Intracellular Fe 2+ plays a crucial role in ferroptosis and other redox-regulated biological processes. However, the direct visualization of its dynamic behavior remains difficult. Herein, we report a resonance Raman-based strategy for imaging the intracellular Fe 2+ via the formation of Fe 2+ –phenanthroline complexes. Initial studies using phenanthroline and bipyridine revealed that Fe 2+ coordination selectively generates resonance Raman signals, but the signal intensity was insufficient for robust intracellular imaging. We developed a series of alkyne-functionalized and deuterated phenanthroline derivatives to improve sensitivity and spectral selectivity. Although resonance enhancement of the alkyne vibrational mode was limited, Fe 2+ coordination of 4,7-diethynyl-1,10-phenanthroline substantially increased visible absorption and aromatic ring-derived resonance Raman intensity. This was attributed to the extension of the conjugated system. This complexation-induced enhancement facilitated sensitive Fe 2+ visualization in living HeLa cells and enabled time-lapse monitoring of Fe 2+ accumulation on the minute timescale. Thus, ligand structural modification can tune the electronic absorption and resonance Raman response of Fe 2+ complexes in living cells. Complexation-induced resonance Raman enhancement provides a versatile platform for visualizing intracellular metal-ion dynamics while preserving the label-free molecular information from spontaneous Raman spectroscopy.

Photoinducible proximity labeling (PL) using photocatalysts offers high spatiotemporal resolution for profiling protein–protein interactions (PPIs). Conventional approaches require genetic installation of a tag to tether the photocatalyst to a protein of interest (POI), which may perturb native protein function and cause artificial labeling. Ligand-directed delivery of photocatalysts to native POIs has emerged as a tag-free alternative; however, this strategy suffers from nonspecific labeling by unbound catalysts and an inherent washing dilemma, where insufficient washing leads to background labeling while excessive washing disrupts ligand binding. These limitations have severely restricted tagfree, ligand-directed PPI profiling. Herein, we report a switchable photoinducible PL strategy based on a nitrobenzoxadiazole (NBD) unit that becomes catalytically active only after covalent conjugation to a POI. This approach consists of a two-step process: introduction of an N -NBD moiety into the POI using a catalytically inactive O -NBD unit, followed by light-induced PL catalyzed by N -NBD. Model experiments identified N -methyl luminol as a selective labeling reagent activated by N -NBD. Using a biotin-conjugated O -NBD unit as a catalytically inactive precursor, biotinconjugated protein A was selectively labeled as a PPI partner of streptavidin. This switchable PL method circumvents the washing dilemma, suppresses nonspecific labeling, and enables high-resolution, tag-free proteomic analyses.

We propose a microfluidic system that enables quantitative nano-artifacts exposure to single cells. Unlike a conventional cell analysis based on pipetting, our system allows precise control of the desired concentration and the time periods, particularly the start timing of the exposure, by encapsulating suspensions containing nano-artifacts and single cells. In this presentation, we evaluate the basic performance of the constructed system. Moreover, we demonstrate an unprecedented cell analysis application: “monitoring of cell death using engineered cells”. The proposed method would open new paradigms for analyses of individual cellular responses.

An alkyne is an unsaturated hydrocarbon characterized by the presence of at least one C≡C bond. Alkyne groups generate a strong Raman peak in the cellular-silent region, a region between 1,800 and 2,800 cm−1 where endogenous molecules do not produce a Raman signal. As a result, alkynes are regularly used as tags to label and visualize small molecules in live cells using Raman microscopy, a method referred to as alkyne-tag Raman imaging (ATRI). ATRI has been applied to various compounds to enable their cellular localization and, recently, alkyne-tagged compounds have been used as Raman sensors to detect intracellular biomolecules, such as metal ions or reactive oxygen species. ATRI has unique advantages over existing methods for localizing small molecules intracellularly, such as enabling super-multiplex detection and incurring a smaller impact on labelled compounds. In this Primer, we describe the principles and key techniques involved in ATRI, including the design of alkyne-tagged molecules, sample preparation and the set-up of Raman microscopes. We showcase the demonstration and application of ATRI, encompassing the development of responsive alkyne-tagged compounds for sensing biomolecules. Finally, we discuss the limitations and potential applications of ATRI, shedding light on the future possibilities of this method. Carbon–carbon triple bonds exhibit a distinct Raman response in the region of 1,800–2,800 cm−1, known as the cellularly silent region. This unique chemical signature, coupled with the small size of alkyne moieties, presents these tags as useful imaging alternatives to bulky fluorescent probes. This Primer discusses the various Raman scattering processes used to image alkyne tags in cells, including the optical set-up required, how to choose an alkyne tag and imaging results from different cellular environments.

Reversible histone acylation is crucial for epigenetic gene expression regulation. Histone acylation is typically mediated by lysine acyltransferases (KATs), which use acyl-CoAs as acyl donors. Here, we revealed the novel role of the histone deacetylases HDAC1 and HDAC2 in histone acylation catalysis. Notably, we show that HDAC1 and HDAC2 directly catalyze sorbylation using sorbic acid, a common food preservative, in addition to facilitating desorbylation. This newly discovered HDAC1/2-driven histone sorbylation function is a distinctive active epigenetic mark that leads to widespread changes in the expression of genes, particularly those involved in cholesterol biosynthesis. Our findings reveal that HDAC1/2 are unique enzymes capable of catalyzing not only the removal but also formation of histone modifications in response to exogenous carboxylic acids such as sorbic acid and benzoic acid. Our results highlight the impact of carboxylic acids found in the environment, such as food additives, on gene expression changes that occur via histone lysine modification regulated by HDAC1 and HDAC2.

Senescent cells, characterized by irreversible cell cycle arrest and inflammatory factor secretion, promote various age-related pathologies. Senescent cells exhibit resistance to ferroptosis, a form of iron-dependent cell death; however, the underlying mechanisms remain unclear. Here, we discovered that lysosomal acidity was crucial for lipid peroxidation and ferroptosis induction by cystine deprivation. In senescent cells, lysosomal alkalinization causes the aberrant retention of ferrous iron in lysosomes, resulting in resistance to ferroptosis. Treatment with the V-ATPase activator EN6 restored lysosomal acidity and ferroptosis sensitivity in senescent cells. A similar ferroptosis resistance mechanism involving lysosomal alkalinization was observed in pancreatic cancer cell lines. EN6 treatment prevented pancreatic cancer development in xenograft and Kras mutant mouse models. Our findings reveal a link between lysosomal dysfunction and the regulation of ferroptosis, suggesting a therapeutic strategy for the treatment of age-related diseases.

We present an on-chip live-cell imaging system for monitoring the cellular responses triggered by molecular stimulation. This system utilizes a non-stationary process performed in microchannels with twin chambers to compare cell types (control and target). First, the gas-liquid interfacial interaction transports the cells to the back of the chamber. Because the streamline barely reaches this area in a steady flow, we can maintain the cells in the observation field even if the solution is replaced. Here, the cells were placed near the thin wall dividing the two chambers, thereby maintaining two cell groups in one microscopic view and completely separating the culture environment from each other. Secondly, molecular diffusion delivers the stimulant to the cells, even in areas not reached by the streamline. In this study, we tested these manipulations using a non-stationary process and achieved the transport of cells and stimulants. In particular, we monitored HL-60 cells engineered to undergo apoptosis in response to a molecular trigger as the programmed cell death. We found that statistical changes in the morphology of engineered cells compared to wild-type cells appear within two hours. Our system not only facilitates live cell imaging for the triggered responses but also contributes to the actuate evaluation of the time scale of cellular robotics.

Raman microscopy is an emerging molecular imaging technology, yet its signal-to-noise ratio (SNR) in measurements of biological specimens is severely limited because of the small cross section of Raman scattering. Here, we present Raman imaging techniques of cryofixed specimens to overcome SNR limitations by enabling long exposure of specimens under highly stabilized low-temperature conditions. The observation of frozen specimens in a cryostat at a constant low temperature immediately after rapid freezing enabled the improvement of SNR and enhanced the spatial and spectral resolution. We also confirmed that the cryofixation can preserve physicochemical states of specimens by observing alkyne-labeled coenzyme Q in cytosol and hemeproteins in acute ischemic myocardium, which cannot be done by fixation using chemical reagents. Last, we applied the technique for multiplex Raman imaging of label-free endogenous molecules and alkyne-tagged molecules in cryofixed HeLa cells, demonstrating its capability of high-content imaging of complex biological phenomena while maintaining physiological conditions.

Raman microscopy enables us to obtain molecular information in biological samples but has suffered from low signal-to-noise ratio (SNR) due to low of Raman scattering cross-section. Here we developed a cryo-stat equipped Raman microscope for low temperature measurement, allowing long time accumulation of Raman signals. We confirmed the SNR improvement in Raman imaging of cryofixed HeLa cells without photodamage under long time observation at low temperature. The reduction of photobleaching in resonant Raman scattering of carotenoid and cytochrome significantly increases the SNR, demonstrated by 7-color high SNR Raman imaging with multiple Raman tags, including EdU, MitoBADY, and alkyne-tagged Coenzyme Q (AltQ2). AltQ2 is a mobile small molecule that cannot be fixed by chemical fixation.

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.

AbstractFlow cytometry is an indispensable tool in biology and medicine for counting and analyzing cells in large heterogeneous populations. It identifies multiple characteristics of every single cell, typically via fluorescent probes that specifically bind to target molecules on the cell surface or within the cell. However, flow cytometry has a critical limitation: the color barrier. The number of chemical traits that can be simultaneously resolved is typically limited to several due to the spectral overlap between fluorescence signals from different fluorescent probes. Here, we present color-scalable flow cytometry based on coherent Raman flow cytometry with Raman tags to break the color barrier. This is made possible by combining a broadband Fourier-transform coherent anti-Stokes Raman scattering (FT-CARS) flow cytometer, resonance-enhanced cyanine-based Raman tags, and Raman-active dots (Rdots). Specifically, we synthesized 20 cyanine-based Raman tags whose Raman spectra are linearly independent in the fingerprint region (400 to 1,600 cm−1). For highly sensitive detection, we produced Rdots composed of 12 different Raman tags in polymer nanoparticles whose detection limit was as low as 12 nM for a short FT-CARS signal integration time of 420 µs. We performed multiplex flow cytometry of MCF-7 breast cancer cells stained by 12 different Rdots with a high classification accuracy of 98%. Moreover, we demonstrated a large-scale time-course analysis of endocytosis via the multiplex Raman flow cytometer. Our method can theoretically achieve flow cytometry of live cells with >140 colors based on a single excitation laser and a single detector without increasing instrument size, cost, or complexity.

TEAD transcription factors are responsible for the transcriptional output of Hippo signaling. TEAD activity is primarily regulated by phosphorylation of its coactivators, YAP and TAZ. In addition, cysteine palmitoylation has recently been shown to regulate TEAD activity. Here, we report lysine long-chain fatty acylation as a posttranslational modification of TEADs. Lysine fatty acylation occurs spontaneously via intramolecular transfer of acyl groups from the proximal acylated cysteine residue. Lysine fatty acylation, like cysteine palmitoylation, contributes to the transcriptional activity of TEADs by enhancing the interaction with YAP and TAZ, but it is more stable than cysteine acylation, suggesting that the lysine fatty-acylated TEAD acts as a "stable active form." Significantly, lysine fatty acylation of TEAD increased upon Hippo signaling activation despite a decrease in cysteine acylation. Our results provide insight into the role of fatty-acyl modifications in the regulation of TEAD activity.

Most Raman sensors are based on Raman tags linked to bulky aromatic molecules that affect the subcellular localization. Therefore, here, we developed a small ratiometric Raman sensor, ThioRas, to effectively detect thiols in live cells. ThioRas has a nitrile group that serves as a Raman tag for the thia-Michael reaction, and its nitrile signal shifts in the presence of an adjacent double bond. The molecular weight of ThioRas (167) was sufficiently small to allow ThioRas distribution throughout cells. ThioRas and its glutathione adduct were simultaneously detected in various subcellular locations, demonstrating its potential applicability as a Raman tag for ratiometric analysis.

Photocatalytic proximity labeling has recently undergone significant advances as a valuable tool for understanding protein–protein and cell–cell interactions. This paper reports the first photocatalytic protein-labeling approach in which the reaction can be controlled using near-infrared (NIR) light (810 nm). Magnetic affinity beads with encapsulated sulfur-substituted silicon (IV) phthalocyanine, which produces singlet oxygen upon NIR irradiation, were prepared. We have developed a method in which the histidine residues of proteins bound to the ligands on the beads are selectively oxidized and labeled by the nucleophilic labeling reagent while minimizing nonspecific adsorption to the dye. Beads with aryl sulfamide, lactose, or CZC-8004 ligands immobilized on their surface can be used to label proteins that bind these ligands, as well as their protein–protein interaction partners.

Palladium enolates derived from β-ketocarbonyl compounds serve as key intermediates in various catalytic asymmetric reactions. We found that the palladium enolate formed from β-ketoamide is stable in air and moisture and we applied this property to develop a peptide purification system using β-ketoamide as a small affinity tag in aqueous media. A solid-supported palladium complex successfully captured β-ketoamide-tagged molecules as palladium enolates and released them in high yield upon acid treatment. Optimum conditions for the catch and release of tagged peptides from a mixture of untagged peptides were established. To demonstrate the value of this methodology in identifying the binding site of a ligand to its target protein, we purified and identified a peptide containing the ligand-binding site from the tryptic digest of cathepsin B labelled with a covalent cathepsin B inhibitor containing a β-ketoamide tag.

Raman microscopy has been used to deduce information about the distributions of endogenous biomolecules without exogenous labeling. Several functional groups, such as alkynes (CC), nitriles (CN), and carbon-deuterium (C–D) bonds, have been employed in recent years as Raman tags to detect target molecules in cells. In this article, we review some recent advances in applications using deuterated fatty acids for lipid analysis, such as investigation of tumor-selective cytotoxicity of γ-linolenic acid (GLA), simultaneous two-color imaging of stearate and oleate using deuterated and protonated alkynes, Raman hyperspectral imaging, and analyses of the physical properties of lipids through spectral unmixing of the C–D vibrational frequencies. In addition, we review some advanced methods for observing intracellular metabolic activities, such as de novo lipogenesis from deuterium-labeled precursors.

Visible light, particularly in the blue region of the spectrum, can cause cell dysfunction through the generation of singlet oxygen, contributing to cellular aging and age-related pathologies. Although photooxidation of nucleic acids, lipids, and amino acids has been extensively studied, the magnitude and span of blue-light-induced protein damages within proteome remain largely unknown. Herein we present a chemoproteomic approach to mapping blue-light-damaged proteins in live mammalian cells by exploiting a nucleophilic alkyne chemical probe. A gene ontology enrichment analysis revealed that cell surface proteins are more readily oxidized than other susceptible sets of proteins, including mitochondrial proteins. In particular, the integrin family of cell surface receptors (ITGs) was highly ranked in the mammalian cells tested, including human corneal endothelial cells. The blue-light-oxidized ITGB1 protein was functionally inactive in promoting cell adhesion and proliferation, suggesting that the photodamage of integrins contributes to the blue-light-induced cell dysfunction. Further application of our method to various cells and tissues should lead to a comprehensive analysis of light-sensitive proteins.

In chemical biology research, various fluorescent probes have been developed and used to visualize target proteins or molecules in living cells and tissues, yet there are limitations to this technology, such as the limited number of colors that can be detected simultaneously. Recently, Raman spectroscopy has been applied in chemical biology to overcome such limitations. Raman spectroscopy detects the molecular vibrations reflecting the structures and chemical conditions of molecules in a sample and was originally used to directly visualize the chemical responses of endogenous molecules. However, our initial research to develop "Raman tags" opens a new avenue for the application of Raman spectroscopy in chemical biology. In this Perspective, we first introduce the label-free Raman imaging of biomolecules, illustrating the biological applications of Raman spectroscopy. Next, we highlight the application of Raman imaging of small molecules using Raman tags for chemical biology research. Finally, we discuss the development and potential of Raman probes, which represent the next-generation probes in chemical biology.

Histone lysine methylation is an epigenetic mark that can control gene expression. In particular, H3K9me3 contributes to transcriptional repression by regulating chromatin structure. Successful mitotic progression requires correct timing of chromatin structure changes, including epigenetic marks. However, spatiotemporal information on histone modifications in living cells remains limited. In this study, we created an FRET-based probe for live-cell imaging based on the HP1α chromodomain (HP1αCD), which binds to H3K9me3. The probe was incorporated into chromatin and the emission ratio decreased after treatment with histone methyltransferase inhibitors, indicating that it successfully traced dynamic changes in H3K9me3. Upon entry into mitosis, the probe's emission ratio transiently increased with a concomitant increase in H3K9me3, then exhibited a stepwise decrease, probably due to loss of HP1αCD binding caused by phosphorylation of H3S10 and demethylation of H3K9me3. This probe will be a useful tool for detecting dynamic changes in chromatin structure associated with HP1α.