Selective-plane-activation structured illumination microscopy.

The background light from out-of-focus planes hinders resolution enhancement in structured illumination microscopy when observing volumetric samples. Here we used selective plane illumination and reversibly photoswitchable fluorescent proteins to realize structured illumination within the focal plane and eliminate the out-of-focus background. Theoretical investigation of the imaging properties and experimental demonstrations show that selective plane activation is beneficial for imaging dense microstructures in cells and cell spheroids.

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.

Multifocal Raman Spectrophotometer for Examining Drug-Induced and Chemical-Induced Cellular Changes in 3D Cell Spheroids.

Cell spheroids offer alternative in vitro cell models to monolayer cultured cells because they express complexities similar to those of in vivo tissues, such as cellular responses to drugs and chemicals. Raman spectroscopy emerged as a powerful analytical tool for detecting chemical changes in living cells because it nondestructively provides vibrational information regarding a target. Although multiple iterations are required in drug screening to determine drugs to treat cell spheroids and assess the inter-spheroid heterogeneity, current Raman applications used in spheroids analysis allow the observation of only a few spheroids owing to the low throughput of Raman spectroscopy. In this study, we developed a multifocal Raman spectrophotometer that enables simultaneous analysis of multiple spheroids in separate wells of a regular 96-well plate. By utilizing 96 focal spots excitation and parallel signal collection, our system can improve the throughput by approximately 2 orders of magnitude compared to a conventional single-focus Raman microscope. The Raman spectra of HeLa cell spheroids treated with anticancer drugs and HepG2 cell spheroids treated with free fatty acids were measured simultaneously, and concentration-dependent cellular responses were observed in both studies. Using the multifocal Raman spectrophotometer, we rapidly observed chemical changes in spheroids, and thus, this system can facilitate the application of Raman spectroscopy in analyzing the cellular responses of spheroids.

Bessel-beam illumination Raman microscopy.

We demonstrate the use of Bessel beams for side illumination slit-scanning Raman imaging for label-free and hyperspectral analysis of cell spheroids. The background elimination by the side illumination and the aberration-resistant Bessel beam drastically improves the image contrast in Raman observation, allowing label-free investigation of intracellular molecules in thick biological samples. Live cell spheroids were observed to confirm the improvement in image contrast and background reduction with Bessel illumination compared to conventional epi-line illumination.

Label-Free Monitoring of Drug-Induced Cytotoxicity and Its Molecular Fingerprint by Live-Cell Raman and Autofluorescence Imaging.

Simultaneous observation of drug distribution at the effector site and subsequent cell response are essential in the drug development process. However, few studies have visualized the drug itself and biomolecular interactions in living cells. Here, we used label-free Raman microscopy to investigate drug-induced cytotoxicity and visualize drug uptake and subcellular localization by its specific molecular fingerprint. A redox-sensitive Raman microscope detected the decrease of reduced cytochrome c (cyt c) after Actinomycin D (ActD) treatment in a time-dependent and dose-dependent format. Immunofluorescence staining of cyt c suggested that the release of cyt c was not the major cause. Combining Raman microscopy with conventional biological methods, we reported that the oxidization of cyt c is an early cytotoxicity marker prior to the release of cyt c. Moreover, as the spectral properties of ActD are sensitive to the surrounding environment, subcellular localization of ActD was visualized sensitively by the weak autofluorescence, and the intercalation of ActD into DNA was detected by shifted Raman peaks, allowing for parallel observation of drug uptake and the mechanism of action. In this research, we achieved simultaneous observation of cytotoxicity and cellular drug uptake by Raman microscopy, which could facilitate a precise understanding of pharmacological effects and predict potential drug toxicity in the future.

Vibration Control of Diamond Nanothreads by Lattice Defect Introduction for Application in Nanomechanical Sensors.

Carbon nanomaterials, such as carbon nanotubes (CNTs) and graphene sheets (GSs), have been adopted as resonators in vibration-based nanomechanical sensors because of their extremely high stiffness and small size. Diamond nanothreads (DNTs) are a new class of one-dimensional carbon nanomaterials with extraordinary physical and chemical properties. Their structures are similar to that of diamond in that they possess sp3-bonds formed by a covalent interaction between multiple benzene molecules. In this study, we focus on investigating the mechanical properties and vibration behaviors of DNTs with and without lattice defects and examine the influence of density and configuration of lattice defects on the two them in detail, using the molecular dynamics method and a continuum mechanics approach. We find that Young's modulus and the natural frequency can be controlled by alternating the density of the lattice defects. Furthermore, we investigate and explore the use of DNTs as resonators in nanosensors. It is shown that applying an additional extremely small mass or strain to all types of DNTs significantly changes their resonance frequencies. The results show that, similar to CNTs and GSs, DNTs have potential application as resonators in nano-mass and nano-strain sensors. In particular, the vibration behaviors of DNT resonators can be controlled by alternating the density of the lattice defects to achieve the best sensitivities.

Quantitative Drug Dynamics Visualized by Alkyne-Tagged Plasmonic-Enhanced Raman Microscopy.

Visualizing live-cell uptake of small-molecule drugs is paramount for drug development and pharmaceutical sciences. Bioorthogonal imaging with click chemistry has made significant contributions to the field, visualizing small molecules in cells. Furthermore, recent developments in Raman microscopy, including stimulated Raman scattering (SRS) microscopy, have realized direct visualization of alkyne-tagged small-molecule drugs in live cells. However, Raman and SRS microscopy still suffer from limited detection sensitivity with low concentration molecules for observing temporal dynamics of drug uptake. Here, we demonstrate the combination of alkyne-tag and surface-enhanced Raman scattering (SERS) microscopy for the real-time monitoring of drug uptake in live cells. Gold nanoparticles are introduced into lysosomes of live cells by endocytosis and work as SERS probes. Raman signals of alkynes can be boosted by enhanced electric fields generated by plasmon resonance of gold nanoparticles when alkyne-tagged small molecules are colocalized with the nanoparticles. With time-lapse 3D SERS imaging, this technique allows us to investigate drug uptake by live cells with different chemical and physical conditions. We also perform quantitative evaluation of the uptake speed at the single-cell level using digital SERS counting under different quantities of drug molecules and temperature conditions. Our results illustrate that alkyne-tag SERS microscopy has a potential to be an alternative bioorthogonal imaging technique to investigate temporal dynamics of small-molecule uptake of live cells for pharmaceutical research.

Dynamic pH measurements of intracellular pathways using nano-plasmonic assemblies.

Plasmonic Ag nano-assemblies moving in a living cell were employed to visualize the spatiotemporal change of intracellular pH by surface-enhanced Raman scattering. Ag nano-assemblies were functionalized with 4-mercaptobenzoic acid (p-MBA) to monitor the variation of the surrounding pH. SERS spectra from the functionalized nano-assemblies that were transported in a cell were recorded to estimate the local pH on the pathways travelled by the nano-assemblies. 3D nanoparticle tracking and SERS measurements were simultaneously performed to trace the pH dynamics with a spatial accuracy of several tens of nanometers and a temporal resolution of 200 ms, which are sufficient to reveal pH changes associated with intracellular transportation. The pH trajectory of the functionalized nanoparticle can be used to visualize the dynamic change of the chemical environment caused by organelle interactions, such as endosomal trafficking and lysosomal fusion. This breakthrough creates a new paradigm for intracellular analysis using SERS and reveals new capability for rapid use of SERS in biological analysis.

Direct visualization of an antidepressant analog using surface-enhanced Raman scattering in the brain.

Detailed spatial information of low-molecular weight compound distribution, especially in the brain, is crucial to understanding their mechanism of actions. Imaging techniques that can directly visualize drugs in the brain at a high resolution will complement existing tools for drug distribution analysis. Here, we performed surface-enhanced Raman scattering (SERS) imaging using a bioorthogonal alkyne tag to visualize drugs directly in situ at a high resolution. Focusing on the selective serotonin reuptake inhibitor S-citalopram (S-Cit), which possesses a nitrile group, we substituted an alkynyl group into its structure and synthesized alkynylated S-Cit (Alk-S-Cit). The brain transitivity and the serotonin reuptake inhibition of Alk-S-Cit were not significantly different as compared with S-Cit. Alk-S-Cit was visualized in the coronal mouse brain section using SERS imaging with silver nanoparticles. Furthermore, SERS imaging combined with fluorescence microscopy allowed Alk-S-Cit to be visualized in the adjacent neuronal membranes, as well as in the brain vessel and parenchyma. Therefore, our multimodal imaging technique is an effective method for detecting low-molecular weight compounds in their original tissue environment and can potentially offer additional information regarding the precise spatial distribution of such drugs.

Quantitative Evaluation of Surface-Enhanced Raman Scattering Nanoparticles for Intracellular pH Sensing at a Single Particle Level.

Intracellular pH is one of the key factors for understanding various biological processes in biological cells. Plasmonic gold and silver nanoparticles (NPs) have been extensively studied for surface-enhanced Raman scattering (SERS) applications for pH sensing as a local pH probe in a living cell. However, the SERS performance of NPs depends on material, size, and shape, which can be controlled by chemical synthesis. Here, we synthesized 18 types of gold and silver NPs with different morphologies such as sphere, rod, flower, star, core/shell, hollow, octahedra, core/satellites, and chainlike aggregates, and quantitatively compared their SERS performance for pH sensing. The SERS intensity from the most commonly utilized SERS probe molecule ( para-mercaptobenzoic acid, p-MBA) for pH sensing was measured at the single nanoparticle level under the same measurement parameters such as low laser power (0.5 mW/µm2), short integration time (100 ms) at wavelengths of 405, 488, 532, 584, 676, and 785 nm. In our measurement, the Ag chain, Ag core/satellites, Ag@Au core/satellites, and Au core/satellites nanoassemblies showed efficient pH sensing at the single particle level. By using p-MBA-conjugated Au@Ag core/satellites, we performed time-lapse pH measurements during apoptosis of HeLa cells. These experimental results confirmed that the pH measurement using p-MBA-conjugated Au@Ag core/satellites can be applied for long-term measurements of intracellular pH during cellular events.

Au-Protected Ag Core/Satellite Nanoassemblies for Excellent Extra-/Intracellular Surface-Enhanced Raman Scattering Activity.

Silver nanoparticles (AgNPs) and their assembled nanostructures such as core/satellite nanoassemblies are quite attractive in plasmonic-based applications. However, one biggest drawback of the AgNPs is the poor chemical stability which also greatly limits their applications. We report fine Au coating on synthesized quasi-spherical silver nanoparticles (AgNSs) with few atomic layers to several nanometers by stoichiometric method. The fine Au coating layer was confirmed by energy-dispersive X-ray spectroscopy elemental mapping and aberration-corrected high-angle annular dark-field scanning transmission electron microscopy. The optimized minimal thickness of Au coating layer on different sized AgNSs (22 nm Ag@0.9 nm Au, 44 nm Ag@1.8 nm Au, 75 nm Ag@2.9 nm Au, and 103 nm Ag@0.9 nm Au) was determined by extreme chemical stability tests using H2O2, NaSH, and H2S gas. The thin Au coating layer on AgNSs did not affect their plasmonic-based applications. The core/satellite assemblies based on Ag@Au NPs showed the comparable SERS intensity and uniformity three times higher than that of noncoated Ag core/satellites. The Ag@Au core/satellites also showed high stability in intracellular SERS imaging for at least two days, while the SERS of the noncoated Ag core/satellites decayed significantly. These spherical Ag@Au NPs can be widely used and have great advantages in plasmon-based applications, intracellular SERS probes, and other biological and analytical studies.

3D SERS (surface enhanced Raman scattering) imaging of intracellular pathways.

We demonstrate dynamic surface-enhanced Raman scattering (SERS) imaging with 3 dimensional mapping in a living cell by combining a confocal Raman spectrometer and dual-focus dark-field microscopy. Gold nanoparticles acting as a probe traveling through the living cell, are tracked by dual-focus dark-field imaging, with position feedback to the confocal Raman spectrometer in order to record evolving SERS signals from the same gold nanoparticle moving through the cell. Simultaneous detection of motion and the SERS spectrum enables the visualization of the SERS pathways. Observation of how the SERS spectra change in space and time during biological functions or by changes of the molecular environment provides exciting opportunities for understanding the molecular basis of the cell dynamics.

Feature-based recognition of surface-enhanced Raman spectra for biological targets.

We propose and compare multiple approaches to automatically process data measured through surface-enhanced Raman scattering (SERS), in the context of intracellular molecule probing. It relies on locally detecting the most relevant spectra to retrieve all data independently through indexing, thus avoiding any pre-filtering which occurs with standard processing methods. We first assess our approach on simulated data of the spectrum of Rhodamine 6G, and then validate high-performing methods on experimental measurements of this compound. The optimized method is then utilized to extract and classify the complex SERS response behavior of gold nanoparticles taken in live cells.

Bio-supramolecular photochirogenesis with molecular chaperone: enantiodifferentiating photocyclodimerization of 2-anthracenecarboxylate mediated by prefoldin.

Photocyclodimerization of 2-anthracenecarboxylate mediated by molecular chaperone protein was performed for the first time to afford chiral syn-head-to-tail and anti-head-to-head dimers (2 and 3) in 10% and 16% enantiomeric excess, respectively, with enhanced yields of sterically and electrostatically less-favored head-to-head dimers (3 and 4).