Observation of the changes in the chemical composition of lipid droplets using Raman microscopy.

We report the dynamics of lipid droplet formation induced by introducing cis- and/or trans-fatty acids into cells. Raman imaging allows the chemical analysis of each droplet, showing that exogenous fatty acids initially enter original endogenous droplets, then induce additional droplets containing endogenous lipids, and finally form their droplets.

Label-Free Imaging of Intracellular Temperature by Using the O-H Stretching Raman Band of Water.

We propose a label-free method for measuring intracellular temperature using a Raman image of a cell in the O-H stretching band. Raman spectra of cultured cells and the medium were first measured at various temperatures using a Raman microscope and the intensity ratio of the two regions of the O-H stretching band was calculated. The intensity ratio varies linearly with temperature in both the medium and cells, and the resulting calibration lines allow simultaneous visualization of both intracellular and extracellular temperatures in a label-free manner. We applied this method to the measurement of temperature changes after the introduction of FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone) in living cells. We observed a temperature rise in the cytoplasm and succeeded in obtaining an image of the change in intracellular temperature after the FCCP treatment.

Raman Imaging Microscopy for Quantitative Analysis of Biological Samples.

Raman imaging microscopy is a powerful tool for label-free imaging of biological samples. It has the advantage of measuring the spatial distribution of endogenous proteins and lipids in cells, as well as obtaining chemical information on these endogenous molecules, such as hydrogen bonding and electrostatic interactions. However, because Raman intensity is very weak compared with fluorescence intensity, obtaining a reliable Raman image requires fast acquisition of a Raman image and rejection of background fluorescence. In this chapter, we describe the procedure for obtaining images of the Raman band of interest using a multipoint technique, which is the fast acquisition method for obtaining an image.

Observation of unusual molecular diffusion behaviour below the lower critical solution temperature of water/2-butoxyethanol mixtures by using fluorescence correlation spectroscopy.

The effect of solute affinity on solute diffusion in binary liquids well below the lower critical solution temperature (LCST) was studied by using fluorescence correlation spectroscopy. We measured the hydrodynamic radii of a hydrophobic and an amphiphilic fluorescent dye under systematic variation of the relative molar fractions of water/2-butoxyethanol and, for comparison, of water/methanol mixtures, which do not show phase separation. We found that the apparent hydrodynamic radius of the hydrophobic dye almost doubled in water/2-butoxyethanol, whereas it remained largely unchanged for the amphiphilic dye and in water/methanol mixtures. Our results indicate that the translational diffusion of solutes is influenced by transient local solution structures, even at temperatures well below the LCST. We conclude that, even far below LCST, different solutes can experience different environments in binary liquid mixtures depending on both the solute and solvent properties, all of which impact their reactivity.

Picosecond dynamics of hydrogen bond rearrangements during phase separation of a triethylamine and water mixture.

The earliest stages of phase separation in a liquid triethylamine (TEA)-water mixture were observed using a picosecond IR laser pulse to produce a temperature jump and ultrafast Raman spectroscopy. Raman spectral changes in the water OH stretching region showed that the temperature rise induced by IR pulses equilibrated within a few tens of picoseconds. Amplitude changes in the TEA CH-stretching region of difference Raman spectra consisted of an initial faster and a subsequent slower process. The faster process within 100 ps is attributed to hydrogen bond weakening caused by the temperature rise. The slower process attributed to phase separation was observed for several nanoseconds, showing the number of hydrogen bond between TEA and water gradually decreased with time. The kinetics of hydrogen bond scission during phase separation indicated a linear growth of the phase-separated component, as observed previously on the nanosecond time scale, rather than the more usual exponential growth. A peak blueshift was observed in the difference Raman spectra during phase separation. This shift implies that hydrogen bond scission of TEA-water aggregates involving very few water molecules took place in the initial stage of phase separation (up to 2 ns), and then was followed by the breaking of TEA-water pairs surrounded by water molecules. This effect may be a result from spatial inhomogeneities associated with the phase separation process: aggregates or clusters existing naturally in solution even below the lower critical soluble temperature.

Highly sensitive Raman measurements of protein aqueous solutions using liquid-liquid phase separation.

A highly sensitive method is proposed for obtaining the Raman spectra of low-concentration proteins and nucleic acids in an aqueous solution using liquid-liquid phase separation. This method uses water droplets formed by adding a large amount of polyethylene glycol into a biomolecular aqueous solution. Ordinary spontaneous Raman spectra are obtained with a high signal-to-noise ratio.

Establishment of a Method for the Introduction of Poorly Water-Soluble Drugs in Cells and Evaluation of Intracellular Concentration Distribution Using Resonance Raman Imaging.

Label-free measurement is essential to understand the metabolism of drug molecules introduced into cells. Raman imaging is a powerful method to investigate intracellular drug molecules because it provides in situ label-free observation of introduced molecules. In this study, we propose that Raman imaging can be used not only to observe the intracellular distribution of drug molecules but also to quantitatively visualize the concentration distribution reflecting each organelle in a single living cell using the Raman band of extracellular water as an intensity standard. We dissolved poorly water-soluble all-trans-retinoic acid (ATRA) in water using a cytocompatible amphiphilic phospholipid polymer, poly[2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate] (PMB) as a solubilizing reagent, introduced it into cells, and obtained the intracellular concentration distribution of ATRA. ATRA was concentrated in the cells and mainly localized to mitochondria and lipid droplets, interacting strongly with mitochondria and weakly with lipid droplets. Poorly water-soluble ß-carotene was also introduced into cells using PMB but was not concentrated intracellularly, indicating that ß-carotene does not interact specifically with intracellular molecules. We established a protocol for the solubilization and intracellular uptake of poorly water-soluble molecules using PMB and obtaining their concentration distribution using Raman microscopy.

Additive-free size-controlled synthesis of gold square nanoplates using photochemical reaction in dynamic phase-separating media.

Ultrafast phase separation of water and 2-butoxyethanol mixture was induced by nanosecond IR laser pulse irradiation. After a certain delay time, a UV laser pulse was introduced to induce photoreduction of aurate ions, which led to the formation of gold nanoparticles in dynamic phase-separating media. The structure and size of the nanoparticles varied depending on the delay time between the IR and UV pulses. For a delay time of 5 and 6 µs, gold square plates having edge lengths of 150 and 100 nm were selectively obtained, respectively. With a delay time of 3 µs, on the other hand, the size of the square plates varied widely from 100 nm to a few micrometers. The size of the gold square plates was also varied by varying the total irradiation time of the IR and UV pulses. The size distribution of the square plates obtained under different conditions suggests that the growth process of the square plates was affected by the size of the nanophases during phase separation. Electron diffraction patterns of the synthesized square plates showed that the square plates were highly crystalline with a Au(100) surface. These results showed that the nanophases formed during laser-induced phase separation can provide detergent-free reaction fields for size-controlled nanomaterial synthesis.

Label-free autofluorescence lifetime reveals the structural dynamics of ataxin-3 inside droplets formed via liquid-liquid phase separation.

Liquid-liquid phase separation is a phenomenon that features the formation of liquid droplets containing concentrated solutes. The droplets of neurodegeneration-associated proteins are prone to generate aggregates and cause diseases. To uncover the aggregation process from the droplets, it is necessary to analyze the protein structure with keeping the droplet state in a label-free manner, but there was no suitable method. In this study, we observed the structural changes of ataxin-3, a protein associated with Machado-Joseph disease, inside the droplets, using autofluorescence lifetime microscopy. Each droplet showed autofluorescence due to tryptophan (Trp) residues, and its lifetime increased with time, reflecting structural changes toward aggregation. We used Trp mutants to reveal the structural changes around each Trp and showed that the structural change consists of several steps on different timescales. We demonstrated that the present method visualizes the protein dynamics inside a droplet in a label-free manner. Further investigations revealed that the aggregate structure formed in the droplets differs from that formed in dispersed solutions and that a polyglutamine repeat extension in ataxin-3 hardly modulates the aggregation dynamics in the droplets. These findings highlight that the droplet environment facilitates unique protein dynamics different from those in solutions.

Label-Free Tracking of Nanoprodrug Cellular Uptake and Metabolism Using Raman and Autofluorescence Imaging.

Nano-DDS, a drug delivery system using nanoparticles, is a promising tool to reduce adverse drug reactions and maximize drug efficiency. Understanding the intracellular dynamics following the accumulation of nanoparticles in tissues, such as cellular uptake, distribution, metabolism, and pharmacological effects, is essential to maximize drug efficiency; however, it remains elusive. In this study, we tracked the intracellular behavior of nanoparticles of a prodrug, cholesterol-linked SN-38 (CLS), in a label-free manner using Raman and autofluorescence imaging. Bright autofluorescent spots were observed in cells treated with CLS nanoparticles, and the color tone of the bright spots changed with incubation time. The Raman spectra of the bright spots showed that the autofluorescence came from the nanoparticles taken into cells, and the change in color of bright spots indicated that CLS turned into SN-38 via hydrolysis inside a cell. It was found that most of the SN-38 were localized in small regions in the cytoplasm even after the conversion from CLS, and only a small amount of SN-38 was dissolved and migrated into other cytoplasm regions and the nucleus. The massive size growth of cells was observed within several tens of hours after the treatment with CLS nanoparticles. Moreover, Raman images of cells using the cytochrome c band and the fluorescence images of cells stained with JC-1 showed that cellular uptake of CLS nanoparticles efficiently caused mitochondrial damage. These results show that the combination of Raman and autofluorescence imaging can provide insight into the intracellular behavior of prodrug nanoparticles and the cell response and facilitate the development of nano-DDSs.

Multiple deuteration of triphenylphosphine and live-cell Raman imaging of deuterium-incorporated Mito-Q.

All aromatic C-H bonds of triphenylphosphine (PPh3) were efficiently replaced by C-D bonds using Ru/C and Ir/C co-catalysts in 2-PrOH and D2O, an inexpensive deuterium source. Furthermore, non-radioactive and safe deuterium-incorporated Mito-Q (drug candidate) was prepared from deuterated PPh3 and used for the live-cell Raman imaging to evaluate the mitochondrial uptake.

Ratiometric analysis of reversible thia-Michael reactions using nitrile-tagged molecules by Raman microscopy.

Ratiometric Raman analysis of reversible thia-Michael reactions was achieved using α-cyanoacrylic acid (αCNA) derivatives. Among αCNAs, the smallest derivative, ThioRas (molecular weight: 167 g mol-1), and its glutathione adduct were simultaneously detected in various subcellular locations using Raman microscopy.

Concentration Quantification of the Low-Complexity Domain of Fused in Sarcoma inside a Single Droplet and Effects of Solution Parameters.

Liquid-liquid phase separation (LLPS) is an important phenomenon in biology, and it is desirable to develop quantitative methods to analyze protein droplets generated by LLPS. This study quantified the change in protein concentration in a droplet in label-free and single-droplet conditions using Raman imaging and the Raman band of water as an intensity standard. Small changes in the protein concentration with variations in pH and salt concentration were observed, and it was shown that the concentration in the droplet decreases as the conditions become less favorable for droplet formation. The effect of exposure to 1,6-hexanediol was also examined, and this additive was found to decrease the protein concentration in the droplet. A model can be proposed in which the addition of 1,6-hexanediol reduces the protein concentration in the droplet, and the droplet disappears when the concentration falls below a certain threshold value.

Regulation of Cell Volume by Nanosecond Pulsed Electric Fields.

Stimulation of cells by nanosecond pulsed electric fields (nsPEFs) has attracted attention as a technology for medical applications such as cancer treatment. nsPEFs have been shown to affect intracellular environments without significant damage to cell membranes; however, the mechanism underlying the effect of nsPEFs on cells remains unclear. In this study, we constructed electrodes for applying nsPEFs and analyzed the change in volume of a single cell due to nsPEFs using fluorescence and Raman microscopy. It was shown that the direction of the change depended on the applied electric field; expansion due to the influx of water was observed at high electric field, and cell shrinkage was observed at low electric field. The change in cell volume was correlated to the change in the intracellular Ca2+ concentration, and nsPEFs-induced shrinking was not observed when the Ca2+-free medium was used. This result suggests that the cell shrinkage is related to the regulatory volume decrease where the cell adjusts the increase in intracellular Ca2+ concentration, inducing the efflux of ions and water from the cell.

Observation of liquid-liquid phase separation of ataxin-3 and quantitative evaluation of its concentration in a single droplet using Raman microscopy.

Liquid-liquid phase separation (LLPS) plays an important role in a variety of biological processes and is also associated with protein aggregation in neurodegenerative diseases. Quantification of LLPS is necessary to elucidate the mechanism of LLPS and the subsequent aggregation process. In this study, we showed that ataxin-3, which is associated with Machado-Joseph disease, exhibits LLPS in an intracellular crowding environment mimicked by biopolymers, and proposed that a single droplet formed in LLPS can be quantified using Raman microscopy in a label-free manner. We succeeded in evaluating the protein concentration and identifying the components present inside and outside a droplet using the O-H stretching band of water as an internal intensity standard. Only water and protein were detected to be present inside droplets with crowding agents remaining outside. The protein concentration in a droplet was dependent on the crowding environment, indicating that the protein concentration and intracellular environment should be considered when investigating LLPS. Raman microscopy has the potential to become a powerful technique for clarifying the chemical nature of LLPS and its relationship with protein aggregation.

Photo-controlled phase separation and mixing of a mixture of water and 2-butoxyethanol caused by photochromic isomerisation of spiropyran.

Photo induced phase separation of a mixture of water and 2-butoxyethanol, in which spiropyran was dissolved as a photoresponsive molecule, was investigated. It was found that the phase separation temperature of the merocyanine (MC) form solution was higher than that of the spiropyrane (SP) form; therefore phase separation was induced by visible light irradiation to the solution which caused the photoisomerization from the MC form to the SP form. The system also exhibits reversible photoinduced phase mixing by irradiation of UV light. The photo-chemical phase separation was also induced by the nanosecond laser pulse irradiation and the dynamics of the phase domain growth were studied.

Time-resolved brewster angle microscopy for photochemical and photothermal studies on thin-films and monolayers.

Transient events in thin films and interfaces have been studied using the technique of time resolved pump-probe nanosecond Brewster angle microscopy. For p-polarized light there is a minimum reflectivity at the Brewster angle. When the interface is viewed with light that is both incident and reflected at the Brewster angle the resulting image is dark. Subsequent small changes is refractive index will then cause an increase in the reflectivity in affected regions providing high contrast images of an altered interface with a dark background level. This is the basis of Brewster angle microscopy. In the present work two synchronized nanosecond pulsed lasers were used in the pump-probe configuration in order to induce changes at an air-liquid interface and to monitor the resulting morphology changes over a range of time delays from nanosecond to milliseconds after laser-excitation. This method can be used to observe morphological changes in phase altering thin-films and molecular monolayers. Further it can be used to obtain information about transient photochemistry even in optically thin materials and nano-films. In the current work the method is used to monitor laser induced processes in phase separating binary liquid mixtures as well as in monolayers of photo-responsive amphiphilic molecules derived from spiropyran on water. The two systems are quite different but provide valuable comparisons.

Real-time observation of X-ray-induced intramolecular and interatomic electronic decay in CH2I2.

The increasing availability of X-ray free-electron lasers (XFELs) has catalyzed the development of single-object structural determination and of structural dynamics tracking in real-time. Disentangling the molecular-level reactions triggered by the interaction with an XFEL pulse is a fundamental step towards developing such applications. Here we report real-time observations of XFEL-induced electronic decay via short-lived transient electronic states in the diiodomethane molecule, using a femtosecond near-infrared probe laser. We determine the lifetimes of the transient states populated during the XFEL-induced Auger cascades and find that multiply charged iodine ions are issued from short-lived (∼20 fs) transient states, whereas the singly charged ones originate from significantly longer-lived states (∼100 fs). We identify the mechanisms behind these different time scales: contrary to the short-lived transient states which relax by molecular Auger decay, the long-lived ones decay by an interatomic Coulombic decay between two iodine atoms, during the molecular fragmentation.

Embedding a Metal-Binding Motif for Copper Transporter into a Lipid Bilayer by Cu(I) Binding.

Peptide-lipid interactions are widely involved with biologically significant phenomena, including the pathogenic mechanisms of protein misfolding diseases and transmembrane protein folding. In this paper, the interaction of the cysteine/tryptophan (Cys/Trp) motif, which is a metal-binding motif of copper transporter (Ctr) proteins, with a lipid bilayer was studied using fluorescence and circular dichroism (CD) spectroscopy. The binding of Cu(I) to the Cys/Trp motif induced a large red-edge excitation shift in the Trp fluorescence, indicating that the Trp residue is located inside the lipid bilayer following complexation of Cu(I) with the Cys/Trp motif. The Stern-Volmer quenching of the Trp fluorescence also supported the Cu(I) binding peptide embedding in the lipid bilayer. The measurement of the CD spectra indicated the increase in ß-sheet content of the Cys/Trp motif peptide as a result of Cu(I) binding. These results lead to the conclusion that complexation with Cu(I) induces the change in the secondary structure of the Cys/Trp motif, which results in the peptide embedding in the lipid bilayer. Cu(I)-induced enhancement of the lipid affinity is discussed in terms of the mechanism for copper transport by Ctr.

Time-Resolved Structured Illumination Microscopy for Phase Separation Dynamics of Water and 2-Butoxyethanol Mixtures: Interpretation of "Early Stage" Involving Micelle-Like Structures.

Phase separation dynamics of a water/2-butoxyethanol (2BE) mixture was studied with newly developed time-resolved structured illumination microscopy (SIM). Interestingly, an employed hydrophobic fluorescent probe for SIM showed spectral shifts up to 500 ns after a laser-induced temperature jump, which suggests 2BE micellar-like aggregates become more hydrophobic at the initial stage of phase separation. This hydrophobic environment in 2BE aggregates, probably due to the ejection of water molecules, continued up to at least 10 µs. Time-resolved SIM and previously reported light scattering data clearly showed that the size of a periodic structure remained constant (ca. 300 nm) from 3 to 10 µs, and then the growth of periodic structures having the self-similarity started. We think that the former and the latter processes correspond to "early stage" (concentration growth) and "late stage" (size growth), respectively, in phase separation dynamics. Here we suggest that, in the early stage, the entity to bear 2BE phase be water-poor 2BE aggregates, and the number density of these aggregates would simply increase in time.