Probing Viscosity of Co-Polymer Hydrogel and HeLa Cell Using Fluorescent Gold Nanoclusters: Fluorescence Correlation Spectroscopy and Anisotropy Decay.

Fluorescence dynamics of gold nanoclusters (Au9 and Au25 ) are studied in the complex and crowded environment of a triblock co-polymer (F127) hydrogel and inside cervical cancer cell, HeLa. In the hydrogel, spherical micelles of F127 remain immobilized with a hydrophobic core (PPO) and a hydrophilic corona (PEO) region. The fluorescence anisotropy decay suggests that the timescale of rotational relaxation in the hydrogel is similar to that in bulk water (viscosity ∼1 cP). From fluorescence correlation spectroscopy (FCS) it is inferred that the local viscosity in the hydrogel is 12 cP for Au9 and 18 cP for Au23 . These results indicate that gold nanoclusters (AuNCs) localize in the corona region of the hydrogel. Evidently, frictions against rotation and translation are different inside the gel. It is suggested that rotation of the AuNCs senses the immediate water-like "void" region while translation motion involves in-and-out movement of the AuNCs at the periphery of the gel. Finally, the gold nanoclusters are used for cell imaging and estimation of intracellular viscosity of HeLa cells.

Time-dependent enhancement of fluorescence from Rhodobacter capsulatus SB1003 and its critical dependence on concentration temperature and static magnetic field.

Continuous exposure of 395 nm light increases the fluorescence emission intensity of photosynthetic purple non-sulphur bacteria, Rhodobacter capsulatus (SB1003). We show that such an increase in fluorescence emission of extracellular pigment complexes (PC) from these photosynthetic bacteria depends on the concentration of the pigment and temperature and can also be modulated by the static magnetic field. The time-dependent enhanced emission disappears either at or below 300 K or below a threshold sample concentration (0.1 mg/ml). The enhanced emission reappears at this condition (T < 278 K) if a static magnetic field (395 mT) is introduced during fluorescence measurement. The time dependence of emission is expressed in terms of a first-order rate constant, k = dF/(Fdt). The sign of k shifts from positive to negative as PC concentration is lowered than a threshold value, implying onset of fluorescence decay (k < 0) rather than amplification (k > 0). At PC concentration higher than a threshold, k becomes negative if the temperature is lowered. But, surprisingly, at low temperature, a static magnetic field reverts the k value to positive. We explain the logical nature of k-switching and photo-dynamics of the aforesaid microbial fluorescence emission by aggregation of protoporphyrin rings present in the PC. While the simultaneous presence of decay in fluorescence and susceptibility to static magnetic field suggests the dominance of triplet states at low temperatures, the process is reversed by SMF-induced removal of spin degeneracy. At higher temperatures, the optical excitability and lack of magnetic response suggest the dominance of singlet states. We propose that the restructuring of the singlet-triplet distribution by intersystem crossing may be the basis of this logical behaviour. In context with microbial function, time-dependent enhancement of fluorescence also implies relay of red photons to the neighbouring microbes not directly exposed to the incident radiation, thus serving as an indirect photosynthetic regulator.

Time Evolution of Local pH Around a Photo-Acid in Water and a Polymer Hydrogel: Time Resolved Fluorescence Spectroscopy of Pyranine.

In this work, we propose a new analysis of the time resolved emission spectra of a photo-acid, HA, pyranine (8-hydroxypyrene-1,3,6-trisulphonic acid, HPTS) based on time resolved area normalized emission spectra (TRANES). Presence of an isoemissive point in TRANES confirms the presence of two emissive species (HA and A- ) inside the system in bulk water and inside a co-polymer hydrogel [F127, (PEO)100 -(PPO)70 -(PEO)100 ]. We show that following electronic excitation, the local pH around HPTS, is much lower than the bulk pH presumably because of ejection of proton from the photo-acid in the excited state. With increase in time, the local pH increases and reaches the bulk value. We further, demonstrate that the excited state pKa of HPTS may be estimated from the emission intensities of HA and A- at long time. The time constant for time evolution of pH is ∼630 ps in water, ∼1300 ps in F127 gel and ∼4700 ps in CTAB micelle. The location and local viscosity sensed by the probe is ascertained using fluorescence correlation spectroscopy (FCS) and fluorescence anisotropy decay. The different values of the local viscosity reported by these two methods are reconciled.

Local environment of organic dyes in an ionic liquid-water mixture: FCS and MD simulation.

The composition dependent local environment of three organic dyes in binary mixtures of a room temperature ionic liquid (1-methyl-3-pentylimidazolium bromide, [pmim][Br]) and water is studied by fluorescence correlation spectroscopy (FCS) and molecular dynamics (MD) simulations. We used three dyes-neutral coumarin 480 (C480), anionic coumarin 343 (C343), and highly hydrophobic 4-(dicyanomethylene)-2-methyl-6-(p-dimethyl-aminostyryl)-4H-pyran (DCM)-to probe different environments in the binary mixtures. The heterogeneity of the [pmim][Br]-water mixture leads to multiple values (i.e., distribution) of diffusion coefficients (Dt). In these binary mixtures, the effective viscosity (ηeff, obtained from FCS) and the local concentration of the [pmim][Br] around the three dyes (revealed by MD simulations) are found to be quite different than that in bulk. The viscosity experienced by the C480 and C343 dyes is almost twice as large as that experienced by DCM dye. Through rigorous MD simulation, we show that in the vicinity of the less hydrophobic coumarin dyes (C480 and C343) compared to DCM dye, the local concentration of the [pmim][Br] is ∼3-7 times larger than that in bulk. In the case of the most hydrophobic dye, DCM, the local concentration of [pmim][Br] is almost similar to bulk-like. Further analysis reveals the formation of hydrogen bond between the imidazolium ring of [pmim][Br] and the carbonyl oxygen atom of the coumarin dyes (C-H[pmim][Br]⋯O=CDye). Finally, computer simulation indicates a slow component of solvation dynamics in the [pmim][Br]-water mixture in the time scale of ∼100-200 ps, which is similar to the experimental observation.

Physical chemistry in a single live cell: confocal microscopy.

A live cell is a complex, yet extremely important container. Understanding the dynamics in a selected intracellular component is a challenging task. We have recently made significant progress in this direction using a confocal microscope as a tool. The smallest size of the focused spot in a confocal microscope is ∼0.2 µm (200 nm). This is nearly one hundred times smaller than the size of a live cell. Thus, one can selectively study different intracellular components/organelles in a live cell. In this paper, we discuss how one can image different intracellular components/organelles, record fluorescence spectra and decay at different locations, ascertain local polarity and viscosity, and monitor the dynamics of solvation, proton transfer, red-ox and other phenomena at specified locations/organelles inside a cell. We will highlight how this knowledge enriched us in differentiating between cancer and non-cancer cells, 3D tumor spheroids and towards drug delivery.

Probing the conformational dynamics of photosystem I in unconfined and confined spaces.

The fluorescence dynamics of Photosystem I (PSI) in bulk water and inside a confined environment like a liposome have been investigated using time resolved confocal microscopy. In bulk water, PSI exhibits a major emission peak at ∼680 nm, while in the liposome it exhibits a markedly blue shifted emission maximum at ∼485 nm. This is indicative of conformational changes due to entrapment and emergence of a stressed conformation of PSI inside the liposome. The observed time constants for the fluorescence lifetime of PSI inside the liposome are significantly high as opposed to PSI in bulk water. More interestingly, the fluorescence intensity of PSI in bulk water exhibits strong fluctuations with many high intensity jumps and these are anti-correlated with the fluorescence lifetime of PSI. In contrast, inside the liposome, no such anti-correlated behaviour is observed. We further demonstrated that PSI exhibits at least two conformational states in bulk water, whereas a single conformation is observed inside the liposome, indicating the conformational rigidity and locking of the PSI complex inside a liposome.

Split-ubiquitin yeast two-hybrid interaction reveals a novel interaction between a natural resistance associated macrophage protein and a membrane bound thioredoxin in Brassica juncea.

Natural resistance associated macrophage proteins (NRAMPs) are evolutionarily conserved metal transporters involved in the transport of essential and nonessential metals in plants. Fifty protein interactors of a Brassica juncea NRAMP protein was identified by a Split-Ubiquitin Yeast-Two-Hybrid screen. The interactors were predicted to function as components of stress response, signaling, development, RNA binding and processing. BjNRAMP4.1 interactors were particularly enriched in proteins taking part in photosynthetic or light regulated processes, or proteins predicted to be localized in plastid/chloroplast. Further, many interactors also had a suggested role in cellular redox regulation. Among these, the interaction of a photosynthesis-related thioredoxin, homologous to Arabidopsis HCF164 (High-chlorophyll fluorescence164) was studied in detail. Homology modeling of BjNRAMP4.1 suggested that it could be redox regulated by BjHCF164. In yeast, the interaction between the two proteins was found to increase in response to metal deficiency; Mn excess and exogenous thiol. Excess Mn also increased the interaction in planta and led to greater accumulation of the complex at the root apoplast. Network analysis of Arabidopsis homologs of BjNRAMP4.1 interactors showed enrichment of many protein components, central to chloroplastic/cellular ROS signaling. BjNRAMP4.1 interacted with BjHCF164 at the root membrane and also in the chloroplast in accordance with its proposed function related to photosynthesis, indicating that this interaction occurred at different sub-cellular locations depending on the tissue. This may serve as a link between metal homeostasis and chloroplastic/cellular ROS through protein-protein interaction.

Cytochrome†c-Capped Fluorescent Gold Nanoclusters: Imaging of Live Cells and Delivery of Cytochrome†c.

Cytochrome c-capped fluorescent gold nanoclusters (Au-NCs) are used for imaging of live lung and breast cells. Delivery of cytochrome c inside the cells is confirmed by covalently attaching a fluorophore (Alexa Fluor 594) to cytochrome c-capped Au-NCs and observing fluorescence from Alexa 594 inside the cell. Mass spectrometry studies suggest that in bulk water, addition of glutathione (GSH) to cytochrome c-capped Au-NCs results in the formation of glutathione-capped Au-NCs and free apo-cytochrome c. Thus glutathione displaces cytochrome c as a capping agent. Using confocal microscopy, the emission spectra and decay of Au-NCs are measured in live cells. From the position of the emission maximum it is shown that the Au-NCs exist as Au8 in bulk water and as Au13 inside the cells. Fluorescence resonance energy transfer from cytochrome c-Au-NC (donor) to Mitotracker Orange (acceptor) indicates that the Au-NCs localise in the mitochondria of live cells.

Fluorescence Dynamics in the Endoplasmic Reticulum of a Live Cell: Time-Resolved Confocal Microscopy.

Fluorescence dynamics in the endoplasmic reticulum (ER) of a live non-cancer lung cell (WI38) and a lung cancer cell (A549) are studied by using time-resolved confocal microscopy. To selectively study the organelle, ER, we have used an ER-Tracker dye. From the emission maximum (λmaxem) of the ER-Tracker dye, polarity (i.e. dielectric constant, ϵ) in the ER region of the cells (≈500 nm in WI38 and ≈510 nm in A549) is estimated to be similar to that of chloroform (λmaxem =506 nm, ϵ≈5). The red shift by 10 nm in λmaxem in the cancer cell (A549) suggests a slightly higher polarity compared to the non-cancer cell (WI38). The fluorescence intensity of the ER-Tracker dye exhibits prolonged intermittent oscillations on a timescale of 2-6 seconds for the cancer cell (A549). For the non-cancer cell (WI38), such fluorescence oscillations are much less prominent. The marked fluorescence intensity oscillations in the cancer cell are attributed to enhanced calcium oscillations. The average solvent relaxation time (<τs >) of the ER region in the lung cancer cell (A549, 250±50 ps) is about four times faster than that in the non-cancer cell (WI38, 1000±50 ps).

Cancer Cell Imaging Using in Situ Generated Gold Nanoclusters.

In situ generated fluorescent gold nanoclusters (Au-NCs) are used for bio-imaging of three human cancer cells, namely, lung (A549), breast (MCF7), and colon (HCT116), by confocal microscopy. The amount of Au-NCs in non-cancer cells (WI38 and MCF10A) is 20-40 times less than those in the corresponding cancer cells. The presence of a larger amount of glutathione (GSH) capped Au-NCs in the cancer cell is ascribed to a higher glutathione level in cancer cells. The Au-NCs exhibit fluorescence maxima at 490-530 nm inside the cancer cells. The fluorescence maxima and matrix-assisted laser desorption ionization (MALDI) mass spectrometry suggest that the fluorescent Au-NCs consist of GSH capped clusters with a core structure (Au8-13). Time-resolved confocal microscopy indicates a nanosecond (1-3 ns) lifetime of the Au-NCs inside the cells. This rules out the formation of aggregated Au-thiolate complexes, which typically exhibit microsecond (≈1000 ns) lifetimes. Fluorescence correlation spectroscopy (FCS) in live cells indicates that the size of the Au-NCs is ≈1-2 nm. For in situ generation, we used a conjugate consisting of a room-temperature ionic liquid (RTIL, [pmim][Br]) and HAuCl4. Cytotoxicity studies indicate that the conjugate, [pmim][AuCl4], is non-toxic for both cancer and non-cancer cells.

Selective Killing of Breast Cancer Cells by Doxorubicin-Loaded Fluorescent Gold Nanoclusters: Confocal Microscopy and FRET.

Fluorescent gold nanoclusters (AuNCs) capped with lysozymes are used to deliver the anticancer drug doxorubicin to cancer and noncancer cells. Doxorubicin-loaded AuNCs cause the highly selective and efficient killing (90 %) of breast cancer cells (MCF7) (IC50 =155 nm). In contrast, the killing of the noncancer breast cells (MCF10A) by doxorubicin-loaded AuNCs is only 40 % (IC50 =4500 nm). By using a confocal microscope, the fluorescence spectrum and decay of the AuNCs were recorded inside the cell. The fluorescence maxima (at ≈490-515 nm) and lifetime (≈2 ns), of the AuNCs inside the cells correspond to Au10-13 . The intracellular release of doxorubicin from AuNCs is monitored by Förster resonance energy transfer (FRET) imaging.

Single-molecule Spectroscopy: Exploring Heterogeneity in Chemical and Biological Systems.

Many chemical and biological systems are heterogeneous in the molecular length scale (∼ 1 nm). Heterogeneity in many chemical systems and organized assemblies may be monitored using single-molecule spectroscopy (SMS). In SMS, the size of the focal spot (i.e., the smallest region to be probed) is nearly half of the excitation wavelength (λ/2, i.e., 200-375 nm) for visible light (400-750 nm). We discuss how one can get spatial resolutions better than 200 nm using molecules as nanometric probes. We show that polymer hydrogels, lipid vesicles, room temperature ionic liquids (RTILs), and binary liquid mixtures exhibit such heterogeneity. Another important observation is solute-dependent friction in RTILs. In an RTIL, diffusion of an ionic solute is slower than that of a neutral solute.

Spectral mapping of 3D multi-cellular tumor spheroids: time-resolved confocal microscopy.

A tumor-like multi-cellular spheroid (3D) differs from a 2D cell in a number of ways. This is demonstrated using time resolved confocal microscopy. Two different tumor spheroids - HeLa (cervical cancer) and A549 (lung cancer) - are studied using 3 different fluorescent dyes - C153 (non-covalent), CPM (covalent) and doxorubicin (non-covalent, anti-cancer drug). The pattern of localization of these three fluorescent probes in the 3D tumor cell exhibits significant differences from that in the conventional 2D cells. For both the cells (HeLa and A549), the total uptake of doxorubicin in the 3D cell is much lower than that in the 2D cell. The uptake of doxorubicin molecules in the A549 spheroid is significantly different compared to the HeLa spheroid. The local polarity (i.e. emission maxima) and solvation dynamics in the 3D tumor cell differ from those in 2D cells. The covalent probe CPM exhibits intermittent fluorescence oscillations in the 1-2 s time scale. This is attributed to redox processes. These results may provide new insights into 3D tumors.

Amyloid beta peptides inside a reconstituted cell-like liposomal system: aggregation, FRET, fluorescence oscillations and solvation dynamics.

Aggregations of amyloid-beta (Aß) peptides were studied inside a reconstituted cell like liposomal system using time-resolved confocal microscopy. Fluorescence correlation spectroscopy (FCS) and confocal images indicate that Aß forms a very large aggregate in bulk and more efficiently, in the bilayer region of the liposome, respectively. The aggregates formed inside the liposome gradually migrate out to bulk water. FRET, from HiLyte Fluor 488 (covalently attached to an Aß peptide) to TRITC (tetramethylrhodamine isothiocyanate) covalently attached to a DHPE lipid present in the bilayer, reveals intermittent oscillations in the time scale of ∼0.5 s. This is attributed to the structural fluctuations of the membrane of the liposome. The solvation dynamics of Aß in monomer and in oligomeric state is studied by monitoring the emission of HiLyte Fluor 488. The solvation dynamics of the Aß monomer is similar to that of oligomeric aggregates in the liposome.

Structural relaxation of acridine orange dimer in bulk water and inside a single live lung cell.

Structural relaxation of the acridine orange (AO) dimer in bulk water and inside a single live lung cell is studied using time resolved confocal microscopy and molecular dynamics (MD) simulations. The emission maxima (λem (max)∼ 630 nm) of AO in a lung cancer cell (A549) and a non-cancer lung fibroblast cell (WI38) suggest that AO exists as a dimer inside the cell. Time-dependent red shift in emission maximum indicates dynamic relaxation of the AO dimer (in the excited state) with a time constant of 500-600 ps, both in bulk water and inside the cell. We have calculated the equilibrium relaxation dynamics of the AO dimer in the ground state using MD simulations and found a slow component of time scale ∼ 350 ps. The intra- and inter-molecular components of the total relaxation dynamics of the AO dimer reveal the presence of a slow component of the order of a few hundred picoseconds. Upon restricting intra-molecular dye dynamics by harmonic constraint between AO monomers, the slow component vanishes. Combining the experimental observations and MD simulation results, we ascribe the slow component of the dynamic relaxation of the AO dimer to the structural relaxation, namely, fluctuations in the distance between the two monomers and associated fluctuation in the number of water molecules.

Effect of alcohol on the structure of cytochrome C: FCS and molecular dynamics simulations.

Effect of ethanol on the size and structure of a protein cytochrome C (Cyt C) is investigated using fluorescence correlation spectroscopy (FCS) and molecular dynamics (MD) simulations. For FCS studies, Cyt C is covalently labeled with a fluorescent probe, alexa 488. FCS studies indicate that on addition of ethanol, the size of the protein varies non-monotonically. The size of Cyt C increases (i.e., the protein unfolds) on addition of alcohol (ethanol) up to a mole fraction of 0.2 (44.75% v/v) and decreases at higher alcohol concentration. In order to provide a molecular origin of this structural transition, we explore the conformational free energy landscape of Cyt C as a function of radius of gyration (Rg) at different compositions of water-ethanol binary mixture using MD simulations. Cyt C exhibits a minimum at Rg ∼ 13 Å in bulk water (0% alcohol). Upon increasing ethanol concentration, a second minimum appears in the free energy surface with gradually larger Rg up to χEtOH ∼ 0.2 (44.75% v/v). This suggests gradual unfolding of the protein. At a higher concentration of alcohol (χEtOH > 0.2), the minimum at large Rg vanishes, indicating compaction. Analysis of the contact map and the solvent organization around protein indicates a preferential solvation of the hydrophobic residues by ethanol up to χEtOH = 0.2 (44.75% v/v) and this causes the gradual unfolding of the protein. At high concentration (χEtOH = 0.3 (58% v/v)), due to structural organization in bulk water-ethanol binary mixture, the extent of preferential solvation by ethanol decreases. This causes a structural transition of Cyt C towards a more compact state.

Unfolding and refolding of a protein by cholesterol and cyclodextrin: a single molecule study.

Unfolding/refolding of a plasma protein, human serum albumin (HSA), is studied using fluorescence correlation spectroscopy (FCS) and single molecule fluorescence resonance energy transfer (sm-FRET). Addition of cholesterol causes unfolding of HSA resulting in an increase in the hydrodynamic diameter (dH = 2rH) from 76 Å in the native state to 120 Å upon addition of 1 mM cholesterol. Addition of ß-cyclodextrin to HSA (unfolded by cholesterol) restores the hydrodynamic diameter back to 78 Å. The cholesterol induced unfolding and ß-cyclodextrin induced refolding are also monitored by measuring the distance between a FRET donor (CPM dye, D) and a FRET acceptor (Alexa 488, A) covalently attached to the protein (HSA). It is observed that the average D-A distance increases from 45 ± 1 Å at 0 mM cholesterol to 51 ± 1 Å at 1 mM cholesterol. Upon addition of ß-cyclodextrin, the D-A distance is restored to 45 ± 1 Å. The binding study indicates that nearly 94% of HSA molecules remain bound to cholesterol in the absence of ß-cyclodextrin and only 5% binds to cholesterol in the presence of ß-cyclodextrin. As much as 57% of the HSA and 99% of the cholesterol molecules bind to ß-cyclodextrin. Thus ß-cyclodextrin removes cholesterol from HSA by hydrophobic binding to cholesterol ("strip off") and also, itself binds to HSA. The conformational dynamics results suggest that addition of ß-cyclodextrin restores native like binding free energy and folding dynamics.

Fluorescence fluctuation of an antigen-antibody complex: circular dichroism, FCS and smFRET of enhanced GFP and its antibody.

The structure and dynamics of an antigen-antibody complex are monitored by circular dichroism (CD) spectroscopy, fluorescence correlation spectroscopy (FCS) and single molecule FRET (smFRET). In this work, the antigen is enhanced GFP (EGFP) and the antibody is anti-EGFP VHH-His6. From FCS measurements, the hydrodynamic radius (rH) of EGFP and its antibody (VHH-His6) is found to be 24 ± 2 Å and 18 ± 2 Å, respectively. For the antigen-antibody complex (EGFP:anti-EGFP VHH-His6), rH is 41 ± 3 Å. CD spectra indicate that the addition of guanidium hydrochloride (GdnHCl) causes unfolding of the antigen, its antibody and their complex, and a consequent increase in size is observed from FCS data. smFRET between EGFP (donor, D) and Alexa 594 (acceptor, A) bound to anti-EGFP VHH-His6 reveals a time dependent fluctuation in donor-acceptor distances. This suggests that the structure of the antigen-antibody complex is dynamic in nature and is not rigid.

Direct observation of the growth and shrinkage of microtubules by single molecule Förster resonance energy transfer.

Single molecule Förster resonance energy transfer (FRET) has been applied, for the first time, to monitor the growth (polymerization) and the shrinkage (depolymerization) of the dynamic microtubules, employing EGFP (attached to Mal3) as a donor and alexa-568 bound to tubulin as an acceptor.

Ionic liquid induced dehydration and domain closure in lysozyme: FCS and MD simulation.

Effect of a room temperature ionic liquid (RTIL, [pmim][Br]) on the structure and dynamics of the protein, lysozyme, is investigated by fluorescence correlation spectroscopy (FCS) and molecular dynamic (MD) simulation. The FCS data indicate that addition of the RTIL ([pmim][Br]) leads to reduction in size and faster conformational dynamics of the protein. The hydrodynamic radius (rH) of lysozyme decreases from 18 Å in 0 M [pmim][Br] to 11 Å in 1.5 M [pmim][Br] while the conformational relaxation time decreases from 65 µs to 5 µs. Molecular origin of the collapse (size reduction) of lysozyme in aqueous RTIL is analyzed by MD simulation. The radial distribution function of water, RTIL cation, and RTIL anion from protein clearly indicates that addition of RTIL causes replacement of interfacial water by RTIL cation ([pmim](+)) from the first solvation layer of the protein providing a comparatively dehydrated environment. This preferential solvation of the protein by the RTIL cation extends up to ∼30 Å from the protein surface giving rise to a nanoscopic cage of overall radius 42 Å. In the nanoscopic cage of the RTIL (42 Å), volume fraction of the protein (radius 12 Å) is only about 2%. RTIL anion does not show any preferential solvation near protein surface. Comparison of effective radius obtained from simulation and from FCS data suggests that the "dry" protein (radius 12 Å) alone diffuses in a nanoscopic cage of RTIL (radius 42 Å). MD simulation further reveals a decrease in distance ("domain closure") between the two domains (alpha and beta) of the protein leading to a more compact structure compared to that in the native state.