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.

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.

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.

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.

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.

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.

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.

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.

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.

Experimental Evaluation of the Density of Water in a Cell by Raman Microscopy.

We report direct observation of a spatial distribution of water molecules inside of a living cell using Raman images of the O-H stretching band of water. The O-H Raman intensity of the nucleus was higher than that of the cytoplasm, indicating that the water density is higher in the nucleus than that in the cytoplasm. The shape of the O-H stretching band of the nucleus differed from that of the cytoplasm but was similar to that of the balanced salt solution surrounding cells, indicating less crowded environments in the nucleus. The concentration of biomolecules having C-H bonds was also estimated to be lower in the nucleus than that in the cytoplasm. These results indicate that the nucleus is less crowded with biomolecules than the cytoplasm.

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.

Conformational changes in inhibitory PAS domain protein associated with binding of HIF-1α and Bcl-xL in living cells.

Inhibitory PAS domain protein (IPAS) is a dual function protein acting as a transcriptional repressor and as a pro-apoptotic protein. Simultaneous dual-color single-molecule imaging of EGFP-IPAS coexpressed with Mit-TagRFP-T in living HeLa cells revealed that fraction of EGFP-IPAS was arrested in the nucleus and on mitochondria. Transiently expressed Cerulean-IPAS in HEK293T cells was present in nuclear speckles when coexpressed with Citrine-HIF-1α or Citrine-HLF. Fluorescence lifetime imaging microscopy (FLIM) analysis of Citrine-IPAS-Cerulean in living CHO-K1 cells clarified the presence of intramolecular FRET. Reduced lifetimes of the donor were partially restored by coexpression of HIF-1α or Bcl-xL, binding proteins of IPAS in the nucleus and mitochondria, respectively. This alteration in lifetimes demonstrates that conformational changes occurred in IPAS by their binding.

Phase behavior of a binary fluid mixture of quadrupolar molecules.

We propose a model molecule to investigate microscopic properties of a binary mixture with a closed-loop coexistence region. The molecule is comprised of a Lennard-Jones particle and a uniaxial quadrupole. Gibbs ensemble Monte Carlo simulations demonstrate that the high-density binary fluid of the molecules with the quadrupoles of the same magnitude but of the opposite signs can show closed-loop immiscibility. We find that an increase in the magnitude of the quadrupoles causes a shrinkage of the coexistence region. Molecular dynamics simulations also reveal that aggregates with two types of molecules arranged alternatively are formed in the stable one-phase region both above and below the coexistence region. String structures are dominant below the lower critical solution temperature, while branched aggregates are observed above the upper critical solution temperature. We conclude that the anisotropic interaction between the quadrupoles of the opposite signs plays a crucial role in controlling these properties of the phase behavior.

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.

Nano-scale characterization of binary self-assembled monolayers under an ambient condition with STM and TERS.

Gold surfaces were modified by benzyl-mercaptan (BM) and then partly replaced with benzenethiol (BT), which formed binary self-assembled monolayers (SAM). Initially BT randomly replaced BM in the monolayer, but at long exchange times >15 nm radius domains were observed with specific relative composition of BT and BM.

A silver nanowire-based tip suitable for STM tip-enhanced Raman scattering.

A chemically synthesized silver nanowire was used for atomic-resolution STM imaging and tip-enhanced Raman scattering (TERS) spectroscopy, yielding excellent reproducibility. This TERS tip will open a new venue to surface analysis, such as molecular finger printing at nanoscales.