Shinsuke Shigeto

Stable Isotope-Labeled Raman Imaging Reveals Dynamic Proteome Localization to Lipid Droplets in Single Fission Yeast Cells

Lipid droplets have been hypothesized to be intimately associated with intracellular proteins. However, there is little direct evidence for both spatiotemporal and functional relations between lipid droplets and proteins provided by molecular-level studies on intact cells. Here, we present in vivo time-lapse Raman imaging, coupled with stable-isotope ((13)C) labeling, of single living Schizosaccharomyces pombe cells. Using characteristic Raman bands of proteins and lipids, we dynamically visualized the process by which (13)C-glucose in the medium was assimilated into those intracellular components. Our results show that the proteins newly synthesized from incorporated (13)C-substrate are localized specifically to lipid droplets as the lipid concentration within the cell increases. We demonstrate that the present method offers a unique platform for proteome visualization without the need for tagging individual proteins with fluorescent probes.

Correlation of Carotenoid Accumulation with Aggregation and Biofilm Development in Rhodococcus sp. SD-74

Aggregation of bacterial populations substantially influences their characteristic properties and functions compared with the planktonic counterpart. It is also involved in the initial stages of biofilm development. Many studies have revealed important roles of bacterial aggregation in microbial production and biodegradation. Nevertheless, mechanistic understanding of bacterial aggregation in vivo and at the molecular level is far from complete. Here, we present a noninvasive, label-free Raman microspectroscopic approach to investigate the aggregation and biofilm development of the biotechnologically important Rhodococcus sp. SD-74. We found that the concentration of intracellular carotenoids increases more than 3-fold within 1 week as the biofilm develops. Raman imaging experiments confirmed that the carotenoid accumulation occurs throughout the Rhodococcus sp. SD-74 biofilm. The correlation between the carotenoid Raman intensities and biofilm development found in the present study provides a new means for quantitative, molecular-level assessment of the level of biofilm development, which is not possible with dye staining assay or electron microscopy. Moreover, our results suggest that microbial production of carotenoids in pigmented bacteria such as Rhodococcus sp. SD-74 may potentially be controlled via bacterial aggregation and biofilm formation.

Ordering, Interaction, and Reactivity of the Low-Lying nπ* and ππ* Excited Triplet States of Acetophenone Derivatives†

The diversity of photophysics and photochemistry of the lowlying excited triplet states of aromatic carbonyl compounds has attracted considerable interest in the field of organic photochemistry. For instance, the intersystem crossing rates, phosphorescence lifetimes, and photoreduction activities of these compounds show a marked dependence on both substituents and solvents. Depending on their electronic configurations, the energy of the low-lying triplet states, namely np*, pp*, and charge transfer (CT), can be influenced by substituents and solvents, with possible alterations in the energy-level ordering of the states. The photoreduction proceeds via the T1 state, [5] which has approximate quantum yields that vary between 1 for the np* T1 state, 0.1 for the pp* state, and 0 for the CT state. The strong substituent and solvent dependence of the photophysics and photochemistry of aromatic carbonyl compounds has thus been discussed in terms of the energy-level ordering of the np*, pp*, and CT excited triplet states. It is known that the photoreduction activity of aromatic carbonyl compounds varies gradually with substituents or with solvents. In particular, the pp* T1 states show varying reactivities that cannot be accounted for solely by energylevel ordering. There have been several arguments about this reactivity variation of the pp* T1 state. It is generally considered that the reactivity arises from mixing of the np* character into the pp* state. A mechanism that involves the thermal excitation to a closely lying np* state has also been suggested. These arguments are not based on direct experimental evidence on the ordering of the excited triplet states and therefore are not conclusive; conventional spectroscopic techniques have not been effective in observing close-lying excited triplet states of aromatic carbonyl compounds. Thus, it is highly important to experimentally clarify the energy-level ordering and the electronic configurations of the low-lying excited triplet states of aromatic carbonyl compounds. We have constructed a nanosecond time-resolved absorption spectrometer that is suitable for observing the triplet–triplet transitions in the near-infrared region as well as the vibrational transitions in themid-infrared region. We have focused on the substituent dependence of both the triplet–triplet absorption spectra and the photoreduction activity of a series of acetophenone derivatives. The time-resolved near-infrared spectra of acetophenone (AP) excited at 325 nm in benzene are shown in Figure 1. Upon photoexcitation, two broad transient absorption bands arise instantaneously within the time resolution of the apparatus, and decay synchronously. One band spans from 2000 to 7000 cm , with a peak at 3500 cm . The other band starts from 7000 cm 1 and extends to the higher-wavenumber region above 12000 cm . The decay of these two bands is completely synchronous with the recovery of the ground-state

Exploring Metabolic Pathways in Vivo by a Combined Approach of Mixed Stable Isotope-Labeled Raman Microspectroscopy and Multivariate Curve Resolution Analysis

Understanding cellular metabolism is a major challenge in current systems biology and has triggered extensive metabolomics research, which in most cases involves destructive analysis. However, the information obtainable only in a nondestructive manner will be required for accurately mapping the global structure of the organisms metabolic network at a given instant. Here we report that metabolic pathways can be explored in vivo by mixed stable isotope-labeled Raman microspectroscopy in conjunction with multivariate curve resolution analysis. As a model system, we studied ergosterol biosynthesis in single living fission yeast cells grown in mixtures of normal and (13)C-labeled glucose as the sole carbon source. The multivariate spectral data analysis of space-resolved Raman spectra revealed the intrinsic spectra and relative abundances of all isotopomers of ergosterol whose carbon atoms in the 5,7-diene moiety of the sterol skeleton are either partly or fully substituted with (13)C. Our approach is applicable to other metabolites and will earn a place in the toolbox of metabolomic analysis.

When cells divide: Label-free multimodal spectral imaging for exploratory molecular investigation of living cells during cytokinesis

In vivo, molecular-level investigation of cytokinesis, the climax of the cell cycle, not only deepens our understanding of how life continues, but it will also open up new possibilities of diagnosis/prognosis of cancer cells. Although fluorescence-based methods have been widely employed to address this challenge, they require a fluorophore to be designed for a specific known biomolecule and introduced into the cell. Here, we present a label-free spectral imaging approach based on multivariate curve resolution analysis of Raman hyperspectral data that enables exploratory untargeted studies of mammalian cell cytokinesis. We derived intrinsic vibrational spectra and intracellular distributions of major biomolecular components (lipids and proteins) in dividing and nondividing human colon cancer cells. In addition, we discovered an unusual autofluorescent lipid component that appears predominantly in the vicinity of the cleavage furrow during cytokinesis. This autofluorescence signal could be utilized as an endogenous probe for monitoring and visualizing cytokinesis in vivo.

Infrared Electroabsorption Spectroscopy of N,N-Dimethyl-p-nitroaniline in Acetonitrile/C2Cl4: Solvation of the Solute and Self-Association of Acetonitrile

Solvated structures of N,N-dimethyl-p-nitroaniline (DMPNA), an analog of p-nitroaniline (PNA), and self-associated structures of acetonitrile (ACN) in mixed solvents of ACN and C(2)Cl(4) were studied using infrared (IR) electroabsorption and FTIR spectroscopies. IR electroabsorption spectroscopy measures changes in IR absorption intensity upon application of external electric field modulation, which are a sensitive probe for permanent dipole moments. In ACN/CCl(4), PNA has been shown to occur as two distinct solvated forms, namely, 1:1 and 1:2 forms, which have one and two ACN molecule(s), respectively, associated with PNA. The IR electroabsorption and FTIR measurements on DMPNA show that, unlike PNA, DMPNA occurs as a monomer in ACN/C(2)Cl(4) rather than as specific solvated structures analogous to the 1:1 and 1:2 forms because of the substitution effect. Not only does the N,N-dimethyl substitution in DMPNA hamper solvation of ACN at the N(CH(3))(2) group, but it also indirectly blocks strong interactions with ACN at the NO(2) group. Furthermore, by using the ΔA signal of DMPNA as an internal intensity standard, it was found that the dipole moment of ACN in the DMPNA/ACN/C(2)Cl(4) system is about 1.5 times larger than that of the ACN monomer in dilute CCl(4) solution. This large value of the dipole moment in the solution studied here is attributable to the formation of a head-to-tail linear dimer of ACN, whereas the antiparallel dimer is energetically more favorable in the gas phase.

Simultaneous Observation of an Intraband Transition and Distinct Transient Species in the Infrared Region for Perovskite Solar Cells

Solar cells based on organometal-halide perovskites such as CH3NH3PbI3 have emerged as a promising next-generation photovoltaic system, but the underlying photophysics and photochemistry remain to be established because of the limited availability of methods to implement the simultaneous and direct measurement of various charge carriers and ions that play a crucial role in the operating device. We used nanosecond time-resolved infrared (IR) spectroscopy to investigate, with high molecular specificity, distinct transient species that are formed in perovskite solar cells after photoexcitation. In CH3NH3PbI3 planar-heterojuction solar cells, we simultaneously observed infrared spectral signatures that are associated with an intraband transition of conduction-band electrons, Fano resonance, and the spiro-OMeTAD cation having an exceptionally short lifetime of 1.0 μs (at ∼1485 cm(-1)). The present results show that the time-resolved IR method offers a unique capability to elucidate these important transients in perovskite solar cells and their dynamic interplay in a comprehensive manner.

Biothiol-triggered, self-disassembled silica nanobeads for intracellular drug delivery

Silica-based nanomaterials have demonstrated great potential in biomedical applications due to their chemical inertness. However, the degradability and endosomal trapping issues remain as rate-limiting barriers during their innovation. In this study, we provide a simple yet novel sol-gel approach to construct the redox-responsive silica nanobeads (ReSiNs), which could be rapidly disassembled upon redox gradient for intracellular drug delivery. The disulfide-linked scaffold of the nanobead was synthesized by employing the dithiobis-(succinimidyl propionate) to bridge (3-aminopropyl)-trimethoxysilane. Such silica matrix could be efficiently disrupted in response to intracellular glutathione, resulting in drug release and collapse of entire nanocarrier. Moreover, the ReSiNs exhibited insignificant cytotoxicity before and after the degradation. These results indicated the potential of using ReSiNs as a novel silica-based, biothiol-degradable nanoplatform for future drug delivery.

Is Our Way of Thinking about Excited States Correct? Time‐Resolved Dispersive IR Study on p‐Nitroaniline

Low-lying excited electronic states of an important class of molecules known as push-pull chromophores are central to understanding their potential nonlinear optical properties. Here we report that a combination of high-sensitivity nanosecond time-resolved dispersive IR spectroscopy and DFT calculations on p-nitroaniline (PNA), a prototypical push-pull molecule, reveals that PNA in the lowest excited triplet state has a partial quinoid structure. In this structure, the quinoid configuration is restricted to a part of the phenyl ring adjacent to the NO(2) group. The partial quinoid structure of PNA cannot be explained by a commonly used hybrid of a neutral form and a zwitterionic charge-transfer form. Our findings not only cast doubt on the general applicability of the classical way of looking at excited states, based exclusively on characteristic resonance structures, but also provide deeper insights into excited-state structure of highly polarizable molecular systems.

In vivo probing of the temperature responses of intracellular biomolecules in yeast cells by label-free Raman microspectroscopy.

Environmental temperature is an essential physical quantity that substantially influences cell physiology by changing the equilibria and kinetics of biochemical reactions occurring in cells. Although it has been extensively used as a readily controllable parameter in genetic and biochemical research, much remains to be explored about the temperature responses of intracellular biomolecules in vivo and at the molecular level. Here we report in vivo probing, achieved with label‐free Raman microspectroscopy, of the temperature responses of major intracellular components such as lipids and proteins in living fission yeast cells. The characteristic Raman band at 1602 cm−1, which has been attributed mainly to ergosterol, showed a significant decrease (≈47 %) in intensity at elevated temperatures above 35 °C. In contrast to this high temperature sensitivity of the ergosterol Raman band, the phospholipid and protein Raman bands did not vary much with increasing culture temperature in the 26–38 °C range. This finding agrees with a previous biochemical study that showed that the initial stages of ergosterol biosynthesis in yeast are hindered by temperature elevation. Moreover, our result demonstrates that Raman microspectroscopy holds promise for elucidation of temperature‐dependent cellular activities in living cells, with a high molecular specificity that the commonly used fluorescence microscopy cannot offer.