Observation of Coherent Perfect Absorption in Oil Film on Water Surface and Sensitive Detection of Refractive Index Anisotropy in the Film.

Sharp reflection dips of 50% were observed when white light was incident from the side of a cell on a 1 µm thick film of silicone oil (polydimethylsiloxane, PDMS, nearly transparent in visible light, with the extinction coefficient κ ≈ 0.0001) above a water surface in the cell so that the total reflection condition was satisfied at the oil-air interface. This is the first observation of a coherent perfect absorption (CPA) phenomenon in liquid. The experimental results can be reproduced by the Fresnel reflectance of the monolayer film, but the wavelength positions at which the dip appears for s-polarized and p-polarized light are reversed if the refractive index of the oil film is assumed to be isotropic. The experimental results were correctly reproduced by assuming that the extraordinary-ray refractive index (light polarized perpendicular to the interface) is 1% larger than the ordinary-ray refractive index (light polarized parallel to the interface). This indicates that the polarization dependence of the CPA phenomenon is extremely sensitive to the difference between the in-plane and out-of-plane refractive indices of the thin film.

Extremely large electro-optic effect of TPPS J-aggregates in the PVA or PVP polymer matrix and aqueous solution.

The molecules of tetra-phenyl porphyrin tetra-sulfonic acid (TPPS) form a J-aggregate via self-organization in aqueous solution. The J-aggregates formed in an aqueous solution with added hydrochloric acid were dispersed in PVP polymer and subjected to electric field modulation spectroscopy. The observed difference in the static polarizability Δαp between the excited and ground states in the TPPS J-aggregates reached a value 2.66 × 104 times and 1.22 × 105 times larger than in the monomer molecules in PVA and PVP polymer matrices, respectively. This large enhancement factor of the electro-optic effect (EOE) may be induced by the higher-order structure of the J-aggregate. The different enhancement factor for each polymer matrix may be introduced by the structural differences between the polymer matrices. Electric field modulation spectroscopy of the J-aggregate in aqueous solution showed an absorbance change of 25% of the magnitude of absorbance peak.

Absorbance spectra of the hematochrome-like granules and eyespot of Euglena gracilis by scan-free absorbance spectral imaging A(x, y, λ) within the live cells.

Euglena gracilis has an organelle resembling hematochrome, with an appearance similar to the eyespot and the absorption band spectrally overlapped with that of the carotenoid. To discriminate the hematochrome-like granules and eyespot, scan-free, non-invasive, absorbance spectral imaging A(x, y, λ) microscopy of single live cells, where A(x, y, λ) means absorbance at a position (x, y) on a two-dimensional image at a specific wavelength λ was applied. This technique was demonstrated to be a powerful tool for basic research on intracellular structural analysis. By this method, characteristic absorption spectra specific to the hematochrome-like granule or eyespot were identified among a variety of spectra observed depending on the location inside the organelles. The hematochrome-like granule was dark orange and deep green in its outline and had a characteristic absorption peak at 620 nm as well as at 676 to 698 nm, suggesting that its origin is a component of chloroplast including chlorophyll a. Furthermore, the representative spectra of these organelles were derived by principal component analysis of the absorbance and its position in absorbance image, indicating that they can be distinguished from each other and other regions. It was also confirmed that even in areas where these organelles and chloroplasts overlap, one can distinguish them from each other. The present research clarified the absorption spectra of the eyespot with 1 × 1 µm spatial resolution and those unpublished of hematochrome-like granules of E. gracilis, and indicated that one can statistically distinguish these organelles by this method.

Orientation Control of Hemispherical Janus Particles and Metal Coating on the Selective Surface To Excite Surface Plasmon Polaritons in the Micro-Kretschmann Geometry.

Asymmetric-shaped particles (the Janus particle) are difficult to be arranged in a uniform orientation on a solid substrate. This difficulty prevents further modification of the selective surface of the particles for fabrication of the Janus particles with anisotropy of the shape and surface. We successfully arranged hemispherical particles in a uniform orientation at the air-water interface. The particles were arranged on the solid substrate in a uniform orientation by transferring the particle film onto the substrate. This arrangement enabled the fabrication of the Janus particles with anisotropy of the shape and surface by selective deposition of a film on either the equatorial plane or the spherical surface. Additionally, we demonstrated the function of the microscopic Kretschmann geometry for excitation of the surface plasmon polaritons of a thin metal film on the equatorial plane of a single hemispherical particle.

Quasi first-order Hermite Gaussian beam for enhanced sensitivity in Sagnac interferometer photothermal deflection spectroscopy.

The detection sensitivity of a Sagnac interferometer photothermal deflection spectroscopy was enhanced by changing the probe beam pattern from zero-order to a quasi-first-order Hermite Gaussian (QHG) beam. The nature of the higher order HG mode, where the beam pattern is preserved during propagation with an increased field gradient, is utilized to enhance the measurement sensitivity. In this spectroscopy, the lateral beam deflection due to the photothermal effect is sensitively detected as a change in the interference light intensity. The change in intensity is amplified due to the higher field gradient of the QHG(1,0) beam at the photodetector. This amplification effect was both numerically and experimentally demonstrated to obtain twofold improvement in the signal-to-noise ratio.

Hydrogen photoproduction in green algae Chlamydomonas reinhardtii sustainable over 2 weeks with the original cell culture without supply of fresh cells nor exchange of the whole culture medium.

Unicellular green algae Chlamydomonas reinhardtii are known to make hydrogen photoproduction under the anaerobic condition with water molecules as the hydrogen source. Since the hydrogen photoproduction occurs for a cell to circumvent crisis of its survival, it is only temporary. It is a challenge to realize persistent hydrogen production because the cells must withstand stressful conditions to survive with alternation of generations in the cell culture. In this paper, we have found a simple and cost-effective method to sustain the hydrogen production over 14 days in the original culture, without supply of fresh cells nor exchange of the culture medium. This is achieved for the cells under hydrogen production in a sulfur-deprived culture solution on the {anaerobic, intense light} condition in a desiccator, by periodically providing a short period of the recovery time (2 h) with a small amount of TAP(+S) supplied outside of the desiccator. As this operation is repeated, the response time of transition into hydrogen production (preparation time) is shortened and the rate of hydrogen production (build up time) is increased. The optimum states of these properties favorable to the hydrogen production are attained in a few days and stably sustained for more than 10 days. Since generations are alternated during this consecutive hydrogen production experiment, it is suggested that the improved hydrogen production properties are inherited to next generations without genetic mutation. The properties are reset only when the cells are placed on the {sulfur-sufficient, aerobic, moderate light} conditions for a long time (more than 1 day at least).

Noise cancellation with phase-detection technique for pump-probe measurement and application to stimulated Raman imaging.

Intensity noise on a probe beam is a serious obstacle to highly sensitive and high-speed pump-probe microscopy. In this report, a reference beam of the probe is prepared and delayed. The intensity modulation by the sample is measured as the phase modulation of the superposition of detected electrical signals of the probe and reference beams, and the intensity noise is canceled. We evaluate performance of the noise cancellation using the super-continuum light from a piece of photonic crystal fiber, and find that the noise is canceled by ∼26 dB. We then apply the method to a stimulated Raman microscope. This method contributes to highly sensitive and high-speed pump-probe imaging with various light sources.

Reddening of the Unicellular Green Alga Euglena gracilis by Dried Bonito Stock and Intense Red Light Irradiation.

This study confirms for the first time that the significant red coloration of Euglena gracilis is induced by bonito stock (BS), a traditional Japanese food, and intense red light exposure (605~660 nm, 1000~1300 µmol photons/m2/s). Under the condition, excessive photosynthetic activity destroyed many chloroplasts, while carotenoids were maintained, resulting in the formation of reddened cells. The HPLC analysis revealed that diadinoxanthin was the primary carotenoid present in reddened cells. Additionally, an undefined xanthophyll, not produced under normal culture conditions, was synthesized and suggested to contain a C=O bond. While it has been reported that strong light stress can increase the total carotenoid content of cells, this study did not verify this claim, and it should be investigated further in future research. Under white light irradiation conditions (90 µmol photons/m2/s) in BS medium, no reddening of cells was observed, and good growth was achieved (over four times the cell density in CM medium on the seventh day). This cell suspension is considered to have a high nutritional value because it is composed of functional food, BS and E. gracilis. The fact that this method does not involve genetic modification suggests the possibility of industrial applications, including food use, even in reddened cells.

Analysis of Unique Motility of the Unicellular Green Alga Chlamydomonas reinhardtii at Low Temperatures down to -8 °C.

Previous studies of motility at low temperatures in Chlamydomonas reinhardtii have been conducted at temperatures of up to 15 °C. In this study, we report that C. reinhardtii exhibits unique motility at a lower temperature range (-8.7 to 1.7 °C). Cell motility was recorded using four low-cost, easy-to-operate observation systems. Fast Fourier transform (FFT) analysis at room temperature (20-27 °C) showed that the main peak frequency of oscillations ranged from 44 to 61 Hz, which is consistent with the 60 Hz beat frequency of flagella. At lower temperatures, swimming velocity decreased with decreasing temperature. The results of the FFT analysis showed that the major peak shifted to the 5-18 Hz range, suggesting that the flagellar beat frequency was decreasing. The FFT spectra had distinct major peaks in both temperature ranges, indicating that the oscillations were regular. This was not affected by the wavelength of the observation light source (white, red, green or blue LED) or the environmental spatial scale of the cells. In contrast, cells in a highly viscous (3.5 mPa·s) culture at room temperature showed numerous peaks in the 0-200 Hz frequency band, indicating that the oscillations were irregular. These findings contribute to a better understanding of motility under lower-temperature conditions in C. reinhardtii.

Excimer luminescence from nonresonantly excited pyrene and perylene molecules in solution.

By nonresonant excitation below the absorption edge of pyrene and perylene molecules in solution, efficient excimer luminescence is observed. For perylene in solution, there are two kinds of nonresonant excimer emission that closely resemble Y- and E-emissions in perylene crystal and perylene in polymer. The concentration dependence of the photon flux density of monomer and excimer emission is reasonably explained by a simple model with a few fitting parameters based on the ordinary excimer formation process, in which the excited-state monomer interacts with a ground-state monomer. Observed quantum efficiency of excimer formation is by orders of magnitude higher than that expected if a monomer is excited from thermally populated vibrational levels. The experimental evidence for direct resonant excitation of excimers in perylene nanocrystals is obtained from the absence of anti-Stokes emission. This is due to a symmetry-breaking effect close to crystal surface, suggesting that excimers are formed through a more efficient process in solution, such as resonant excitation of weakly interacting molecules with a symmetry-broken intermolecular configuration.

Vibrational energy flow between modes by dynamic mode coupling in THIATS J-aggregates.

We performed ultrafast pump-probe spectroscopy of J-aggregates of 3,3'-disulfopropyl-5,5'-dichloro-9-ethyl thiacarbocyanine triethylammonium (THIATS), one of the most typical cyanine dyes, and detected excited molecular vibrations, using a sub-10 fs pulse laser. The time-resolved two-dimensional difference absorption (ΔA) spectra are observed between -314 and 1267 fs. By performing the Fourier transform and spectrogram analysis, vibrational modes in THIATS are observed at 285, 485, 555, 824, and 1633 cm(-1) and there was a modulation of the vibrational frequencies around 1633 cm(-1) which depend on the delay time, respectively. By the analysis of the modulation, energy flow is found to take place from other modes to the 1633 cm(-1) mode through the low frequency mode with ∼50 cm(-1). Also, by fitting the real-time traces of ΔA with the sum of two exponential functions and a constant term, the average lifetimes of three electronically excited states were found to be τ1 = 52 ± 5 fs and τ2 = 540 ± 78 fs. By performing single-exponential fitting around the stationary absorption peak at 1.990 eV, in the negative time range, the electronic dephasing time, T2(ele), is determined to be 18.30 fs.

Spatial Distribution of Flagellated Microalgae Chlamydomonas reinhardtii in a Quasi-Two-Dimensional Space.

Although the phenomenon of collective order formation by cell-cell interactions in motile cells, microswimmers, has been a topic of interest, most studies have been conducted under conditions of high cell density, where the space occupancy of a cell population relative to the space size ϕ>0.1 (ϕ is the area fraction). We experimentally determined the spatial distribution (SD) of the flagellated unicellular green alga Chlamydomonas reinhardtii at a low cell density (ϕ≈0.01) in a quasi-two-dimensional (thickness equal to cell diameter) restricted space and used the variance-to-mean ratio to investigate the deviation from the random distribution of cells, that is, do cells tend to cluster together or avoid each other? The experimental SD is consistent with that obtained by Monte Carlo simulation, in which only the excluded volume effect (EV effect) due to the finite size of cells is taken into account, indicating that there is no interaction between cells other than the EV effect at a low cell density of ϕ≈0.01. A simple method for fabricating a quasi-two-dimensional space using shim rings was also proposed.

Sagnac interferometer for photothermal deflection spectroscopy.

Photothermal deflection spectroscopy is combined with a Sagnac interferometer to enhance the sensitivity of the absorption measurement by converting the photothermal beam deflection effect into the light intensity change by the interference effect. Because of stable light interference due to the common path, the signal intensity can be amplified without increasing the noise by extending the optical path length between a sample and a photodetector. The sensitivity is further improved by the use of focusing optics and double-pass geometry. This makes photothermal deflection spectroscopy applicable to any kind of material in the whole visible region with a xenon lamp for excitation and water or air as a deflection medium.

Algorithm of auto-balancing noise-canceling based on noise correlation for high-speed balancing, high-dynamic range, and robustness against DC-offset drift.

The influence of the light source noise can be reduced by subtracting the signal of the light source noise (reference signal) from that of the probe light (probe signal). Here, it is essential that the intensities of the signals are equated. To equate the intensities, an auto-balancing method is widely employed, where the gain of the probe signal is feedback-controlled, regarding the DC component in the subtraction as an error signal. However, DC-offset drift causes a deviation from the optimal intensity balance. Additionally, the DC component is often several orders of magnitude larger than the sample signal, which requires a high-dynamic range in the circuitry. Furthermore, if the feedback control is too fast, it cancels out the sample signal. In this study, we formulate a noise correlation auto-balancing method, where the correlation of the reference signal and residual noise in the subtraction is employed as the error signal. With this scheme, all the above problems are avoided. The feasibility of the algorithm was demonstrated by a prototype circuitry and signals emulating the probe and reference signals. It did not suffer from the DC-offset drift, while a 44-dB canceling rate with auto-balancing of a 1.3-MHz cutoff frequency was demonstrated. We foresee, such as in pump/probe measurements, that this scheme improves the robustness, dynamic range, and response time required to follow changes in transmittance and the measurement position of the sample while employing a light source that is advantageous in wavelength selectivity, coherence, and cost but is noisy.

Development of a Simple Fabrication Method for Magnetic Micro Stir Bars and Induction of Rotational Motion in Chlamydomonas reinhardtii.

A magnetic micro stirrer bar (MMSB) is used in the mixing operation of microfluidic devices. We have established a low-cost and easy method to make MMSBs using magnetic (neodymium magnets, magnet sheets) or non-magnetic powders (SUS304) as materials. We demonstrated three kinds of MMSB have respective advantages. To confirm the practical use of this MMSB, a cell suspension of the motile unicellular green alga Chlamydomonas reinhardtii was stirred in microwells. As a result, the number of rotating cells increased with only one of the two flagella mechanically removed by the shear force of the rotating bar, which facilitates the kinetic analysis of the flagellar motion of the cell. The rotational motion of the monoflagellate cell was modeled as translational (orbital) + spinning motion of a sphere in a viscous fluid and the driving force per flagellum was confirmed to be consistent with previous literature. Since the present method does not use genetic manipulations or chemicals to remove a flagellum, it is possible to obtain cells in a more naturally viable state quickly and easily than before. However, since the components eluted from the powder material harm the health of cells, it was suggested that MMSB coated with resin for long-term use would be suitable for more diverse applications.

Real-time vibrational dynamics in chlorophyll a studied with a few-cycle pulse laser.

We use a 6.8-fs laser as the light source for broad-band femtosecond pump-probe real-time vibrational spectroscopy to investigate both electronic relaxation and vibrational dynamics of the Q(y)-band of Chl-a at 293 K. More than 25 vibrational modes coupled to the Q(y) transition are observed. Eleven of them have been clarified predominantly due to the excited state, and six of them are concluded to be nearly exclusively resulting from the ground-state wave-packet motion. Moreover, thanks to the broad-band detection over 5000 cm⁻¹, the modulated signals due to the excited state vibrational coherence are observed on both sides of the 0-0 transition with equal separation. The corresponding nonlinear process has been studied using a three-level model, from which the probe wavelength dependence of the phase of the periodic modulation can be calculated. The probe wavelength dependence of the vibrational amplitude is interpreted in terms of the interaction between the "pump" or "laser," Stokes, and anti-Stokes field intermediated by the molecular vibrations. In addition, an excited state absorption peak at ~709 nm has been observed. To the best of our knowledge, this is the first study of broad-band real-time vibrational spectroscopy in Chl-a.

Spectral modulation observed in Chl-a by ultrafast laser spectroscopy.

Broadband real-time dynamic vibronic coupling in Chl-a were experimentally studied using few cycle laser pulses of 6.8fs duration and a 128-channnel lock-in amplifier. Thanks to the extreme temporal resolution benefitting from the ultrashort laser pulse, the real-time modulation of the electronic transition energy induced by the molecular vibrations were calculated by the time dependent first moments of the bleaching band. The transition energy was found to be modulated periodically with the same frequencies of molecular vibration found in the Fourier amplitude spectrum of the difference absorbance real-time traces. This was interpreted to be due to the difference in the effective transition energy associated with the wavepacket motion induced by the equilibrium positions of potential curves between the ground state and the excited state. Using the values, Huang-Rhys factors for several vibrational modes involved in the spectral modulation at the room-temperature have been determined.

Electric field-controlled dissociation and association of porphyrin J-aggregates in aqueous solution.

The electrooptic effects of porphyrin J-aggregates of tetraphenyl porphyrin tetrasulfonic acid (TPPS) in aqueous solution were studied using electroabsorption (EA) spectroscopy. When the J-aggregates were three-dimensionally distributed, the EA spectra exhibited broadening in the exciton band. When a DC or AC electric field was applied for a long time, the J-aggregates with KCl were dissociated into monomers via N-mers (N = 2-4) as intermediate states, while those without KCl had an increase in aggregation. The EA spectra showed a red shift in the exciton band for N-mers, which indicates that N-mers are isolated microaggregates with a coherent aggregation number N, and isolated microaggregates have not been microscopically or spectrally observed until now. The estimated third-order nonlinear optical susceptibility χ((3)) for EA spectra in aqueous solution was 10(4) times larger than that in a polymer film. The molecular rearrangement model was applied to a variety of orientational distributions and the results were explained fairly well. The contribution of the electric double layer is the most probable reason for the large enhancement of χ((3)) for the solution sample. The dynamic equilibrium between two types of monomers, J-aggregates of various aggregation numbers and cations such as K(+) and H(+) was investigated to reveal that K(+) is more loosely bound to the constituent monomers in J-aggregates than H(+). Equilibrium equations also show that well-grown aggregates with N > 15 tend to dominate in a solution of J-aggregates, which explains why only well-developed aggregates can be observed spectroscopically.

Absorbance spectroscopy of light scattering samples placed inside an integrating sphere for wide dynamic range absorbance measurement.

In the absorbance measurement of a sample that scatters light significantly, it is necessary to consider the effect of the attenuation of incident light due to scattering on the measured absorbance. Since the usual absorbance measurement with an integrating sphere (IS) cannot remove the influence of backscattering, we performed the absorbance measurement considering the light scattered to almost all solid angles by placing the sample inside the IS. Ni(NO3)2 and Co(NO3)2 aqueous solutions were used as non-scattering samples, and Ni(NO3)2 solutions mixed with submicrometer polystyrene spheres as scatterers were used as scattering samples. The sample-concentration dependence of the measured absorbance was investigated for the cell containing the sample placed at the entrance of or inside the IS. It was found that even inside the IS, the measured absorbance does not match the true absorbance because light is partially multiply transmitted through the sample or detected without being transmitted through the sample. Due to the latter reason, the saturated absorbance inside the IS was lower than that at the entrance. We derived the formula with three fitting parameters relating the measured and true absorbance taking these factors into account, which quantitatively reproduced the concentration dependence of the absorbance in the non-scattering sample. When the scattering samples were placed at the entrance and inside of the IS, the measured absorbance increased and decreased, respectively, compared to those without scatterers. This decrease in absorbance for the scattering samples inside the IS was also explained by the proposed formula slightly modified.

Noninvasive and safe cell viability assay for Paramecium using natural pigment extracted from food.

Noninvasive, safe and cost-effective cell viability assay is important in many fields of biological research such as cell culture and counting. We examined ten typical natural pigments extracted from food to find that Monascus pigment (MP) or anthocyanin pigment (AP: purple sweet potato and purple cabbage) with Tris (Trimethylolaminomethane) works as a good indicator of viability assay for dye exclusion test (DET) of Paramecium. This was confirmed spectrally by scan-free, non-invasive absorbance spectral imaging A (x, y, λ) microscopy. We developed a new method of cell capture using a metal mesh to confine live Paramecium in a restricted space. This has the advantage that a low-cost and robust capture can be fabricated without using special equipment, compared to a conventional lab-on-a-chip. As a result, MP and AP stained dead cells as quick as methylene blue (MB), a synthetic dye conventionally used in DET within 1 min when treated with microwave and benzalkonium chloride. The natural pigments with Tris had little effect on inhibiting the growth of Paramecium, but MB killed all the cells within 1 h. MP is most useful because it allows non-invasive DET without Tris. This approach provides less invasive and safe DET.