Real-time three-dimensional micro-imaging of liquid front in spreading sessile droplet under transient spreading states.

Dynamic wetting behaviors of sessile droplets on substrates play crucial roles for various industrial chemical processes. In the case of complete wetting, it has been proposed that a precursor film which is nanometer-order thickness and micrometer-order length further expands outside the macroscopic contact line of the sessile droplet. While the time evolution of the precursor film is believed to strikingly affect the macroscopic wetting behavior (e.g., spreading velocity), it had been hard to visualize the three-dimensional shape of the precursor film at the early stage of wetting. Further, although the spreading velocity rapidly decreases upon time at the early stage of the wetting (transient state) and then converged to be a constant at later time (steady state), conventional fluid mechanics theories generally describe only the wetting behavior at the steady state. Therefore, experimental observation of time evolution of the precursor film shape in the transient state is essential to proceed the theories to the next step. Here, a monochromatic laser interference microscope was developed to visualize three-dimensional shape of the wetting front of sessile droplets in real time. By detecting an interference of the laser reflected at the liquid and substrate surfaces, the precursor film was successfully visualized with a time resolution of 20 ms and a thickness resolution of about 3.5 nm at worst. For 10 cSt silicone oil on Si substrate, a 60-nm-thick and 70-µm-long precursor film was observed for 2-10 min after dropping and its spreading velocity which decreased with time was quantitatively analyzed.

Analysis of Cation Composition in Dolomites on the Intact Particles Sampled from Asteroid Ryugu.

Characterization of the elemental distribution of samples with rough surfaces has been strongly desired for the analysis of various natural and artificial materials. Particularly for pristine and rare analytes with micrometer sizes embedded on specimen surfaces, non-invasive and matrix effect-free analysis is required without surface polishing treatment. To satisfy these requirements, we proposed a new method employing the sequential combination of two imaging modalities, i.e., microenergy-dispersive X-ray fluorescence (micro-XRF) and Raman micro-spectroscopy. The applicability of the developed method is tested by the quantitative analysis of cation composition in micrometer-sized carbonate grains on the surfaces of intact particles sampled directly from the asteroid Ryugu. The first step of micro-XRF imaging enabled a quick search for the sparsely scattered and micrometer-sized carbonates by the codistributions of Ca2+ and Mn2+ on the Mg2+- and Fe2+-rich phyllosilicate matrix. The following step of Raman micro-spectroscopy probed the carbonate grains and analyzed their cation composition (Ca2+, Mg2+, and Fe2+ + Mn2+) in a matrix effect-free manner via the systematic Raman shifts of the lattice modes. The carbonates were basically assigned to ferroan dolomite bearing a considerable amount of Fe2+ + Mn2+ at around 10 atom %. These results are in good accordance with the assignments reported by scanning electron microscopy-energy-dispersive X-ray spectroscopy, where the thin-sectioned and surface-polished Ryugu particles were applicable. The proposed method requires neither sectioning nor surface polishing; hence, it can be applied to the remote sensing apparatus on spacecrafts and planetary rovers. Furthermore, the non-invasive and matrix effect-free characterization will provide a reliable analytical tool for quantitative analysis of the elemental distribution on the samples with surface roughness and chemical heterogeneity at a micrometer scale, such as art paintings, traditional crafts with decorated shapes, as well as sands and rocks with complex morphologies in nature.

Local elasticity evaluation of acid-denatured collagen by photoacoustic spectroscopy.

While there are various analytical methods for elasticity evaluation, those with micrometer-order spatial resolution are still under developing. As some of biological tissues such as capillary vessels and cochlea are very small and/or highly heterogeneous, development of analytical techniques with such high spatial resolution has been desired for biological and medical purposes. Especially, the elasticity of capillary vessels (several micrometer in diameter) would be an important indicator to find out early diseases. To measure the local elasticity for such small and/or heterogeneous samples, we have proposed an approach based on a temporal waveform of photoacoustic (PA) signal, i.e., time-domain PA. As the time-domain PA contains both the vibrating frequency and the sound propagation time after the excitation, it provides the information on the local elasticity (from the frequency) at a specific depth (from the propagation time) of samples. In the present study, the signal from collagen sheets were obtained and analyzed as models of blood vessel walls and scaffolds for regenerative medicine. In contrast to previous studies using the agarose gel which showed a single frequency peak, the signal from the collagen sheets was mainly composed of two frequency peaks, assignable to surface and bulk vibration. Further, the bulk vibration was found to sensitively reflect the elasticity of the samples. Since the PA effect can be induced only at the position where the light absorber exists, the analytical method proposed here would allow us to measure the local elasticity and its spatial distribution in blood vessels and other tissues.

New analytical method for the evaluation of heterogeneity in cation compositions of dolomites by micro-XRF and Raman spectroscopies.

Dolomite (CaMg(CO3)2) is an abundant carbonate mineral contained in sedimentary rocks and plays significant roles in water and carbon cycle in geo/cosmochemical environments. Since the cation compositions of carbonates are sensitive to the aqueous environment where they were precipitated and persisted, quantitative analysis of their cation compositions provides valuable information on the aqueous environments and their changes. The difficulty for the analysis of natural dolomite is that Mg2+ is continuously substituted by Fe2+ or Mn2+, and hence they sometimes possess micrometer-scale heterogeneity. Such heterogeneity carries quite important information on the gradual changes in aqueous environments due to changes in thermodynamic conditions and/or aqueous chemical compositions. In the present study, we explored a new quantitative scale to assess such heterogeneity of cation composition in natural dolomite and ferroan dolomite by combining X-ray fluorescence (XRF) and Raman spectroscopy. While the Fe + Mn content differed spot-by-spot, it was found that the Raman wavenumber and Fe + Mn content linearly correlated with each other. Since the spatial resolution of micro-Raman spectroscopy is as high as 1 µm, it does not require vacuum conditions, and is free from so-called matrix effect faced in other methods utilizing X-Rays and electron beams, the proposed qualitative analytical scale can provide a useful tool to assess the cation compositions in dolomites found in nature.

Real-time observation of the precursor film for low viscosity liquids spreading on solid substrates by a customized differential laser interference microscopy.

While static wettability is well treated with Young's equation via its static contact angle, theoretical analyses for wetting dynamics are not yet reaching consensus due to a singularity of the spreading forces worked at the vapor/liquid/solid contact line. One plausible explanation to overcome the singularity problem is that there is a so-called precursor film spreading outside the apparent contact line. After its first finding in 1919, many researchers have attempted to visualize its shape. However, because its length and thickness are as small as micrometer and nanometer-order, respectively, its visualization still remains a challenging issue especially for low-viscosity liquids. In the present study, we developed a differential laser interference microscope, which has a thickness resolution of approximately 2 nm at the best, and applied it to the wetting front of 10 cSt of silicone oil spreading on a silicon wafer with an almost constant spreading velocity. As a result, the precursor film of 14 µm long and 108 nm thick was clearly visualized. While the macro contact line has a finite advancing contact angle of 4.0°, the gradient of the precursor film surface gradually decreased and converged to ~ 0.1° at the micro-contact angle. The shape of the precursor film was independent of the time after the dropping for the range of 600 s ± 10%, which is consistent to theoretical estimation. The present study demonstrated that our interferometer simultaneously achieved nanometer thickness resolutions, micrometer in-plane spatial resolution, and at least a millisecond temporal resolution with a simple optical setup.

Non-destructive estimation of the cation composition of natural carbonates by micro-Raman spectroscopy.

Carbonates play a crucial role in the water and carbon cycles of both geochemical and cosmochemical environments. As carbonates do not exist homogeneously with other minerals in rocks and sands of various sizes, an analytical method that simultaneously satisfies non-destructivity and high spatial resolution has been desired. Further, the ability of semi-quantitative analysis with carbonates-selectivity and without any pre-treatments is added, for its applicability would be extended to remote sensing for deep sea and outer spaces. Here, we focused on the application of micro-Raman spectroscopy, where the vibrational wavenumbers of the translational (T) and librational (L) modes of carbonates are sensitively related to their cation composition. By comparing the semi-quantitative information obtained by X-ray fluorescence spectroscopy, it was found that these vibrational wavenumbers are approximately linearly related to the cation composition. Consequently, a conversion matrix was proposed to estimate the cation composition from the T and L mode vibrational wavenumbers. This method is universally applicable to any cation composition in carbonates, with no background information on the analyte required. To improve the accuracy, conversion matrices were further optimized to three solid-solution series of carbonates. It is worth noting that the proposed conversion matrices are free from matrix effects and do not depend on the total amount of carbonate in a sample. Therefore, the proposed method provides a useful analytical basis for remote sensing of the cation composition of carbonates, both in terrestrial and extra-terrestrial environments.

Micro-Raman spectroscopic analysis on natural carbonates: linear relations found via biaxial plotting of peak frequencies for cation substituted species.

Carbonates are ubiquitous minerals carrying important information on aqueous environments where they precipitated on the Earth and space. While their ideal chemical formulae are denoted as simple as MCO3 or M1M2(CO3)2 (M: metal cations), natural carbonates generally form solid-solution series and their compositions deviate from the ideal formulae. Since their cation composition due to the substitution provides a sensitive indicator for chemical and thermodynamic environments of aqueous solutions where they precipitated, their composition analysis has been widely carried out from the environmental/geochemical/astrochemical aspects. However, in widely used back-scattered electron and energy dispersion X-Ray analyses, samples should be generally sliced and/or their surface be polished prior to the measurements. For analyzing rare samples with small sizes, such as ones sampled from deep-sea and/or meteorites and asteroids, a non-destructive method without any pretreatments has been strongly desired. Here, a novel analytical method for discriminating various carbonates with Raman micro-spectroscopy is demonstrated, showing that the biaxial plot of the peak frequencies of their lattice modes linearly moves upon partial substitution of the cations. The cation substitution leads to linear movement in the biaxial map, and the slopes of the movement were different for Mg2+-Fe2+ and Mn2+-Fe2+ substitutions. This finding suggests that the micro-Raman analysis would be a non-destructive analytical method for evaluating the relative amount of Mg2+, Fe2+, and Mn2+ in dolomite-ankerite-kutnohorite solid-solution series, as well as Mg2+/Fe2+ ratio for magnesite-breunnerite-siderite. It would be helpful for analyzing the present and past terrestrial and cosmochemical environments.

Development of dark-field dynamic light scattering microscopy and its application: Tracking dynamics of particles in condensed slurries spreading on planar/nonplanar substrates.

HYPOTHESIS: While dynamics of particles in slurries is usually evaluated by dynamic light scattering measurements, this technique had only been applicable to particles in the bulk slurries. Because this limitation is mainly owing to strong reflection of light, the dynamics of particles in slurries spreading/drying on solid substrates is to be obtained by spatially separating the reflection light from scattering (signal) light. This may allow us to track the particles in practical samples such as cosmetics or inks spreading on solid surfaces. EXPERIMENT: We developed novel "dark-field dynamic light scattering microscopy". The system was evaluated with test samples of polystyrene beads dispersed in several viscosities of bulk glycerol aqueous solutions. This setup was then applied to slurries spreading/drying on planar and nonplanar substrates. FINDINGS: The results for planar surface indicate that origin of coffee-ring are the particles flowing into the edge of the droplet just before complete drying. On a skin-modelled nonplanar substrate, the slurry on bumps was found to maintain semi-dry condition longer than that at dents. This suggests that the dispersive medium was supplied to bumps from dents. This unique flow was explained as effective drying from the bumps increased surface tension at the bumps to pull up the liquid around.

A hydrogen-bonding structure in self-formed nanodroplets of water adsorbed on amorphous silica revealed via surface-selective vibrational spectroscopy.

Water adsorption onto a material surface is known to change macroscopic surface properties such as wettability and friction coefficient. While the role of the adsorbed water has been discussed for a long time, the interfacial structure of the adsorbed water has not been fully recognized in many cases. In this study, the hydration structure of water adsorbed on a vapor/silica interface at room temperature was studied via heterodyne-detected vibrational sum-frequency generation spectroscopy. The vibrational spectra of the interfacial molecules obtained here were different from those estimated via conventional sum-frequency generation spectroscopy. Interestingly, our results suggest that, at low humidity, the adsorbed water on silica forms nanodroplets instead of a uniform film. Because no silanol group was found to be hydrogen-bonding free, it was concluded that water molecules gather around the silanol group to form strongly hydrogen-bonded droplets. At high humidity, while the adsorbed water partially behaves like a bulk liquid, deprotonation of the silanol was not observed, unlike the case of silica surfaces in contact with bulk liquid water.

Characterization of the Elasticity of Small Objects Buried in Media Based on the Measurements of Photoacoustic Temporal Waveforms.

Since the elasticity of biological tissues is related to their pathological states, the development of new methods allowing for non-invasive measurements of the elasticity has been desired in the medical field. We present a characterization of the elasticity of objects buried in media from the temporal waveforms of photoacoustic signals. As the increment in Young's moduli of the objects, the frequency corresponding to the gravitational center of the power spectra obtained by the Fourier-transformation of the waveforms is increased. In our experiment configuration, the elasticity of buried objects is able to be identified up to about 1 MPa of Young's modulus from the frequency. These results suggest that measurements on the temporal waveforms of photoacoustic signals and the resultant power spectra would provide a useful method for evaluating the elasticity of deeply-situated microscopic pathological lesions, such as stage 0 or 1 mammary gland cancer, which is difficult by conventional ultrasound elastography.

Dynamic Light Scattering Measurements for Soft Materials on Solid Substrates: Employing Evanescent-wave Illumination and Dark-field Collection with a High Numerical Aperture Microscope Objective.

We developed an instrument that allows us to measure dynamic light scattering from soft materials on solid substrates by avoiding strong background due to the reflection light from substrates. In the instrument, samples on substrates are illuminated by evanescent-light field and the resultant scattered light from the samples is collected with a dark-field optical configuration by employing a high numerical aperture microscope objective. We applied the instrument to measure the dynamic properties of supported lipid bilayers (SLBs), which have been widely utilized in industries as functional materials such as biosensors. From the time course of the scattered light from the SLBs, the power spectrum with the broad peak ranging from 10 to 20 kHz is observed. The use of the microscope objectives enables us to apply the instrument to future light scattering imaging for dynamic properties of soft materials supported on various substrates by combining with conventional microscope systems.

Development of a Near-infrared Laser-induced Surface Deformation Microscope and Its Application to the Dynamic Viscoelastic Measurements of Single Living Plant Cell Surfaces.

A near-infrared laser-induced surface deformation (NIR-LISD) microscope is developed and is applied to the dynamic viscoelastic measurements of the surface of a living plant cell. In the microscope, the deformation of the surface is induced by an NIR laser beam, and then the change in intensity of the probe beam reflected from the surface reflects its viscoelasticity. The application of the NIR laser beam has a great advantage for the prevention of damage to the plant cell compared to the irradiation of a visible laser beam in LISD measurements. The NIR-LISD microscope allows for discriminating the differences in power spectra between the subapical and lateral regions of single rhizoids. It is a useful method for the dynamic viscoelastic measurements of cells, such as plant cells, that are damaged due to the strong absorption of ultraviolet or visible light.

Measurement of Water Distribution on Micro-structured Surface Buried in Water as a Model of Super Water Repellent Surface by Stimulated Raman Scattering Interferometer.

Water repellency of surfaces is realized by the roughness of the surfaces and the coating compounds on the surfaces. It is known that the water repellent surfaces are due to invasion of water into the trench structures on the surface, or due to air remaining in the trench structures. However, for more effective designing and fabrication of water-repellent surfaces, it is essential to measure the spatial distribution of water around the microstructures. In the present study, the SRS interferometer developed by the authors is applied to distinguish whether water or air is present in the trenches on the water-substrate interface buried by water. By obtaining the SRS interference signal generated from water while scanning the sample position, the phase shift of the SRS interference signal due to the microstructure on the interface was observed. From the quantitative analysis of the phase difference, it was revealed that the trench was filled with water. It is concluded that SRS interferometry is an effective method to observe experimentally the interface condition with chemical contrast along microstructures.

Programmable RNA detection with a fluorescent RNA aptamer using optimized three-way junction formation.

RNAs play essential roles in various cellular processes and can be used as biomarkers. Hence, it is important to detect endogenous RNA for understanding diverse cellular functions and diagnosing diseases. To construct a low-cost and easy-to-use RNA detection probe, a chemically unmodified RNA aptamer that binds to a pro-fluorophore to increase its fluorescence is desirable. Here, we focused on Broccoli, a superior variant of Spinach, which is a well-known fluorescent RNA aptamer that binds to DFHBI-1T and emits green fluorescence. We experimentally characterized Broccoli and predicted that it forms a G-quadruplex-based DFHBI-1T recognition region sandwiched between two stems. Based on this, we designed a Broccoli-based RNA detection probe (BRD probe) composed of a sequence of destabilized Broccoli fused with complementary sequences against target RNA. The resulting probe with its target RNA formed a stable three-way junction, named the MT2 three-way junction, which contributed to efficient refolding of the Broccoli structure and allowed for programmable RNA detection with high signal-to-noise ratio and sensitivity. Interestingly, the MT2 three-way junction also could be applied to probe construction of a truncated form of Spinach (Baby Spinach). The BRD and Baby Spinach-based RNA detection probes (BSRD probe) exhibited up to 48- and 140-fold fluorescence enhancements in the presence of their target RNAs and detected small amounts of target RNA that were as low as 160 and 5 nM, respectively. Thus, we experimentally characterized the higher order structure of Broccoli and developed structure-switching aptamer probes for highly sensitive, programmable, RNA detection using an MT2 three-way junction.

Mechanical Properties of the Coat Protein Layer and Cortex in Single Bacillus subtilis Spores Studied with an Atomic Force Microscope and Laser-induced Surface Deformation Microscope.

The differences in the mechanical properties between the cortex and coat protein layer in a Bacillus subtilis spore were clarified using an atomic force microscope (AFM) and an originally developed laser-induced surface deformation (LISD) microscope. AFM force curve measurements show that the Young's modulus of the coat protein layer is ca. 66% lower compared with that of the cortex. It has been experimentally clarified that the cortex makes a greater contribution to the rigidity of spores than the coat protein layer. From comparisons of the LISD power spectra, it is revealed that the coat protein layer has two different viscoelastic regions, and that the cortex relatively has a higher viscous nature than the coat protein layer. Furthermore, the LISD power spectrum above 1 × 105 Hz in the coat protein layer suggests that the local region in the coat protein layer behaves as a more elastic body compared with the cortex.

Cytotoxicity against cancer cells of chitosan oligosaccharides prepared from chitosan powder degraded by electrical discharge plasma.

Chitosan oligosaccharides, which obtain from degradation of chitosan, possess some interesting molecular weight-dependent biological properties, especially anticancer activity. Therefore, the conversion of chitosan to chitosan oligosaccharides with specific molecular weight has been continuously investigated in order to find effective strategies that can achieve both economic feasibility and environmental concerns. In this study, a novel process was developed to heterogeneously degrade chitosan powder by highly active species generated by electrical discharge plasma in a dilute salt solution (0.02 M) without the addition of other chemicals. The degradation rate obtained from the proposed process was comparable to that obtained from some other methods with the addition of acids and oxidizing agents. Separation of the water-soluble degraded products containing chitosan oligosaccharides from the reaction solution was simply done by filtration. The obtained chitosan oligosaccharides were further evaluated for an influence of their molecular weights on cytotoxicity against cancer cells and the selectivity toward cancer and normal cells.

Estimation of G-quartet-forming guanines in parallel-type G-quadruplexes by optical spectroscopy measurements of their single-nucleobase substitution sequences.

Since much attention has been paid to in vivo biological functions of G-quadruplexes, structural analyses of G-quadruplexes are essential for understanding their functional mechanisms. Here, we established a simple optical-spectroscopy-based method for the estimation of G-quartet-forming guanines in parallel-type G-quadruplexes using measurements of circular dichroism and the thermal melting temperature.

Discrimination between Normal and Cancerous Cells from Dynamic Viscoelastic Properties with a Laser-induced Surface Deformation Microscope.

We have clarified the differences in the power-law between normal and corresponding cancerous cells from dynamic viscoelastic measurements in a frequency range of 102 to 105 Hz with a laser-induced surface deformation (LISD) microscope. From the differences in the power spectra at higher frequencies, it has been clarified that a normal cell obeys the power-law with a single exponent, while a cancer cell with two exponents, indicating that the plasma membrane in the cancerous cell has at least two layers with different viscoelastic properties. In LISD measurements, the extension of the upper limit of the applied frequency up to 105 Hz allows us to clarify the existence of the two power-law exponents in the cancerous cell. Understanding the differences between normal and cancerous cells from the power-law in addition to conventional elasticity would be useful for the identification of cancerous cells and for the construction of a mechanical model for their invasion.

Laser-induced surface deformation microscope for the study of the dynamic viscoelasticity of plasma membrane in a living cell.

A laser-induced surface deformation (LISD) microscope is developed and applied to measurement of the dynamic relaxation responses of the plasma membrane in a living cell. A laser beam is tightly focused on an optional area of cell surface and the focused light induces microscopic deformation on the surface via radiation pressure. The LISD microscope not only allows non-contact and destruction-free measurement but provides power spectra of the surface responses depending on the frequency of the intensity of the laser beam. An optical system for the LISD is equipped via a microscope, allowing us to measure the relaxation responses in sub-cellular-sized regions of the plasma membrane. In addition, the forced oscillation caused by the radiation pressure for surface deformation extends the upper limit of the frequency range in the obtained power spectra to 106 Hz, which enables us to measure relaxation responses in local regions within the plasma membrane. From differences in power-law exponents at higher frequencies, it is realized that a cancerous cell obeys a weaker single power-law than a normal fibroblast cell. Furthermore, the power spectrum of a keratinocyte cell obeys a power-law with two exponents, indicating that alternative mechanical models to a conventional soft glassy rheology model (where single power-laws explain cells' responses below about 103 Hz) are needed for the understanding over a wider frequency range. The LISD microscope would contribute to investigation of microscopic cell rheology, which is important for clarifying the mechanisms of cell migration and tissue construction.

Thermoresponsive PEG-Coated Nanotubes as Chiral Selectors of Amino Acids and Peptides.

A series of nanotubes with a dense layer of short poly(ethylene glycol) (PEG) chains on the inner surface are prepared by means of a coassembly process using glycolipids and PEG derivatives. Dehydration of the PEG chains by heating increases the hydrophobicity of the nanotube channel and fluorescent-dye-labeled amino acids are extracted from bulk solution. Rehydration of the PEG chains by cooling results in back-extraction of the amino acids into the bulk solution. Because of the supramolecular chirality of the nanotubes, amino acid enantiomers can be separated in the back-extraction procedure, which is detectable with the naked eye as a change in fluorescence as the amino acids are released from the nanotubes. The efficiency and selectivity of the chiral separation are enhanced by tuning the chemical features and inner diameter of the nanotube channels. For example, compared with wide nanotube channels (8 nm), narrow nanotube channels (4 nm) provide more effective electrostatic attraction and hydrogen bond interaction environments for the transporting amino acids. Introduction of branched alkyl chains to the inner surface of the nanotubes enables chiral separation of peptides containing hydrophobic amino acids. The system described here provides a simple, quick, and on-site chiral separation in biological and medical fields.