonfluorescent in aqueous solution with fluorescent quantum yields of 0.03, but exhibited fluorescence enhancement after noncovalently binding with bovine serum albumin (BSA). Upon addition of BSA, the fluorescence intensity increased by ca. a factor of 10, along with a 10-fold extension in the fluorescence lifetime. SQ-1 and SQ-2 interacted with BSA efficiently and appeared to show a preference for binding at site II, which involves combinational effects of electrostatic and hydrophobic interactions. The fluorogenic squaraine dyes were then used to label BSA, forming BSA-based nanoparticles (NPs) through noncovalent binding. The resulting BSA-SQ NPs exhibited enhanced near-IR fluorescence and reduced aggregation of the squaraine moiety. The BSA-SQ NPs were used for cell incubation and bioimaging studies. Confocal fluorescent images were obtained for HCT 116 cells incubated with the BSA-SQ NPs and LysoSensor Green, demonstrating the utility of the NP probes for intracellular imaging. This strategy ovecomes the generally low fluorescence emission of SQ dyes in water and aggregation-reduced fluorescence, providing a versatile strategy for sensing and imaging in biological environments.

Near-Infrared-Emitting Nanoparticles for Lifetime-Based Multiplexed Analysis and Imaging of Living Cells

The increase in information content from bioassays and bioimaging requires robust and efficient strategies for the detection of multiple analytes or targets in a single measurement, thereby addressing current health and security concerns. For fluorescence techniques, an attractive alternative to commonly performed spectral or color multiplexing presents lifetime multiplexing and the discrimination between different fluorophores based on their fluorescence decay kinetics. This strategy relies on fluorescent labels with sufficiently different lifetimes that are excitable at the same wavelength and detectable within the same spectral window. Here, we report on lifetime multiplexing and discrimination with a set of nanometer-sized particles loaded with near-infrared emissive organic fluorophores chosen to display very similar absorption and emission spectra, yet different fluorescence decay kinetics in suspension. Furthermore, as a first proof-of-concept, we describe bioimaging studies with 3T3 fibroblasts and J774 macrophages, incubated with mixtures of these reporters employing fluorescence lifetime imaging microscopy. These proof-of-concept measurements underline the potential of fluorescent nanoparticle reporters in fluorescence lifetime multiplexing, barcoding, and imaging for cellular studies, cell-based assays, and molecular imaging.

A rhodamine-based fluorescent probe for detecting Hg2+ in a fully aqueous environment

A water-soluble fluorescent probe for Hg(2+) based on a rhodamine B derivative was designed and synthesized. The new probe showed reversible colorimetric and fluorescent response to Hg(2+) in a fully aqueous solution. The probe exhibited real-time detection of Hg(2+) with high selectivity in media containing less than 1% organic cosolvent. Furthermore, bioimaging studies indicated that the new probe was cell permeable and suitable for the real-time imaging of Hg(2+) in living cells by confocal microscopy.

Meta – Material Brain Biology for Information Gain

Optical properties of brain materials can be investigated based on concepts derived from physical models for signal communication, while relevantly relating them to bio systems applications. Bio system responses to ultra weak photo optic signals can prompt environment assisted behavior; and coherent cellular communication. Evolution of bio structures and materials occur as adaptations, in response to cues from the current environment, so as to enable efficient energy and information transfer functions. This is significant in an environment of ultra-weak photo emissions (UPE) and its optic signals in brain, which is reportedly related to bio energetic processes of energy metabolism; intrinsic optic signals have been associated with oscillatory functional rhythms involving calcium activity, and cellular communication. Such non–molecular signaling could couple to molecular recognition for biocontrol, and observed to have a role in bioprocess monitoring and bio imaging studies. In the case of UPE, the signals are of an ultra-weak nature and its long range propagation is hampered by bio-system absorption and scattering, despite theoretical possibilities for transmission in the mitochondrial reticular network, probably based on more simplistic assumptions. However, this possibility is limited, under physiological conditions, when bio system dissipation losses are taken into account. Some prevalent models tend to suggest limited propagation of photo-optic signals of a weak nature. It is postulated here that evolution of meta materials in brain biology in response to environment cues from UPE, would help to realize optic NIMs [Negative Refraction Index Materials]with tunability and information gain. The paper investigates the theoretical possibility for this, while relating to brain biology. The application potential of physical models for NIR and optic signaling communication, in a bio system context, is taken into account. Optical properties of brain materials are discussed in the light of concepts related to non-linear effects like negative refraction and coherent resonances and their potential application in brain biology.Such effects and behavior in brain might help to reduce biosystem scatter and dissipative losses to facilitate signal communication with long-range propagation, using ultra-weak photo optic signals.

Quantum Dots: A Quantum Dot Photoswitch for DNA Detection, Gene Transfection, and Live‐Cell Imaging (Small 22/2012)

The preparation of a protein-derived biopolymer coating for encapsulating quantum dots (QDs) by multivalent interactions is described on page 3465 by T. Weil and co-workers. Coated QDs undergo fast endocytosis, and they are highly stable and noncytotoxic in live cells. The formation of complexes containing QDs and plasmid DNA leads to proportionally decreased photoluminescence of the QDs. Efficient gene transfection is achieved, which is attractive for live-cell bioimaging and mechanistic studies of non-viral gene delivery.

A Quantum Dot Photoswitch for DNA Detection, Gene Transfection, and Live‐Cell Imaging

Quantum dots (QDs) coated with an albumin-derived copolymer shell exhibit significant photoresponsiveness to DNA loading and have great potential for investigating gene delivery processes. The QDs reported herein are positively charged, have attractive optical properties, and are noncytotoxic and notably stable in live cells. Their complex formation with plasmid DNA leads to proportionally decreased photoluminescence and efficient gene transfection is observed. Therefore, they are suitable for live-cell bioimaging and mechanistic studies of nonviral gene delivery. Fluorescence correlation spectroscopy is applied for the first time to investigate individual QDs diffusing in large endosomes inside living cells, and serves as a valuable tool to study the physical properties of QDs inside live cells. The data obtained in this study strongly support the notable stability of these QDs, even in cell endosomes.

Bio-imaging Studies on Host Pathogen Interactions

Extended abstract of a paper presented at Microscopy and Microanalysis 2012 in Phoenix, Arizona, USA, July 29 – August 2, 2012.