Transient Raman Snapshots of the Twisted Intramolecular Charge Transfer State in a Stilbazolium Dye.

Optically triggered twisted intramolecular charge transfer (TICT) states in donor-acceptor chromophores form the molecular basis for designing bioimaging probes that sense polarity, microviscosity, and pH in vivo. However, a lack of predictive understanding of the "twist" localization precludes a rational design of TICT-based dyes. Here, using femtosecond stimulated Raman spectroscopy, we reveal a distinct Raman signature of the TICT state for a stilbazolium-class mitochondrial staining dye. Resonance-selective probing of 4-N,N-diethylamino-4″-N'-methyl-stilbazolium tosylate (DEST) tracks the excited-state structure of the dye as it relaxes to a TICT state on a picosecond time scale. The appearance of a remarkably blue-shifted C=C stretching mode at 1650 cm-1 in the TICT state is attributed to the "twist" of a single bond adjacent to the ethylenic π-bridge in the DEST backbone based on detailed electronic structure calculations and vibrational mode analysis. Our work demonstrates that the π-bridge, connecting the donor and acceptor moieties, influences the spatial location of the "twist" and offers a new perspective for designing organelle-specific probes through cogent tuning of backbone dynamics.

Super continuum generation in water doped with Homeopathic medicines

We have carried out systematic studies to investigate the effect on supercontinuumgeneration in water using 40 fs laser pulses when doped with Homeopathic medicines. We perform these studies using five series of medications with different levels of dilution (10-30 to 10-100000). We measure supercontinuum spectra that span from 400-1050nm. We monitor the area under the curve in the range 450-750 nm for each sample at a fixed incident laser energy. Our observations indicate that the yield of supercontinuum generation, in water containing Homeopathic medicine is significantly different from that obtained in water containing plain ethanol. The measurement for different dilutions shows up to 7 times standard deviation variation in the yield of supercontinuum generationEven though linear absorption in the UV-visible region does not show any significant difference for different Homeopathic medicines, the supercontinuum yield which depends on the effective nonlinear refractive index changes with different samples. (AU)

Effect of nucleants in photothermally assisted crystallization.

Laser-induced crystallization is emerging as a promising technique to crystallize biomolecules like amino acids and proteins. The use of external materials as nucleants and novel seeding methods open new paths for protein crystallization. We report here the results of experiments that explore the effect of nucleants on laser-based crystallization of microlitre droplets of small molecules, amino acids, and proteins. The role of parameters like solute concentration, droplet volume, type and size of the nucleant, and laser power, are systematically investigated. In addition to crystallization of standard molecules like NaCl, KCl, and glycine, we demonstrate the crystallization of negatively (l-histidine), and positively (l-aspartic acid) charged amino acids and lysozyme protein. Single crystal X-ray diffraction and Raman spectroscopy studies unequivocally indicate that the nucleants do not alter the molecular structure of glycine, hydrogen bonding patterns, and packing. Localized vaporization of the solvent near the nucleant due to photothermal heating has enabled us to achieve rapid crystallization - within 3 s - at laser intensities of 0.1 MW cm-2, significantly lower than those reported earlier, with both saturated and unsaturated solutions. The outcome of the current experiments may be of utility in tackling various crystallization problems during the formation of crystals large enough to perform X-ray crystallography.

Varying coordination modes of amide ligand in group 12 Hg(II) and Cd(II) complexes: synthesis, crystal structure and nonlinear optical properties.

Reactions of the amide ligand, H2L (H2L = N,N'-bis[2-(2-pyridyl)methyl]pyridine-2,6-dicarboxamide) with CdCl2 and Hg(CH3COO)2, in 1 : 1 ratio, at 298 K yield dimeric [Hg(L)]2 (1) and trimeric [Cd3(H2L)4Cl6] (2), respectively. In 1, the H2L is coordinated to Hg(II) via six N-atoms of central and terminal pyridines as well as of deprotonated amido groups, whereas the carbonyl groups remain free. However, in 2, the H2L is coordinated to Cd(II) through terminal pyridine N atoms and O atoms from carbonyl groups, whereas the nitrogen atoms of the central pyridine, two terminal pyridine and of all amido groups remain free. Molecular structures of 1 and 2 are confirmed by single crystal X-ray studies. The varying coordination modes of H2L give rise to different electrochemical behavior of 1 and 2, which has also been rationalized by theoretical calculations. Moreover, nonlinear optical (NLO) behavior of both complexes has been investigated using ultra-short femtosecond laser pulses, which ensures that the NLO response is exclusively from their electronic component.

A search for the sulphur hexafluoride cation with intense, few cycle laser pulses.

It is well established that upon ionization of sulphur hexafluoride, the SF6(+) ion is never observed in mass spectra. Recent work with ultrashort intense laser pulses has offered indications that when strong optical field are used, the resulting "bond hardening" can induce changes in the potential energy surfaces of molecular cations such that molecular ions that are normally unstable may, indeed, become metastable enough to enable their detection by mass spectrometry. Do intense, ultrashort laser pulses permit formation of SF6(+)? We have utilized intense pulses of 5 fs, 11 fs, and 22 fs to explore this possibility. Our results are negative: no evidence is discovered for SF6(+). However, multiply charged sulphur and fluorine ions from highly charged SF6(q+) ions are observed that enable us to resolve the controversy regarding the kinetic energy release accompanying formation of F(+) fragment ions. Quantum chemical computations of field-distorted potential energy curves of SF6 and its molecular ion enable us to rationalize our non-observation of SF6(+). Our findings have implications for high harmonic generation from SF6 in the few-cycle regime.

On the birefringence of healthy and malaria-infected red blood cells.

The birefringence of a red blood cell (RBC) is quantitatively monitored as it becomes infected by a malarial parasite. Large changes occur in the cell's refractive index at different stages of malarial infection. The observed rotation of an optically trapped, malaria-infected RBC is not a simple function of shape distortion: the malarial parasite is found to itself exercise a profound influence on the rotational dynamics by inducing stage-specific birefringence. Our measurements shed new light on the competition between shape- and form-birefringence in RBCs. We demonstrate the possibility of using birefringence to establish very early stages of infected parasites and of assessing various factors that contribute to birefringence in normal and infected cells. Our results have implications for the development and use of noninvasive techniques of quantifying changes in cell properties induced by malaria disease pathology.

Seventh-harmonic generation from tightly focused 2 µm ultrashort pulses in air.

We report generation of third, fifth and seventh harmonics from air by using tightly focused, ultrashort pulses of short-wave infrared (2 µm) radiation. We have measured the third- and fifth-harmonic efficiencies to be 5×10(-5) and ~1.4×10(-5), respectively, with the ratio of fifth-to-third-harmonic efficiency being close to 0.28. Our experimental results provide confirmation of expectations of the higher-order Kerr effect model.

Anomalies in the motion dynamics of long-flagella mutants of Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii has long been used as a model organism in studies of cell motility and flagellar dynamics. The motility of the well-conserved '9+2' axoneme in its flagella remains a subject of immense curiosity. Using high-speed videography and morphological analyses, we have characterized long-flagella mutants (lf1, lf2-1, lf2-5, lf3-2, and lf4) of C. reinhardtii for biophysical parameters such as swimming velocities, waveforms, beat frequencies, and swimming trajectories. These mutants are aberrant in proteins involved in the regulation of flagellar length and bring about a phenotypic increase in this length. Our results reveal that the flagellar beat frequency and swimming velocity are negatively correlated with the length of the flagella. When compared to the wild-type, any increase in the flagellar length reduces both the swimming velocities (by 26-57%) and beat frequencies (by 8-16%). We demonstrate that with no apparent aberrations/ultrastructural deformities in the mutant axonemes, it is this increased length that has a critical role to play in the motion dynamics of C. reinhardtii cells, and, provided there are no significant changes in their flagellar proteome, any increase in this length compromises the swimming velocity either by reduction of the beat frequency or by an alteration in the waveform of the flagella.

Enhancing the strength of an optical trap by truncation.

Optical traps (tweezers) are beginning to be used with increasing efficacy in diverse studies in the biological and biomedical sciences. We report here results of a systematic study aimed at enhancing the efficiency with which dielectric (transparent) materials can be optically trapped. Specifically, we investigate how truncation of the incident laser beam affects the strength of an optical trap in the presence of a circular aperture. Apertures of various sizes have been used by us to alter the beam radius, thereby changing the effective numerical aperture and intensity profile. We observe significant enhancement of the radial and axial trap stiffness when an aperture is used to truncate the beam compared to when no aperture was used, keeping incident laser power constant. Enhancement in trap stiffness persists even when the beam intensity profile is modulated. The possibility of applying truncation to multiple traps is explored; to this end a wire mesh is utilized to produce multiple trapping that also alters the effective numerical aperture. The use of a mesh leads to reduction in trap stiffness compared to the case when no wire mesh is used. Our findings lead to a simple-to-implement and inexpensive method of significantly enhancing optical trapping efficiency under a wide range of circumstances.

Axicon-based writing of waveguides in BK7 glass.

We report on the direct writing of waveguide structures using an axicon lens to focus a 40 fs laser pulse within BK7 glass. The written structures are characterized for waveguiding action: waveguiding action for 635 and 1550 nm light and propagation loss as low as 0.19 dB/cm is measured. Loss values decrease with laser exposure time for incident energy of 300 µJ, indicating enhancement of index contrast. At higher energies, a reverse trend is obtained: higher loss values are obtained as index contrast degrades with an increase in exposure time.

Tank treading of optically trapped red blood cells in shear flow.

Tank-treading (TT) motion is established in optically trapped, live red blood cells (RBCs) held in shear flow and is systematically investigated under varying shear rates, temperature (affecting membrane viscosity), osmolarity (resulting in changes in RBC shape and cytoplasmic viscosity), and viscosity of the suspending medium. TT frequency is measured as a function of membrane and cytoplasmic viscosity, the former being four times more effective in altering TT frequency. TT frequency increases as membrane viscosity decreases, by as much as 10% over temperature changes of only 4°C at a shear rate of ∼43 s(-1). A threshold shear rate (1.5 ± 0.3 s(-1)) is observed below which the TT frequency drops to zero. TT motion is also observed in shape-engineered (spherical) RBCs and those with cholesterol-depleted membranes. The TT threshold is less evident in both cases but the TT frequency increases in the latter cells. Our findings indicate that TT motion is pervasive even in folded and deformed erythrocytes, conditions that occur when such erythrocytes flow through narrow capillaries.

Shape anisotropy induces rotations in optically trapped red blood cells.

A combined experimental and theoretical study is carried out to probe the rotational behavior of red blood cells (RBCs) in a single beam optical trap. We induce shape changes in RBCs by altering the properties of the suspension medium in which live cells float. We find that certain shape anisotropies result in the rotation of optically trapped cells. Indeed, even normal (healthy) RBCs can be made to rotate using linearly polarized trapping light by altering the osmotic stress the cells are subjected to. Hyperosmotic stress is found to induce shape anisotropies. We also probe the effect of the medium's viscosity on cell rotation. The observed rotations are modeled using a Langevin-type equation of motion that takes into account frictional forces that are generated as RBCs rotate in the medium. We observe good correlation between our measured data and calculated results.

Flagella-generated forces reveal gear-type motor in single cells of the green alga, Chlamydomonas reinhardtii.

Optically trapped single cells of the biflagellated, green alga, Chlamydomonas reinhardtii, rotate. The rotational dynamics of trapped wild-type and mutant cells show that functional flagella play a decisive role: the entire flagellar apparatus (central microtubules, radial spokes, and dynein arms) is involved. Any aberration in this apparatus leads to non-functionality, indicating a gear-type mechanism. The translational and rotational motions of the wild-type and mutant cells do not differ significantly. Optical forces alone do not play a vital role in the rotational dynamics of this cellular motor, making them useful as probes of the internal dynamics without external influence.

Measuring erythrocyte deformability with fluorescence, fluid forces, and optical trapping.

A laser-based method has been developed for experimentally probing single red blood cell (RBC) buckling and determining RBC membrane rigidity. Our method combines a liquid flow cell, fluorescence microscopy, and an optical-trap to facilitate simple measurements of the shear modulus and buckling properties of single RBCs, under physiological conditions. The efficacy of the method is illustrated by studying buckling behavior of normal and Plasmodium-infected RBCs, and the effect of Plasmodium falciparum-conditioned medium on normal, uninfected cells. Our simple method, which quantifies single-RBC deformability, may ease detection of RBC hematological disorders.

White-light-induced fragmentation of methane.

We experimentally probe molecular ionization and dissociation of methane molecules in the gas phase upon their irradiation by intense pulses of white light that spans the wavelength range 500-850 nm. White light pulses are generated upon irradiation of BK7 glass by 36-fs-long pulses of intense 820 nm laser light. Comparison is made of the molecular fragmentation patterns obtained using white light that is depolarized with those obtained using single-color (820 nm) light that is highly chirped. On the basis of such comparison, we make hitherto-unavailable estimates of the in situ intensity of white light pulses. Results obtained using white light also indicate that resonances apparently do not play any role in the ionization dynamics that ensue upon irradiation by intense, broadband light; neither are the dynamics affected by the polarization properties of the 820 nm light that is used to generate the white light.

Suppression of ultrafast supercontinuum generation in a salivary protein.

The first studies of the propagation of ultrafast (<45 fs) pulses of intense infrared light through protein media reveal that supercontinuum (white light) generation is severely suppressed in the presence of the protein alpha-amylase, a potential stress marker in human saliva. The continuum suppression capacity is attributed to the electron scavenging property of the protein.

Study of P. falciparum-infected erythrocytes and induced anisotropies under optical and fluid forces.

BACKGROUND & OBJECTIVES: The effect of P. falciparum on erythrocytes has been studied for a long time at the population level but actual studies at the single cell level remain largely unexplored. The aim of this study was to address the host-parasite relationship at the single cell level under two different kinds of forces, an optical force and a fluid force. The questions addressed were about the basic host-parasite interactions, but our findings have larger implications in diverse fields of parasite biology. METHODS: Erythrocytes were monitored under optical forces (using optical tweezers) and fluid forces (using microfluidic chambers) and dynamical images were captured in real-time video clips. These videos were then split into their respective frames so as to yield temporal information and various parameters pertaining to membrane structure, ionic imbalance and interaction with different forces were studied. RESULTS: The results of this study mainly bring to fore the inherent differences between infected and normal cell populations at the single cell level under various external forces. We probed three different criteria folding times, rotation speeds and rolling frequency to show inherent difference in various cell populations and also the dependence of the above to the cycle of the parasite. INTERPRETATION & CONCLUSION: This study portrays the importance of single cell observations pertaining to the host-parasite relationship. It shows the effect the malarial parasite has on erythrocytes and how the intrinsic property of the infected and its neighbouring uninfected cells change as compared to normal erythrocytes. There are thus implications in the fields of cytoadherence, parasite invasions and host immune evasion.

Enhanced optical limiting and nonlinear absorption properties of azoarene-appended phosphorus (V) tetratolylporphyrins.

Optical limiting performance, third-order nonlinearity chi(3), and nonlinear absorption properties have been investigated in a new class of azoarene phosphorus (V) porphyrins with charge transfer (CT) states. The introduction of axial azoarene groups into the phosphorus porphyrin structure is found to reduce the limiting threshold by a factor of 2 and lead to a rise in the second hyperpolarizability by 1 order of magnitude in the picosecond time regime and by 2 orders of magnitude in the nanosecond regime. The experimental data show reverse saturation of absorption in the nanosecond time regime and a saturation of the nonlinear absorption above a fluence of 0.5 J/cm2 in the picosecond regime. The presence of the CT state reduces saturation of excited-state absorption (ESA) in the S1 --> Sn transition through the S1 --> CT transition. Faster CT --> T1 transition increases the ESA from T1 --> Tn states in the nanosecond regime. A self-consistent theoretical analysis based on rate equations is used to estimate the high-lying excited-state lifetimes and absorption cross sections from the experimental results.