The morphology and topology of mitochondria provide useful information about the physiological function of skeletal muscle. Previous studies of skeletal muscle mitochondria are based on observation with transmission, scanning electron microscopy or fluorescence microscopy. In contrast, photothermal (PT) microscopy has advantages over the above commonly used microscopic techniques because of no requirement for complex sample preparation by fixation or fluorescent-dye staining. Here, we employed the PT technique using a simple diode laser to visualize skeletal muscle mitochondria in unstained and stained tissues. The fine mitochondrial network structures in muscle fibers could be imaged with the PT imaging system, even in unstained tissues. PT imaging of tissues stained with toluidine blue revealed the structures of subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria and the swelling behavior of mitochondria in damaged muscle fibers with sufficient image quality. PT image analyses based on fast Fourier transform (FFT) and Grey-level co-occurrence matrix (GLCM) were performed to derive the characteristic size of mitochondria and to discriminate the image patterns of normal and damaged fibers. (C) 2017 Optical Society of America

We present a method for improving the signal-to-noise ratio in photothermal microscopy by optimizing the detection aperture in the spatially segmented balanced detection scheme. Experimental studies show a 1.44-fold improvement in the signal-to-noise ratio compared with the previous scheme. Consequently, the ratio is 9.6 times larger than that in conventional detection. The proposed method is implemented in a laser scanning photothermal microscope using low-cost compact laser diodes as the light source.

We have introduced a pupil filter, an inverse-annular pupil filter in a pump-probe photothermal microscope, which provides resolution enhancement in three dimensions. The resolution is probed to be improved in lateral and axial resolution by imaging experiment using 20 nm gold nanoparticles. The improvement in X (perpendicular to the common pump and probe polarization direction), Y (parallel to the polarization direction), and Z (axial direction) are by 15 +/- 6, 8 +/- 8, and 21 +/- 2 % from the resolution without a pupil filter. The resolution enhancement is even better than the calculation using vector field, which predicts the corresponding enhancement of 11, 8, and 6 %. The discussion is made to explain the unexpected results. We also demonstrate the photothermal imaging of thick biological samples (cells from rabbit intestine and kidney) stained with hematoxylin and eosin dye with the inverse-annular filter. (C) 2016 Author(s).

A fast, high-sensitivity photothermal microscope was developed by implementing a spatially segmented balanced detection scheme into a laser scanning microscope. We confirmed a 4.9 times improvement in signal-to-noise ratio in the spatially segmented balanced detection compared with that of conventional detection. The system demonstrated simultaneous bi-modal photothermal and confocal fluorescence imaging of transgenic mouse brain tissue with a pixel dwell time of 20 mu s. The fluorescence image visualized neurons expressing yellow fluorescence proteins, while the photothermal signal detected endogenous chromophores in the mouse brain, allowing 3D visualization of the distribution of various features such as blood cells and fine structures probably due to lipids. This imaging modality was constructed using compact and cost-effective laser diodes, and will thus be widely useful in the life and medical sciences. (C) 2016 Optical Society of America

A scheme for reducing image distortion in photothermal microscopy is presented. In photothermal microscopy, the signal shape exhibits twin peaks corresponding to the focusing or defocusing of the probe beam when a sample is scanned in the axial direction. This causes a distortion when imaging a structured sample in the axial plane. Here, we demonstrate that image distortion caused by the twin peaks is effectively suppressed by providing a small offset between two the focal planes of the pump and the probe beams. Experimental results demonstrate improvement in resolution, especially in the axial direction, over conventional optical microscopy-even with the focal offset. When a dry objective lens with a numerical aperture of 0.95 is used, the full width at half the maximum of the axial point spread function is 0.6 mu m, which is 50% (62%) smaller than the focal spot sizes of the pump (probe) beam. Herein, we present high-resolution three-dimensional imaging of thick biological tissues based on the present scheme. (C) 2015 Optical Society of America

Pixel-by-pixel processed fluorescence difference microscopy is experimentally demonstrated by multiplexing excitation laser beams with Gaussian and donut spot shapes and then demultiplexing the fluorescent signals using lock-in amplifiers. With this scheme, a fixed sample of fluorescent spheres and a slice of mouse brain tissue are imaged with resolutions that exceed the diffraction limit. Compared to previously reported subtraction imaging techniques, this pixel-by-pixel scan can be applied to improve the resolution of a moving sample without introducing subtraction errors. The synchronized signal detection feature makes this method extendible to various applications. (C) 2014 Elsevier B.V. All rights reserved.

Nonlinear photothermal microscopy, in which the intensity of the pump heating beam is modulated at f and the photothermal signal is extracted from the probe beam with a lock-in amplifier referred to 2f, is applied to the imaging of mouse melanoma without any staining. The pump and probe pulses, with central wavelengths of 488 and 632 nm, and a pulse duration of similar to 100 ps, are filtered from a compact commercial supercontinuum fiber laser source. An auto-balanced detector is applied to accumulate the signal and remove the laser noise of the probe. The spatial resolution of the nonlinear photothermal imaging is enhanced by similar to 18% in both theoretical calculations and experiments, compared with a linear photothermal mechanism, and the resolution enhancement is theoretically similar to 42% compared with conventional optical microscopy. This imaging technique shows possibilities for the clinical evaluation of melanoma with a high contrast and spatial resolution. (C) 2015 Optical Society of America

Nonlinear photothermal microscopy is applied in the imaging of biological tissues stained with chlorophyll and hematoxylin. Experimental results show that this type of organic molecules, which absorb light but transform dominant part of the absorbed energy into heat, may be ideal probes for photothermal imaging without photochemical toxicity. Picosecond pump and probe pulses, with central wavelengths of 488 and 632 nm, respectively, are spectrally filtered from a compact supercontinuum fiber laser source. Based on the light source, a compact and sensitive super-resolution imaging system is constructed. Further more, the imaging system is much less affected by thermal blurring than photothermal microscopes with continuous-wave light sources. The spatial resolution of nonlinear photothermal microscopy is similar to 188 nm. It is similar to 23% higher than commonly utilized linear photothermal microscopy experimentally and similar to 43% than conventional optical microscopy theoretically. The nonlinear photothermal imaging technology can be used in the evaluation of biological tissues with high-resolution and contrast. (C) 2015 Optical Society of America

A novel detection method is proposed for highly sensitive photothermal microscopic imaging. This method is based on the characteristics of an angular-dependent photothermal signal; it improves signal intensity by up to two times and rejects the intensity noise of the probe beam. The subdiffraction resolution photothermal imaging of mouse skin melanoma is demonstrated using a laser diode-based photothermal microscopy system to evaluate this method. We confirm that the signal intensity is enhanced 1.7 times compared with the conventional detection method. Moreover, the intensity noise of the laser diode used for the probe beam is effectively reduced by approximately 31 dB, even for a sample with non-uniformity of the refractive index and stationary absorption. This method is implemented by means of a commonly used balanced detector and is thus potentially useful for high-speed imaging. (C) 2015 Optical Society of America

Multi-wavelength microscopic imaging is essential to visualize a variety of nanoscale cellular components with high specificity and high spatial resolution. However, previous techniques are based on fluorescence, and thus cannot visualize nonfluorescent species, which are much less suffered from photodamage or photobleaching and hence are intrinsically useful in wider range of optical microscopy. Here, we show that simultaneous multi-wavelength imaging of nonfluorescent species can be achieved with the use of a photothermal microscope. Dual-wavelength subdiffraction imaging of biological tissues stained with hematoxylin and eosin is demonstrated. Three-dimensional label-free imaging of mouse melanoma tissue section is also presented to demonstrate the effectiveness of the enhanced spatial resolution. Our technique can be implemented using cost-effective and compact laser diodes and is applicable for various types of both fluorescent and nonfluorescent tissues. (C) 2015 Optical Society of America

Simultaneous two-color subtraction microscopy using mode multiplexing is realized experimentally. The samples are irradiated with single laser diode at wavelength of 445 nm. Then the beam split laser spots generate separate solid and donut spatial modes and are multiplexed with modulators for simultaneous excitation. The produced fluorescence signals are back collected and further divided into two color bands with dichroic mirrors. Then they are detected with two photomultipliers and demultiplexed in four lock-in amplifiers. Four fluorescence images are recorded in every scan and resolution enhanced images are obtained in two color channels after applying the subtraction strategy. With this method, imaging results of microspheres stained with organic dyes and mesenteric lymph nodes of a mouse labeled with quantum dots (Q525/650) are realized. Improvement of 20% ∼ 30% in resolving power of the two color channels compared with confocal microscopy is achieved in with corresponding subtraction factor of about 0.3.

We present a scheme for time-resolved pump-probe microscopy using intensity modulated laser diodes. The modulation frequencies of the pump and probe beams are varied up to 500 MHz with fixed frequency detuning typically set at 15 kHz. The frequency response of the pump-probe signal is detected using a lock-in amplifier referenced at the beat frequency. This frequency domain method is capable of characterizing the nanosecond to picosecond relaxation dynamics of sample species without the use of a high speed detector or a high frequency lock-in amplifier. Furthermore, as the pump-probe signal is based on the nonlinear interaction between the two laser beams and the sample, our scheme provides better spatial resolution than the conventional diffraction-limited optical microscopes. Time-resolved pump-probe imaging of fluorescence beads and aggregates of quantum dots demonstrates that this method is useful for the microscopic analysis of optoelectronic devices. The system is implemented using compact and low-cost laser diodes, and thus has a broad range of applications in the fields of photochemistry, optical physics, and biological imaging. (C) 2014 AIP Publishing LLC.

K-TCNQ crystals show photoinduced phase transition (PIPT) between a Mott insulator and a spin Peierls (SP) phase, having T-c = 395 K (= and the crystal has T-c = 395 K of the PIPT). We report the intra-molecular vibrations in the excited K-TCNQ crystal after impulsive excitation using a sub 9.4 fs pulse laser in the reflection spectrum. The frequencies of the vibrations are close to the Raman frequencies of molecules. The frequencies increase with melting of the SP phase, and then decrease with rearrangement of the dimerization. In addition, the frequencies of the vibrations are time-resolved to be modulated by the intermolecular optical phonon with 510 fs period corresponding to 65 cm(-1). This modulation is associated with the periodical change of coupling strengths of intermolecular charge transfer and/or the dipolar coupling between the counterpart molecules in the neighboring chains.

We evaluated the optimal detection angle for maximizing the signal to noise ratio (SNR) in sub-diffraction resolution photothermal microscopy. The angular dependent photothermal signal was calculated based on scattering theory using the temporally modulated Yukawa potential, and its detection angle and modulation frequency dependencies were analyzed. We verified the theoretical findings by imaging gold nanoparticles using laser diode based photothermal microscopy with balanced detection scheme. High-sensitivity (SNR similar to 40) photothermal biological imaging of a mouse brain was also demonstrated.

We experimentally demonstrate the use of annular beams to improve lateral resolution in laser-diode-based pump-probe microscopy. We found that the width of the point-spreading function in the case of the annular pump-probe beams is 162 nm, which is 30% smaller than that of the circular beams (232 nm). Furthermore, side lobes were efficiently suppressed at the focal plane since the pump-probe signal is proportional to the product of the two beam intensities. This scheme is demonstrated for the photothermal signal of nonfluorescent gold nanoparticles and the stimulated emission signal of fluorescence beads. (C) 2014 Optical Society of America

We demonstrate the use of intensity-modulated laser diodes to implement pump-probe microscopy and achieved sub-diffraction resolution imaging with shot-noise limited sensitivity with a scheme of balanced detection. This technique has several applications for various types of induced transmission change, including excited-state absorption, ground state absorption bleaching and stimulated emission. By using our technique, biological imaging of mouse T cells and the axons of neurons in the cerebral cortex was demonstrated. (C) 2014 Optical Society of America

We present an analytical method for quantifying exciton hopping in an energetically disordered system with quenching sites. The method is subsequently used to provide a quantitative understanding of exciton hopping in a quantum dot (QD) array. Several statistical quantities that characterize the dynamics (survival probability, average number of distinct sites visited, average hopping distance, and average hopping rate in the initial stage) are obtained experimentally by measuring time-resolved fluorescence intensities at various temperatures. The time evolution of these quantities suggests in a quantitative way that at low temperature an exciton tends to be trapped at a local low-energy site, while at room temperature, exciton hopping occurs repeatedly, leading to a large hopping distance. This method will serve to facilitate highly efficient optoelectronic devices using QDs such as photovoltaic cells and light-emitting diodes, since exciton hopping is considered to strongly influence their operational parameters. The presence of a dark QD (quenching site) that exhibits fast decay is also quantified.

Time-resolved transient absorption spectroscopy for water solution of cytosine with sub-10fs deep ultraviolet laser pulse is reported. Ultrafast electronic excited state dynamics and coherent molecular vibrational dynamics are simultaneously observed and their relaxation mechanisms are discussed.

We investigate the dynamics of exciton hopping in a CdSe/ZnS quantum dot (QD) array composed of an inhomogeneously broadened ensemble. Time- and spectrally resolved fluorescence intensities are measured by varying the excitation photon energy at the absorption edge. This method allows us to observe fluorescence from only the subdistribution of the QD ensemble, thereby allowing the dynamics of exciton hopping, which depends on the initial (donor) exciton energy, to be elucidated. Experimental results along with numerical calculations using a model of a coupled QD array show that when high-energy QDs are selectively excited, exciton energy transfer occurs repeatedly to a site of low energy, leading to a large exciton hopping length. In contrast, when the low-energy end of the QD ensemble is excited, the exciton tends to be trapped in the initial QD. Furthermore, from the analysis of the decay time of fluorescence intensities, it is suggested that there are dark QDs associated with the defect and/or off state of blinking QDs in the ensemble and energy transfer to such a site is mainly followed by quenching.

The dynamics of exciton hopping in an array of inhomogeneously broadened CdSe/ZnS quantum dot (QD) ensembles is examined by measuring time-and spectrally resolved fluorescence intensities. We have found a decrease in the fluorescence decay time as well as a dynamic redshift of the fluorescence spectrum originating from exciton transfer. Both show the characteristic temperature dependence reflecting the peculiar exciton dynamics in the QD ensemble. We propose a model of coupled QD arrays where inhomogeneous distribution and dark QDs that are related to a long-lasting off-state of blinking QDs are taken into account. Experimental results together with numerical calculations based on this model suggest that at low temperatures, an exciton transfers to a local low-energy site and tends to be trapped, whereas at high temperatures, thermally activated hopping of the exciton occurs repeatedly. Furthermore, we show that the decay time decrease of the QD array is attributable to exciton hopping to dark QDs.

We describe the principles of two spectroscopic methods of non-resonance light scattering (LS) and optical-Kerr-effect (OKE) spectroscopies in detail, and review basic but truly important phenomena observed by these two methods. Particularly, we focus on the following three experiments: 1) Response functions determined by frequency-domain LS and time-domain OKE spectroscopies, almost completely agree with each other, indicating that the quantum-mechanical fluctuation-dissipation theorem (QM-FDT) holds well in this system. 2) Femtosecond time responses of liquids clearly show an initial rise process even though they show an exponential response at a later time. This indicates that the Debye relaxation model does not hold in such an early time region. 3) From the measurement of the Stokes and anti-Stokes LS intensity ratios, it is found that the LS spectra of relaxation modes in liquids and solids are symmetric with respect to the spectral origin of the scattered light and hence the ratio does not satisfy the Boltzmann distribution rule expected from QM-FDT. These experimental results which contain apparently contradictory data are closely related with the nature of relaxation modes examined, which more or less assume a macroscopic character, and also with the physical basis of relaxation, which is inevitably connected to the observation problem of quantum mechanics. We discuss these points in relation to the physical reality of macroscopic quantity.

We investigated the origin of the iridescent violet-bluish feathers of the adult Jungle Crow, Corvus macrorhynchos, using microscopic and optical techniques. A single layer of melanin granules was found below the surface of the barbules in the feathers of male crows. Although the barbule microstructure was clearly sexually dimorphic, neither the appearance nor the optical measurements were notably different between the sexes, which indicated that the single layer of melanin granules did not contribute to the iridescent color of the feathers. We also found a thin layer, which we refer to as the epicuticle, at the surfaces of the barbules of both male and female feathers, indicating thin-film interference as the most likely cause of the iridescent color. We investigated this possibility by measuring reflection patterns and spectra. Our results suggest that the weak violet-bluish feather color of the feathers of the Jungle Crow is caused by thin-film interface due to the presence of an epicuticle on the feather barbules.

Excitation energy transfer (EET) processes in CdSe/CdZnS quantum dot (QD) clusters have been investigated in this study by measuring their time-resolved and spectrally resolved fluorescence intensities. The contributions of radiative and non-radiative exciton recombination through EET are evaluated, where the latter is expected to occur in a large class of QD ensembles because of the presence of nonluminescent QDs. It appears that the fluorescence decay in larger QDs serving as acceptor does not show an initial rise, in addition the lifetime of the acceptor QD is independent of the excitation wavelength, suggesting that an EET is followed mostly by non-radiative recombination. (C) 2010 Elsevier B.V. All rights reserved.

We studied the excitation energy transfer (EET) process in CdSe/CdZnS quantum dot (QD) clusters by measuring time-and spectrally-resolved fluorescence intensities. Radiative and non-radiative exciton recombinations that occurs after EET were evaluated: the latter occurred in a large class of QD ensembles because of the presence of nonluminescent (dark) QDs. Numerical simulation confirmed the effect of the dark QDs on time-dependent fluorescence intensities, and the results were compared with that of the experiment. Nonradiative recombination occurring after EET was found to be dominant in the low quantum yield of QDs. The EET rate as a function of QD energy was successfully calculated on the basis of these findings. (C) 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

An actively stabilized Michelson interferometer with a long optical-path-length difference is developed to generate a phase-locked pulse pair on the order of nanoseconds. A rigid frame and a piezoelectric feedback loop enable us to stabilize an interferometer to the optical-path-length difference of up to 5.8 m within lambda/20 for 1 h. It is demonstrated that this interferometer is useful for generating a phase-tunable pulse pair up to an interval of 19 ns, which opens new possibilities for measuring the longtime coherence in the nanosecond region in the field of coherent control spectroscopy.

A quantum dot-based ratiometric pH sensor that responds to the pH range 6-8 was developed using FITC (fluorescein isothiocyanate) conjugated CdSe/CdZnS nanocrystals.

In recent years, structural colors have attracted great attention in a wide variety of research fields. This is because they are originated from complex interaction between light and sophisticated nanostructures generated in the natural world. In addition, their inherent regular structures are one of the most conspicuous examples of non-equilibrium order formation. Structural colors are deeply connected with recent rapidly growing fields of photonics and have been extensively studied to clarify their peculiar optical phenomena. Their mechanisms are, in principle, of a purely physical origin, which differs considerably from the ordinary coloration mechanisms such as in pigments, dyes and metals, where the colors are produced by virtue of the energy consumption of light. It is generally recognized that structural colors are mainly based on several elementary optical processes including thin-layer interference, diffraction grating, light scattering, photonic crystals and so on. However, in nature, these processes are somehow mixed together to produce complex optical phenomena. In many cases, they are combined with the irregularity of the structure to produce the diffusive nature of the reflected light, while in some cases they are accompanied by large-scale structures to generate the macroscopic effect on the coloration. Further, it is well known that structural colors cooperate with pigmentary colors to enhance or to reduce the brilliancy and to produce special effects. Thus, structure-based optical phenomena in nature appear to be quite multi-functional, the variety of which is far beyond our understanding. In this article, we overview these phenomena appearing particularly in the diversity of the animal world, to shed light on this rapidly developing research field.

The dynamics of a chemical pulse at the interface of excitable media having different diffusion properties is presented experimentally using the Belousov-Zhabotinsky reaction system. When a chemical pulse propagates from a larger diffusion region to a smaller diffusion region, it stops and forms a steady excitation band at the interface. Furthermore, a pulse train stimulated by this band appears subsequently. A simple one-dimensional model with discontinuous diffusion is proposed, and a numerical simulation is performed that shows good agreement with the experiment. Unidirectional pulse propagation across the interface is strongly suggested on the basis of the proposed model.

A coupling function that describes the interaction between self-sustained oscillators in a phase equation is derived and applied experimentally to Belousov-Zhabotinsky (BZ) oscillators. It is demonstrated that the synchronous behavior of coupled BZ reactors is explained extremely well in terms of the coupling function thus obtained. This method does not require comprehensive knowledge of either the oscillation mechanism or the interaction among the oscillators, both of these being often difficult to elucidate in an actual system. These facts enable us to accurately analyze the weakly coupled entrainment phenomenon through the direct measurement of the coupling function.

A new method to determine a coupling function in a phase model is theoretically derived for coupled self-sustained oscillators and applied to Belousov-Zhabotinsky (BZ) oscillators. The synchronous behavior of two coupled BZ reactors is explained extremely well in terms of the coupling function thus obtained. This method is expected to be applicable to weakly coupled multioscillator systems, in which mutual coupling among nearly identical oscillators occurs in a similar manner. The importance of higher-order harmonic terms involved in the coupling function is also discussed.

Unidirectionally coupled reactors in Belousov-Zhabotinsky reaction is studied experimentally under the bistable state between oscillation and resting. Irregular transitions between the two states are observed (intermittent oscillation). Phase analysis reveals that the transitions occur in a timely manner, which will be discussed on the analogy of stochastic resonance using a two-dimensional potential surface.

We have constructed a small ferroin-catalyzed Belousov-Zhabotinsky reactor connected with a main reactor through continuous mass flow. When each reactor is in an independent oscillating state. the activation energy of the oscillation frequency differs considerably from each other, which enables us to investigate the oscillating state of the former under precise control of the latter through coupling strength and frequency difference. With increasing flow rate. both synchronized and intermittent oscillations appear according to the sign of the frequency difference. A precursor exists near the synchronization, while the transition from resting to oscillating state takes place in a timely manner for the intermittent oscillation. (C) 2004 Published by Elsevier B.V.

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