Opalescence Arising from Network Assembly in Antibody Solution.

Opalescence of therapeutic antibody solutions is one of the concerns in drug formulation. However, the mechanistic insights into the opalescence of antibody solutions remain unclear. Here, we investigated the assembly states of antibody molecules as a function of antibody concentration. The solutions of bovine gamma globulin and human immunoglobulin G at around 100 mg/mL showed the formation of submicron-scale network assemblies. The network assembly resulted in the appearance of opalescence with a transparent blue color without the precipitates of antibodies. Furthermore, the addition of trehalose and arginine, previously known to act as protein stabilizers and protein aggregation suppressors, was able to suppress the opalescence arising from the network assembly. These results will provide an important information for evaluating and improving protein formulations.

Vapor-Induced Conversion of a Centrosymmetric Organic-Inorganic Hybrid Crystal into a Proton-Conducting Second-Harmonic-Generation-Active Material.

Chemical responsivity in materials is essential to build systems with switchable functionalities. However, polarity-switchable materials are still rare because inducing a symmetry breaking of the crystal structure by adsorbing chemical species is difficult. In this study, we demonstrate that a molecular organic-inorganic hybrid crystal of (NEt4)2[MnN(CN)4] (1) undergoes polarity switching induced by water vapor and transforms into a rare example of proton-conducting second-harmonic-generation-active material. Centrosymmetric 1 transforms into noncentrosymmetric polar 1·3H2O and 1·MeOH by accommodating water and methanol molecules, respectively. However, only water vapor causes a spontaneous single-crystal-to-single-crystal transition. Moreover, 1·3H2O shows proton conduction with 2.3 × 10-6 S/cm at 298 K and a relative humidity of 80%.

Visualizing intra-medulla lipids in human hair using ultra-multiplex CARS, SHG, and THG microscopy.

We performed label-free imaging of human-hair medulla using multi-modal nonlinear optical microscopy. Intra-medulla lipids (IMLs) were clearly visualized by ultra-multiplex coherent anti-Stokes Raman scattering (CARS) spectroscopic imaging. Two groups of IMLs were found: second harmonic generation (SHG) active and inactive. By combining SHG analysis with CARS, the two groups were identified as free fatty acids and wax esters, respectively.

Visualization of intracellular lipid metabolism in brown adipocytes by time-lapse ultra-multiplex CARS microspectroscopy with an onstage incubator.

We visualized a dynamic process of fatty acid uptake of brown adipocytes using a time-lapse ultra-broadband multiplex coherent anti-Stokes Raman scattering (CARS) spectroscopic imaging system with an onstage incubator. Combined with the deuterium labeling technique, the intracellular uptake of saturated fatty acids was traced up to 9 h, a substantial advance over the initial multiplex CARS system, with an analysis time of 80 min. Characteristic metabolic activities of brown adipocytes, such as resistance to lipid saturation, were elucidated, supporting the utility of the newly developed system.

Characterization of Intra/Extracellular Water States Probed by Ultrabroadband Multiplex Coherent Anti-Stokes Raman Scattering (CARS) Spectroscopic Imaging.

Detailed knowledge of the water status in living organisms is crucial for understanding their physiology and pathophysiology. Here, we developed a technique to spectroscopically image water at high resolution using ultrabroadband multiplex coherent anti-Stokes Raman scattering (CARS) microscopy equipped with a supercontinuum light source. This system allows for the visualization of a wide spectrum of CARS signals from the fingerprint to the end of O-H stretching at a spectral resolution of ∼10 cm-1. Application of the system to living mammalian cells revealed a spectral red shift of the O-H stretching vibrational band inside compared to outside the cells, suggesting the existence of stronger hydrogen bonds inside the cells. Furthermore, potential changes in spectra were examined by adding mannitol to the extracellular solution, which increases the osmolality outside the cells and thereby induces dehydration of the cells. Under this treatment, the red shift of the O-H stretching band was further enhanced, revealing the effects of mannitol on water states inside the cells. The methodology developed here should serve as a powerful tool for the chemical imaging of water in living cells in various biological and medical contexts.

Raman optical activity spectroscopy by visible-excited coherent anti-Stokes Raman scattering.

We developed a Raman optical activity (ROA) spectroscopic system with visible-excited coherent anti-Stokes Raman scattering (CARS). A supercontinuum within the visible region was generated with a photonic crystal fiber pumped with both 532 and 1064 nm excitation, generating a multiplexed CARS-ROA spectrum covering the whole fingerprint region. In visible excitation, the CARS-ROA spectrum of (-)-ß-pinene shows a higher contrast ratio of the chirality-induced signal to the achiral background than that of the previously reported near-infrared CARS-ROA spectrum.

Active involvement of micro-lipid droplets and lipid-droplet-associated proteins in hormone-stimulated lipolysis in adipocytes.

The regulation of lipolysis in adipocytes involves coordinated actions of many lipid droplet (LD)-associated proteins such as perilipin, hormone sensitive lipase (HSL), adipose triglyceride lipase (ATGL), and its activator protein, CGI-58. Here, we describe the cellular origin and physiological significance of micro LDs (mLDs) that emerge in the cytoplasm during active lipolysis, as well as the roles of key lipolytic proteins on mLDs in differentiated 3T3-L1 adipocytes. Multiplex coherent anti-Stokes Raman scattering (CARS) microscopy demonstrated that mLDs receive the fatty acid (FA) moiety of triglyceride from pre-existing LDs during lipolysis. However, when FA re-esterification was blocked, mLDs did not emerge. Time-lapse imaging of GFP-tagged LD-associated proteins and immunocytochemical analyses showed that particulate structures carrying LD-associated proteins emerged throughout the cells upon lipolytic stimulation, but not when FA re-esterification was blocked. Overall lipolysis, as estimated by glycerol release, was significantly lowered by blocking re-esterification, whereas release of free FAs was enhanced. ATGL was co-immunoprecipitated with CGI-58 from the homogenates of lipolytically stimulated cells. Following CGI-58 knockdown or ATGL inhibition with bromoenol lactone, release of both glycerol and FA was significantly lowered. AICAR, an activator of AMP-activated protein kinase, significantly increased FA release, in accordance with increased expression of ATGL, even in the absence of CGI-58. These results suggest that, besides on the surface of pre-existing central LDs, LD-associated proteins are actively involved in lipolysis on mLDs that are formed by FA re-esterification. Regulation of mLDs and LD-associated proteins may be an attractive therapeutic target against lipid-associated metabolic diseases.

Electronically resonant third-order sum frequency generation spectroscopy using a nanosecond white-light supercontinuum.

Third-order sum frequency generation (TSFG) is one of the third-order nonlinear optical processes, and has the generation mechanism analogous to third harmonic generation (THG). By using a white-light supercontinuum, we can obtain broadband multiplex TSFG spectra. In the present study, we developed an electronically resonant TSFG spectrometer, and applied it to obtain TSFG spectra of hemoproteins. Analyzed TSFG ratio spectra clearly showed the resonant enhancement attributable to the electronic state of hemoproteins. This is a promising method for the imaging of electronic states of molecules inside living cells or tissues.

Raman optical activity by coherent anti-Stokes Raman scattering spectral interferometry.

We demonstrate a method to measure Raman optical activity (ROA) by using coherent anti-Stokes Raman scattering (CARS) spectral interferometry. An extremely weak chirality-induced CARS field is amplified through the interference with a strong CARS field generated from an external reference and is extracted by the Fourier transformation. In this interferometric coherent Raman optical activity (iCROA), both the sign and the magnitude of optical active non-resonant background susceptibility can be directly determined. Measurement of a CARS-ROA spectrum with less artifact is obtained because a broad offset artifact due to optical rotatory dispersion is clearly distinguished in iCROA.

Label-Free Identification of Spore-Forming Bacteria Using Ultrabroadband Multiplex Coherent Anti-Stokes Raman Scattering Microspectroscopy.

Spore-forming bacteria accumulate dipicolinic acid (DPA) to form spores to survive in extreme environments. Vibrational spectroscopy is widely used to detect DPA and elucidate the existence of the bacteria, while vegetative cells, another form of spore-forming bacteria, have not been studied extensively. Herein, we applied coherent anti-Stokes Raman scattering (CARS) microscopy to spectroscopically identify both spores and vegetative cells without staining or molecular tagging. The spores were identified by the strong CARS signals due to DPA. Furthermore, we observed bright spots in the vegetative cells in the CARS image at 1735 cm-1. The vegetative cells contained molecular species with C=O bonds because this vibrational mode was associated with the carbonyl group. One of the candidate molecular species is diketopimelic acid (DKP), a DPA precursor. This hypothesis was verified by comparing the spectrum obtained by the vegetative cells with that of the DKP analogue (ketopimelic acid) and with the result obtained by DFT calculation. The results indicate that the observed vegetative cell is in the sporulation process. CARS spectra can be used to monitor the maturation and preformation of spores.

Label-free tetra-modal molecular imaging of living cells with CARS, SHG, THG and TSFG (coherent anti-Stokes Raman scattering, second harmonic generation, third harmonic generation and third-order sum frequency generation).

We have developed a new multimodal molecular imaging system that combines CARS (coherent anti-Stokes Raman scattering), SHG (second harmonic generation), THG (third harmonic generation) and multiplex TSFG (third-order sum frequency generation) using a subnanosecond white-light laser source. Molecular composition and their distribution in living cells are clearly visualized with different contrast enhancements through different mechanisms of CARS, SHG, THG and TSFG. A correlation image of CARS and TSF reveals that the TSF signal is generated predominantly from lipid droplets inside a cell as well as the peripheral cell wall.

Time-frequency resolved analysis of a nanosecond supercontinuum source dedicated to multiplex CARS application.

In this paper, we describe and investigate the properties of a broadband source designed from a nanosecond microchip laser operating at high repetition rate and dedicated to multiplex-CARS application. We demonstrate that a strong reshaping of the initial pulse profile drastically affects the Stokes wave and therefore represents an important limitation in CARS experiment. In particular, we emphasize the saturation effect of the peak power of the Stokes wave resulting from supercontinuum generation. However, we show that this type of compact system can be particularly suitable for achieving CARS measurement.

Observation of Raman optical activity by heterodyne-detected polarization-resolved coherent anti-Stokes Raman scattering.

We report the first observation of Raman optical activity (ROA) by coherent anti-Stokes Raman scattering. Thanks to the more freedom of polarization configurations in coherent anti-Stokes Raman scattering than in spontaneous Raman spectroscopy, the contrast ratio of the chiral signal to the achiral background has been improved markedly. For (-)-ß-pinene, it is 2 orders of magnitude better than that in the reported spontaneous ROA measurement. This is also the first measurement of ROA signal using a pulsed laser source.

Label-free enzymatic reaction monitoring in water-in-oil microdroplets using ultra-broadband multiplex coherent anti-Stokes Raman scattering spectroscopy.

We propose a system for monitoring an enzymatic reaction, i.e., dehydrogenation of ethanol catalyzed by alcohol dehydrogenase, in microdroplets using ultra-broadband multiplex coherent anti-Stokes Raman scattering (CARS) spectroscopy. The reaction solution was encapsulated in water-in-oil microdroplets with diameters of 50 µm. The reaction was monitored by measuring the concentration of coenzymes from the CARS spectrum obtained in one-second exposure time. The results obtained using our system was consistent with those of the conventional fluorescence measurement system and indicate the potential of CARS spectroscopy for droplet-based high-throughput screening of enzymes.

Calibration for a count rate-dependent time correlation function and a random noise reduction in pulsed dynamic light scattering.

A pulsed dynamic light scattering (DLS) system, which would be potentially applied to nonlinear DLS with molecular selectivity, was developed by combining a sub-nanosecond pulsed laser with a software-based detection system. The distortion of the time correlation function due to the clipping effect in the photon counting module, and the resulting underestimation of the particle size, were successfully calibrated based on a theoretical simulation. The effective removal of random noises was also demonstrated via time gating synchronized to the laser pulses.

Spectroscopic second and third harmonic generation microscopy using a femtosecond laser source in the third near-infrared (NIR-III) optical window.

In this study, second harmonic generation (SHG) and third harmonic generation (THG) spectroscopic imaging were performed on biological samples using a femtosecond laser source in the third near-infrared (NIR) optical window (NIR-III). Using a visible-NIR spectrometer, the SHG and THG signals were simultaneously detected and were extracted using spectral analysis. Visualization of biological samples such as cultured cells (HEK293 T), mouse brain slices, and the nematode Caenorhabditis elegans was performed in a label-free manner. In particular, in an SHG image of an entire coronal brain section (8 × 6 mm2), we observed mesh-like and filamentous structures in the arachnoid mater and wall of the cerebral ventricle, probably corresponding to the collagen fibers, cilia, and rootlet. Moreover, the THG images clearly depicted the densely packed axons in the white matter and cell nuclei at the cortex of the mouse brain slice sample and lipid-rich granules such as lipid droplets inside the nematode. The observations and conclusions drawn from this technique confirm that it can be utilized for various biological applications, including in vivo label-free imaging of living animals.

Coherent anti-Stokes Raman scattering cell imaging and segmentation with unsupervised data analysis.

Coherent Raman imaging has been extensively applied to live-cell imaging in the last 2 decades, allowing to probe the intracellular lipid, protein, nucleic acid, and water content with a high-acquisition rate and sensitivity. In this context, multiplex coherent anti-Stokes Raman scattering (MCARS) microspectroscopy using sub-nanosecond laser pulses is now recognized as a mature and straightforward technology for label-free bioimaging, offering the high spectral resolution of conventional Raman spectroscopy with reduced acquisition time. Here, we introduce the combination of the MCARS imaging technique with unsupervised data analysis based on multivariate curve resolution (MCR). The MCR process is implemented under the classical signal non-negativity constraint and, even more originally, under a new spatial constraint based on cell segmentation. We thus introduce a new methodology for hyperspectral cell imaging and segmentation, based on a simple, unsupervised workflow without any spectrum-to-spectrum phase retrieval computation. We first assess the robustness of our approach by considering cells of different types, namely, from the human HEK293 and murine C2C12 lines. To evaluate its applicability over a broader range, we then study HEK293 cells in different physiological states and experimental situations. Specifically, we compare an interphasic cell with a mitotic (prophase) one. We also present a comparison between a fixed cell and a living cell, in order to visualize the potential changes induced by the fixation protocol in cellular architecture. Next, with the aim of assessing more precisely the sensitivity of our approach, we study HEK293 living cells overexpressing tropomyosin-related kinase B (TrkB), a cancer-related membrane receptor, depending on the presence of its ligand, brain-derived neurotrophic factor (BDNF). Finally, the segmentation capability of the approach is evaluated in the case of a single cell and also by considering cell clusters of various sizes.

Optical coherence tomography-based tissue dynamics imaging for longitudinal and drug response evaluation of tumor spheroids.

We present optical coherence tomography (OCT)-based tissue dynamics imaging method to visualize and quantify tissue dynamics such as subcellular motion based on statistical analysis of rapid-time-sequence OCT signals at the same location. The analyses include logarithmic intensity variance (LIV) method and two types of OCT correlation decay speed analysis (OCDS). LIV is sensitive to the magnitude of the signal fluctuations, while OCDSs including early- and late-OCDS (OCDS e and OCDS l , respectively) are sensitive to the fast and slow tissue dynamics, respectively. These methods were able to visualize and quantify the longitudinal necrotic process of a human breast adenocarcinoma spheroid and its anti-cancer drug response. Additionally, the effects of the number of OCT signals and the total acquisition time on dynamics imaging are examined. Small number of OCT signals, e.g., five or nine suffice for dynamics imaging when the total acquisition time is suitably long.

Multiplex coherent anti-Stokes Raman scattering highlights state of chromatin condensation in CH region.

Coherent Raman microscopy has become a powerful tool in label-free, non-destructive and fast cell imaging. Here we apply high spectral resolution multiplex coherent anti-Stokes Raman scattering (MCARS) microspectroscopy in the high wavenumber region to the study of the cell cycle. We show that heterochromatin - the condensed state of chromatin - can be visualised by means of the vibrational signature of proteins taking part in its condensation. Thus, we are able to identify chromosomes and their movement during mitosis, as well as structures like nucleoli and nuclear border in interphase. Furthermore, the specific organization of the endoplasmic reticulum during mitosis is highlighted. Finally, we stress that MCARS can reveal the biochemical impact of the fixative method at the cellular level. Beyond the study of the cell cycle, this work introduces a label-free imaging approach that enables the visualization of cellular processes where chromatin undergoes rearrangements.