Proteins function not only through intramolecular folding and intermolecular complex formation but also through phase transitions driven by intermolecular interactions. Such phases, including liquid-like condensates, amorphous aggregates (AAs), and amyloid fibrils, are linked to distinct biological functions and pathologies. Although the transition from liquid-like condensates to amyloids has been extensively studied, the kinetic relationships between amyloids and other metastable solid states under cell-sized confinement remain unclear, which may hinder the establishment of effective therapeutic strategies. This knowledge gap arises because bulk-scale experiments inevitably lead to the conversion of metastable phases into the most stable phase. We developed a droplet-based microfluidic system that quantifies amyloid nucleation and metastable AA formation. Using the yeast prion protein Sup35, we found that condensates convert into both amyloids and AAs and that AA formation imposes a kinetic barrier that suppresses amyloid formation in a size-dependent manner at the micrometer scale, highlighting the importance of size effects in condensate-to-amyloid transitions. Furthermore, we demonstrated that the well-known amyloid inhibitor (-)-epigallocatechin-3-gallate paradoxically promoted amyloid formation at low concentrations by modulating the AA and amyloid nucleation kinetics. This phenomenon cannot, in principle, be observed in bulk-scale experiments and became apparent only under micrometer-scale confinement in the present system. These findings provide fundamental insights into protein phase transitions in cellular environments and may guide the development of novel therapeutic strategies targeting the metastable aggregates of amyloidogenic proteins.

Physical stimuli such as electrical and thermal cues have emerged as promising tools for regulating various biological phenomena. Here, we show that nanosecond pulsed electric fields (nsPEFs) can efficiently and noninvasively induce and enhance neuronal differentiation in PC-12 cells. The application of nsPEFs at 4.5 kV/cm with a 50 ns pulse width markedly increased the differentiation rate in the absence of nerve growth factor (NGF) and promoted neurite elongation, both in the absence and presence of NGF, demonstrating a synergistic effect between electrical and biochemical signalling. In situ Raman microscopy revealed an increase in the C-H stretching band intensity in PC-12 cells following exposure to nsPEFs, indicating increased intracellular biomolecular density associated with transient cell shrinkage, with cell volume reduced to 91%. Temperature analysis based on the O-H stretching Raman band of water further showed that the nsPEFs application induced an intracellular temperature rise of approximately 1 degrees C. Such biophysical alterations are likely to promote neuronal differentiation.

Liquid-liquid phase separation (LLPS) generates high-concentration biomolecular liquid droplets and contributes to diverse intracellular processes. Intracellular droplets contain not only proteins that drive LLPS but also various coexisting components and abundant hydration water, and their properties depend strongly on both the constituent components and the surrounding intracellular environments. However, the quantitative understanding of these factors within living cells remains limited. Raman microscopy is well suited for this challenge, enabling label-free, direct analysis of structures and concentrations of constituent molecules and hydration water in droplets, while also providing information on intracellular molecular crowding environments. In this review, we summarize recent Raman microscopic studies on intracellular droplets, focusing on coexisting components, structural characterization, hydration water, and their relationships with molecular crowding.

Cellular senescence is accompanied by complex changes in nuclear mechanics. Here, integrated Raman, Brillouin, and refractive index imaging quantitatively revealed that senescent nuclei exhibit an increased high-frequency viscoelastic response, despite the increase in nuclear water content and volume and the decrease in biomolecular density. This discrepancy indicates that the nuclear mechanical alteration in senescent cells is not simply due to molecular condensation, as discussed in the context of chromatin reorganization.

Chromatin is a dynamic, higher-order structure that compacts genomic DNA within the nucleus and regulates genome organization and accessibility. Chromatin is broadly classified into transcriptionally inert heterochromatin and active euchromatin based on their condensation states; however, the molecular basis underlying this condensation difference remains unresolved. In this study, we show that phosphatidylcholine-type lipid molecules are associated with heterochromatin in living cells and contribute to its physical condensation. Using label-free Raman-Brillouin microscopy, we simultaneously mapped molecular concentrations and high-frequency viscoelastic properties in a living cell at subcellular resolution. Raman imaging revealed that lipid molecules are selectively enriched in heterochromatin, and their local concentrations are correlated with the mechanical response of heterochromatin. These lipids were absent from mitotic chromosomes and reappeared in DNA-rich regions during the early G1 phase, indicating that lipid incorporation is a regulated step in the formation of heterochromatin. These results expand the molecular view of chromatin and demonstrate how simultaneous Raman-Brillouin imaging can link chemical constituents with the mechanical properties of a cell.

In this study, stable tetrazene radical cation salts were synthesized and characterized for the first time. The radical cation derived from 1,2-di(2-azaadamantan-2-yl)diazene (DAD) was isolated as an air-stable solid, retaining its integrity for at least 120 days at ambient temperature (∼25 °C) and pressure. X-ray crystallography and electron spin-resonance spectroscopy revealed the delocalization of the unpaired electron over the tetrazene core and into the adamantane framework. DAD undergoes two well-separated, reversible redox processes and displays high catalytic activity for alcohol oxidation under mild conditions. Systematic structural modifications identified the key framework features governing the radical cation stability and catalytic performance.

We present the results of Raman imaging of molecular crowding environments in a living cell and a liquid droplet formed by liquid-liquid phase separation. Using the Raman band of water as an internal intensity standard, we propose an in situ quantification method for evaluating biomolecular concentrations. Based on these concentration measurements, intracellular crowding environments can be quantitatively assessed. A single liquid droplet in a buffer solution is found to have a very high biomolecular concentration, exceeding a few millimolar concentrations, and the concentration within a droplet varies depending on the surrounding environment. The biomolecular concentration in a droplet increases as conditions facilitate droplet formation, and we propose a model in which changes in the surrounding environment lead to the formation of dense droplets, which subsequently transform into aggregates. Concentration quantification in a single droplet was also performed in a living cell, demonstrating that the biomolecular concentration in a droplet is not much different from that in the surrounding intracellular environment. This result indicates that within a droplet in a cell, certain molecules are highly concentrated, while other molecules are excluded, and the overall concentration is comparable to that of the surrounding intracellular environment. The droplet formation within a cell can be regarded as a redistribution of biomolecules constituting a molecular crowding environment.

Liquid-liquid phase separation (LLPS) is a phenomenon where homogeneous solutions of biomacromolecules separate into two liquid phases and generate liquid droplets enriched in specific biomolecules. LLPS of neurodegeneration-related proteins, including fused in sarcoma (FUS), promotes their aggregation, causing fatal diseases such as amyotrophic lateral sclerosis (ALS). Recent studies showed that RNAs regulate LLPS of these proteins and inhibit their aggregation, which may play an important role in preventing the disease onset; however, the underlying molecular mechanisms remain elusive. It is also unknown whether endogenous RNAs regulate LLPS and subsequent aggregation in cells. In this study, we investigated features of RNAs that enable their entrance into FUS droplets and inhibition of FUS aggregation via droplets and clarified the underlying mechanisms using Raman microscopy. We found that RNA length is one of the primary factors governing both the aggregation-inhibition effect and the localization of RNAs in the droplets in buffer solutions. Short (<50-nt) RNAs were concentrated inside the droplets and inhibited the aggregation. Our quantification method using Raman microscopy revealed that the short RNAs are enriched in FUS droplets by binding to FUS proteins through electrostatic interactions. On the other hand, long (>1000-nt) RNAs were not concentrated and dissolved the droplets. Raman imaging of living cells revealed that intracellular FUS droplets are enriched with endogenous RNAs at levels comparable to in vitro droplets and exhibit high fluidity, confirming that endogenous RNAs play a crucial role in suppressing droplet-to-aggregate transition of FUS in cells. These findings indicate that short RNAs stabilize FUS droplets through heterotypic RNA-FUS interactions that compete with homotypic FUS-FUS direct contacts responsible for aggregation, whereas binding of long RNAs enhances FUS solubility and promotes droplet dissolution. Our study highlights the protective role of RNAs against pathogenic aggregation of neurodegeneration-related proteins via droplets.

Chromatin, a fundamental component of eukaryotic genomes, is categorized into euchromatin and heterochromatin, which play distinct roles in gene regulation. Although these two chromatin states are distinguished by their degree of condensation, quantitatively measuring the degree of chromatin condensation, as well as the physical properties of chromatin in living cells, remains challenging. In this study, label-free in situ quantitative imaging was performed using a Raman-Brillouin microscope to visualize the spatial distribution of molecular concentration and viscoelasticity within the nuclear environment of a living cell. A quantitative concentration distribution image of each intracellular biomolecule was obtained by combining Raman imaging with multivariate curve resolution analysis, using a water Raman band as an internal standard. Simultaneous Raman-Brillouin imaging enables the quantitative visualization of viscoelasticity within a cell. Using this approach, we found that, in addition to DNA, heterochromatin is enriched in lipids and that lipids play a critical role in heterochromatin formation, determining its mechanical properties. These findings provide new insights into the mechanism of heterochromatin formation and its chemical and physical properties, leading to a comprehensive understanding of gene regulation and nuclear organization. ### Competing Interest Statement The authors have declared no competing interest.

The endoplasmic reticulum (ER) plays crucial roles in maintaining protein quality control and regulating dynamic Ca2+ storage in eukaryotic cells. However, the proteostasis system involved in ER-mediated protein quality control has not been fully characterized. Here we show that Ca2+ triggers the condensation of PDIA6, an ER-resident disulfide isomerase and molecular chaperone, into quality control granules. In contrast to the condensation mechanism observed for proteins containing low-complexity domains, our results indicate that transient but specific electrostatic interactions occur between the first and the third folded thioredoxin-like domains of PDIA6. We further show that the PDIA6 condensates recruit proinsulin, thereby accelerating the oxidative proinsulin folding and suppressing the proinsulin aggregation inside quality control granules, essential for secretion of insulin.

We developed a label-free method for visualizing intracellular temperature distribution using Raman image analysis combined with deep learning. Analysis of the entire region of the O-H stretching band of water using deep learning achieves an accuracy of similar to 0.2 degrees C, allowing intracellular temperature mapping without any pretreatments such as fluorescent labeling. Experiments using photoexcitation of carbon nanotubes attached to living cells demonstrate that this method can obtain transient changes in the temperature distribution within a cell.

High-concentration PEG-based liquid–liquid phase separation concentrates biomolecules into droplets, enabling highly sensitive, label-free Raman analysis of enzymatic reactions and protein–small-molecule interactions in dilute, small-volume samples.

Accurate quantification of intracellular thiols in live cells remains challenging owing to the inherent properties of fluorescent probes, such as signal saturation, photobleaching, and dye aggregation, despite the development of numerous thiol-detecting probes. Here, we present (E)-2-cyano-3-isopropylacrylamide (iPrCAA) as a ratiometric Raman probe for quantifying and visualizing intracellular thiol concentrations. iPrCAA is the smallest thiol-detecting probe currently available for live-cell detection (molecular weight 138 g/mol). It exhibits high reactivity toward thiol-adduct formation and solubility in water, which enhance its sensitivity in relation to that of existing Raman probes. Thus, iPrCAA enables the real-time monitoring of thiol depletion in live cells, demonstrating that a thiol-free medium reduces thiol levels by approximately 3-fold. Through its ability to visualize endogenous molecules, iPrCAA revealed that the thiol distribution closely resembles that of proteins. This Raman probe provides a practical means for efficiently quantifying endogenous thiols and dynamically tracking thiol fluctuations under stress conditions.

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.

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 β-carotene was also introduced into cells using PMB but was not concentrated intracellularly, indicating that β-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.

Liquid droplets, formed by intracellular liquid-liquid phase separation (LLPS), are called membraneless organelles. They provide transient enzymatic reaction fields for maintaining cellular homeostasis, although they might transform into aggregates, leading to neurodegenerative diseases. To understand the nature of intracellular droplets, it is crucial to quantify the liquid droplets inside a living cell as well as to elucidate the underlying biological mechanism. In this study, we performed near-infrared fluorescence and Raman imaging to quantify chemical components inside stress granules (SGs) formed by LLPS in living cells. The Raman images reveal that the nucleic acid concentration inside the SGs was more than 20% higher than the surrounding cytoplasm, whereas the lipid concentration was lower. Quantitative Raman intensity analysis using a water Raman band as an internal standard enables in situ concentration determination of nucleic acids in the SGs and other organelles. The intensity of the biomolecular C-H bands relative to the water band indicates that the crowding environment inside the SGs depends on the stress type; under oxidative stress, the inside of the SGs was nearly identical to the outside, whereas it was sparser in hyperosmotic stressed cells, suggesting that the high concentrations of nucleic acids play a pivotal role in maintaining the environments inside the SGs. These results demonstrate that intracellular droplets are not always highly condensed.

The Raman spectra of droplets of the low-complexity domain of an RNA-binding protein (Pbp1 LC) were measured to investigate the mechanism of the disappearance of Pbp1 LC droplets in oxidative environments. Raman quantification analysis indicated that the Pbp1 LC concentration in the droplets decreased with the addition of hydrogen peroxide. It was directly confirmed from the methionine band that the methionine residues were oxidized by hydrogen peroxide. We quantitatively evaluated the relationship between the degree of the methionine oxidation and the Pbp1 LC concentration in the droplets and showed that the droplet dissipation is induced by the methionine oxidation.

Liquid-liquid phase separation (LLPS), which results in the formation of highly concentrated droplets of biomolecules, is involved in various physiological phenomena. We have performed a label-free quantification of the concentration of a G-quadruplex-forming RNA and an RGG peptide in a single droplet using Raman microscopy. The concentration ratio of the RNA to the peptide within the droplet was found to maintain even when the prepared concentration ratio of these two species was varied. This result indicates that electrostatic interactions between the RNA and the peptide induced the droplet formation. It was also shown that the RNA maintains its Gquadruplex structure inside the droplets.