Hiroyuki Yamakoshi

Raman and SERS microscopy for molecular imaging of live cells

Raman microscopy is a promising technology for visualizing the distribution of molecules in cells. A challenge for live-cell imaging using Raman microscopy has been long imaging times owing to the weak Raman signal. Here we present a protocol for constructing and using a Raman microscope equipped with both a slit-scanning excitation and detection system and a laser steering and nanoparticle-tracking system. Slit scanning allows Raman imaging with high temporal and spatial resolution, whereas the laser beam steering system enables dynamic surface-enhanced Raman imaging using gold nanoparticles. Both features enable mapping of the distributions of molecules in live cells and visualization of cellular transport pathways. Furthermore, its utility can be expanded to small-molecule imaging by using tiny Raman-active tags such as alkyne. For example, DNA synthesis in a cell can be visualized by detecting 5-ethynyl-2′-deoxyuridine (EdU), a deoxyuridine derivative with an alkyne moiety. We describe the optics, hardware and software to construct the Raman microscope, and discuss the conditions and parameters involved in live-cell imaging. The whole system can be built in ∼8 h.

Alkyne-Tag Raman Imaging for Visualization of Mobile Small Molecules in Live Cells

Alkyne has a unique Raman band that does not overlap with Raman scattering from any endogenous molecule in live cells. Here, we show that alkyne-tag Raman imaging (ATRI) is a promising approach for visualizing nonimmobilized small molecules in live cells. An examination of structure-Raman shift/intensity relationships revealed that alkynes conjugated to an aromatic ring and/or to a second alkyne (conjugated diynes) have strong Raman signals in the cellular silent region and can be excellent tags. Using these design guidelines, we synthesized and imaged a series of alkyne-tagged coenzyme Q (CoQ) analogues in live cells. Cellular concentrations of diyne-tagged CoQ analogues could be semiquantitatively estimated. Finally, simultaneous imaging of two small molecules, 5-ethynyl-2-deoxyuridine (EdU) and a CoQ analogue, with distinct Raman tags was demonstrated.

Imaging of EdU, an alkyne-tagged cell proliferation probe, by Raman microscopy.

Click-free imaging of the nuclear localization of an alkyne-tagged cell proliferation probe, EdU, in living cells was achieved for the first time by means of Raman microscopy. The alkyne tag shows an intense Raman band in a cellular Raman-silent region that is free of interference from endogenous molecules. This approach may eliminate the need for click reactions in the detection of alkyne-labeled molecules.

Sphingomyelin distribution in lipid rafts of artificial monolayer membranes visualized by Raman microscopy

Significance Phase separation in lipid rafts has been observed with fluorescently labeled lipids, but they are often excluded from the ordered domain because of the steric effect of the bulky fluorophore on lipid packing, making it difficult to analyze the interior of the raft domain. Here, we synthesized an analog of sphingomyelin tagged with a small Raman active diyne moiety, which provides high chemical selectivity without affecting the membrane properties. Raman microscopy successfully visualized, at single lipid-layer sensitivity, a heterogeneous spatial distribution of this probe within raft-like ordered domains, which was different from the generally accepted raft model. This approach provides both chemical selectivity and quantitative imaging capability and is useful for functional studies of lipid rafts. Sphingomyelin (SM) and cholesterol (chol)-rich domains in cell membranes, called lipid rafts, are thought to have important biological functions related to membrane signaling and protein trafficking. To visualize the distribution of SM in lipid rafts by means of Raman microscopy, we designed and synthesized an SM analog tagged with a Raman-active diyne moiety (diyne-SM). Diyne-SM showed a strong peak in a Raman silent region that is free of interference from intrinsic vibrational modes of lipids and did not appear to alter the properties of SM-containing monolayers. Therefore, we used Raman microscopy to directly visualize the distribution of diyne-SM in raft-mimicking domains formed in SM/dioleoylphosphatidylcholine/chol ternary monolayers. Raman images visualized a heterogeneous distribution of diyne-SM, which showed marked variation, even within a single ordered domain. Specifically, diyne-SM was enriched in the central area of raft domains compared with the peripheral area. These results seem incompatible with the generally accepted raft model, in which the raft and nonraft phases show a clear biphasic separation. One of the possible reasons is that gradual changes of SM concentration occur between SM-rich and -poor regions to minimize hydrophobic mismatch. We believe that our technique of hyperspectral Raman imaging of a single lipid monolayer opens the door to quantitative analysis of lipid membranes by providing both chemical information and spatial distribution with high (diffraction-limited) spatial resolution.

A sensitive and specific Raman probe based on bisarylbutadiyne for live cell imaging of mitochondria

We previously showed that bisarylbutadiyne (BADY), which has a conjugated diyne structure, exhibits an intense peak in the cellular Raman-silent region. Here, we synthesized a mitochondria-selective Raman probe by linking bisphenylbutadiyne with triphenylphosphonium, a well-known mitochondrial targeting moiety. This probe, named MitoBADY, has a Raman peak 27 times more intense than that of 5-ethynyl-2-deoxyuridine. Raman microscopy using submicromolar extracellular probe concentrations successfully visualized mitochondria in living cells. A full Raman spectrum is acquired at each pixel of the scanned sample, and we showed that simultaneous Raman imaging of MitoBADY and endogenous cellular biomolecules can be achieved in a single scan. MitoBADY should be useful for the study of mitochondrial dynamics.

Alkyne-Tag SERS Screening and Identification of Small-Molecule-Binding Sites in Protein

Identification of small-molecule-binding sites in protein is important for drug discovery and analysis of protein function. Modified amino-acid residue(s) can be identified by proteolytic cleavage followed by liquid chromatography-mass spectrometry (LC-MS), but this is often hindered by the complexity of the peptide mixtures. We have developed alkyne-tag Raman screening (ATRaS) for identifying binding sites. In ATRaS, small molecules are tagged with alkyne and form covalent bond with proteins. After proteolysis and HPLC, fractions containing the labeled peptides with alkyne tags are detected by means of surface-enhanced Raman scattering (SERS) using silver nanoparticles and sent to MS/MS to identify the binding site. The use of SERS realizes high sensitivity (detection limit: ∼100 femtomole) and reproducibility in the peptide screening. By using an automated ATRaS system, we successfully identified the inhibitor-binding site in cysteine protease cathepsin B, a potential drug target and prognostic marker for tumor metastasis. We further showed that the ATRaS system works for complex mixtures of trypsin-digested cell lysate. The ATRaS technology, which provides high molecular selectivity to LC-MS analysis, has potential to contribute in various research fields, such as drug discovery, proteomics, metabolomics and chemical biology.

Novel Raman-tagged sphingomyelin that closely mimics original raft-forming behavior

Three Raman probes of sphingomyelin (SM) were synthesized and evaluated for their applicability to imaging experiments. One probe containing a hydroxymethyl-1,3-butadiyne moiety in the polar head group showed strong scattering. The solid-state (2)H NMR spectra of this probe in oriented bilayer membrane revealed excellent compatibility with natural SM in phase behavior since the probe undergoes phase separation to form raft-like liquid ordered (Lo) domains in the raft-mimicking mixed bilayers.

Dual function of coronatine as a bacterial virulence factor against plants: possible COI1–JAZ-independent role

Coronatine (COR, 1) is a phytotoxin and structural mimic of the plant hormone (+)-7-iso-jasmonoyl-L-isoleucine (2). COR (1) functions as a ligand of the COI1–JAZ co-receptor, which is the exclusive receptor of 2. Recently, a new role for 1 as a plant virulence factor for Pseudomonas syringae has attracted the attention of plant scientists. Bacteria invade the plant apoplast through stomatal pores. The host plant then responds to the bacterial invasion by closing the stomatal pores (stomatal defense). COR (1) functions as a bacterial chemical weapon that secures the path of infection by reopening the closed stomata. The mechanism is thought to involve inhibition of abscisic acid-signaling through the COI1–JAZ pathway. Thus, 1 plays an important role in plant–microbe interactions by abrogating the plant immune response. In this study, we synthesized seven analogues of 1 with naturally occurring α-amino acids and assessed their effect on stomata in a model plant, Arabidopsis thaliana. Structure–activity relationship studies of the analogues coupled with genetic studies and in silico docking analyses with COI1–JAZ strongly suggested that stomatal reopening induced by 1 may not rely on the COI1–JAZ signaling pathway. Our results suggest that stomatal reopening is triggered by 1 in conjunction with the conventional COI1–JAZ mode of action.

Noncanonical Function of a Small-Molecular Virulence Factor Coronatine against Plant Immunity: An In Vivo Raman Imaging Approach

Coronatine (1), a small-molecular virulence factor produced by plant-pathogenic bacteria, promotes bacterial infection by inducing the opening of stomatal pores, the major route of bacterial entry into the plant, via the jasmonate-mediated COI1-JAZ signaling pathway. However, this pathway is also important for multiple plant functions, including defense against wounding by herbivorous insects. Thus, suppression of the COI1-JAZ signaling pathway to block bacterial infection would concomitantly impair plant defense against herbivorous wounding. Here, we report additional, COI1-JAZ-independent, action of 1 in Arabidopsis thaliana guard cells. First, we found that a stereoisomer of 1 regulates the movement of Arabidopsis guard cells without affecting COI1-JAZ signaling. Second, we found using alkyne-tagged Raman imaging (ATRI) that 1 is localized to the endoplasmic reticulum (ER) of living guard cells of Arabidopsis. The use of arc6 mutant lacking chloroplast formation was pivotal to circumvent the issue of autofluorescence during ATRI. These findings indicate that 1 has an ER-related action on Arabidopsis stomata that bypasses the COI1-JAZ signaling module. It may be possible to suppress the action of 1 on stomata without impairing plant defense responses against herbivores.

Raman imaging of alkyne as a small tag for biological molecules

Role of small molecules such as drugs or metabolites in cells is commonly studied by fluorescence microscopy in which a fluorescent label is attached to the molecule. However, fluorescent labels are typically large that often interfere with the normal cellular function of the molecule. To avoid the use of bulky fluorescent labels, we introduce a technique that uses a simple small chemical tag called alkyne consisting of two carbons connected by a triple bond. The alkyne-tagged molecule is imaged using Raman microscopy that detects the strong Raman signal from the CC triple bond stretching vibration (~2120 cm-1). Because the alkyne signal is located in the silent region of the cell (1800-2700 cm-1), it does not interfere with any intrinsic cellular Raman signals. Here, we demonstrate this technique by showing Raman images of an alkyne-tagged component of DNA in a living cell using a slit-scanning confocal Raman microscope. This fast imaging technique is based on a line-shaped focus illumination and simultaneous detection of the Raman spectra from multiple points of the sample. Using this microscope, we obtained time-course Raman images of the incorporation of EdU in the DNA of HeLa cells in just several tens of minutes.