Supermultiplexed optical imaging and barcoding with engineered polyynes.

Optical multiplexing has a large impact in photonics, the life sciences and biomedicine. However, current technology is limited by a 'multiplexing ceiling' from existing optical materials. Here we engineered a class of polyyne-based materials for optical supermultiplexing. We achieved 20 distinct Raman frequencies, as 'Carbon rainbow', through rational engineering of conjugation length, bond-selective isotope doping and end-capping substitution of polyynes. With further probe functionalization, we demonstrated ten-color organelle imaging in individual living cells with high specificity, sensitivity and photostability. Moreover, we realized optical data storage and identification by combinatorial barcoding, yielding to our knowledge the largest number of distinct spectral barcodes to date. Therefore, these polyynes hold great promise in live-cell imaging and sorting as well as in high-throughput diagnostics and screening.

Background-free imaging of chemical bonds by a simple and robust frequency-modulated stimulated Raman scattering microscopy.

Being able to image chemical bonds with high sensitivity and speed, stimulated Raman scattering (SRS) microscopy has made a major impact in biomedical optics. However, it is well known that the standard SRS microscopy suffers from various backgrounds, limiting the achievable contrast, quantification and sensitivity. While many frequency-modulation (FM) SRS schemes have been demonstrated to retrieve the sharp vibrational contrast, they often require customized laser systems and/or complicated laser pulse shaping or introduce additional noise, thereby hindering wide adoption. Herein we report a simple but robust strategy for FM-SRS microscopy based on a popular commercial laser system and regular optics. Harnessing self-phase modulation induced self-balanced spectral splitting of picosecond Stokes beam propagating in standard single-mode silica fibers, a high-performance FM-SRS system is constructed without introducing any additional signal noise. Our strategy enables adaptive spectral resolution for background-free SRS imaging of Raman modes with different linewidths. The generality of our method is demonstrated on a variety of Raman modes with effective suppressing of backgrounds including non-resonant cross phase modulation and electronic background from two-photon absorption or pump-probe process. As such, our method is promising to be adopted by the SRS microscopy community for background-free chemical imaging.

Probe design for super-multiplexed vibrational imaging.

Optical microscopy has served biomedical research for decades due to its high temporal and spatial resolutions. Among various optical imaging techniques, fluorescence imaging offers superb sensitivity down to single molecule level but its multiplexing capacity is limited by intrinsically broad bandwidth. To simultaneously capture a vast number of targets, the newly emerging vibrational microscopy technique draws increasing attention as vibration spectroscopy features narrow transition linewidth. Nonetheless, unlike fluorophores that have been studied for centuries, a systematic investigation on vibrational probes is underemphasized. Herein, we reviewed some of the recent developments of vibrational probes for multiplex imaging applications, particularly those serving stimulated Raman scattering (SRS) microscopy, which is one of the most promising vibrational imaging techniques. We wish to summarize the general guidelines for developing bioorthogonal vibrational probes with high sensitivity, chemical specificity and most importantly, tunability to fulfill super-multiplexed optical imaging. Future directions to significantly improve the performance are also discussed.

A New Approach to Sensitize Antitumor Monofunctional Platinum(II) Complexes via Short Time Photo-Irradiation.

Sensitizing the antitumor activity of monofunctional PtII complexes is a reliable approach to developing antitumor agents different from the classic Pt-based drugs. Considering the poor intracellular accumulation of monofunctional PtII complexes, in this study, the photosensitizing monofunctional PtII complex Pt-BA was derived from a weak BODIPY (boron-dipyrromethene)-derived photosensitizer BA, with the purpose to improve its antitumor cytotoxicity via enhancing its intracellular accumulation with a short time photo-irradiation. Photoinduced reactive oxygen species (ROS) determination indicated that the PtII center in Pt-BA is able to improve the photoinduced ROS production ability of BA, which makes Pt-BA a mild photosensitizer. Fluorescence imaging disclosed that dark incubation makes Pt-BA accumulate mainly on the surface of cell membrane, and the later short time photo-irradiation (5 min) promotes distinctly the intracellular accumulation of Pt-BA, which has been confirmed by inductively coupled plasma-mass spectrometry determination. Flow cytometric Annexin V-FITC assay indicated that the short time irradiation of Pt-BA induces in situ the cell membrane damage, which might finally enhance the intracellular accumulation of this monofunctional complex. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay confirmed that the short time photo-irradiation promotes distinctly the antitumor cytotoxicity of Pt-BA against MCF-7, SGC-7901, A549, and HeLa cell lines. The photopromoted antitumor activity of Pt-BA implies that modifying monofunctional PtII complex as a mild photosensitizer to promote its cell accumulation is a useful approach to sensitizing the antitumor activity of monofunctional PtII complex and renders the possibility of monofunctional PtII prodrugs for precise chemotherapy via only short time photoactivation.

Super-Resolution Vibrational Imaging Using Expansion Stimulated Raman Scattering Microscopy.

Stimulated Raman scattering (SRS) microscopy is an emerging technology that provides high chemical specificity for endogenous biomolecules and can circumvent common constraints of fluorescence microscopy including limited capabilities to probe small biomolecules and difficulty resolving many colors simultaneously. However, the resolution of SRS microscopy remains governed by the diffraction limit. To overcome this, a new technique called molecule anchorable gel-enabled nanoscale Imaging of Fluorescence and stimulated Raman scattering microscopy (MAGNIFIERS) that integrates SRS microscopy with expansion microscopy (ExM) is described. MAGNIFIERS offers chemical-specific nanoscale imaging with sub-50 nm resolution and has scalable multiplexity when combined with multiplex Raman probes and fluorescent labels. MAGNIFIERS is used to visualize nanoscale features in a label-free manner with CH vibration of proteins, lipids, and DNA in a broad range of biological specimens, from mouse brain, liver, and kidney to human lung organoid. In addition, MAGNIFIERS is applied to track nanoscale features of protein synthesis in protein aggregates using metabolic labeling of small metabolites. Finally, MAGNIFIERS is used to demonstrate 8-color nanoscale imaging in an expanded mouse brain section. Overall, MAGNIFIERS is a valuable platform for super-resolution label-free chemical imaging, high-resolution metabolic imaging, and highly multiplexed nanoscale imaging, thus bringing SRS to nanoscopy.

Highly-multiplexed volumetric mapping with Raman dye imaging and tissue clearing.

Mapping the localization of multiple proteins in their native three-dimensional (3D) context would be useful across many areas of biomedicine, but multiplexed fluorescence imaging has limited intrinsic multiplexing capability, and most methods for increasing multiplexity can only be applied to thin samples (<100 µm). Here, we harness the narrow spectrum of Raman spectroscopy and introduce Raman dye imaging and tissue clearing (RADIANT), an optical method that is capable of imaging multiple targets in thick samples in one shot. We expanded the range of suitable bioorthogonal Raman dyes and developed a tissue-clearing strategy for them (Raman 3D imaging of solvent-cleared organs (rDISCO)). We applied RADIANT to image up to 11 targets in millimeter-thick brain slices, extending the imaging depth 10- to 100-fold compared to prior multiplexed protein imaging methods. We showcased the utility of RADIANT in extracting systems information, including region-specific correlation networks and their topology in cerebellum development. RADIANT will facilitate the exploration of the intricate 3D protein interactions in complex systems.

9-Cyanopyronin probe palette for super-multiplexed vibrational imaging.

Multiplexed optical imaging provides holistic visualization on a vast number of molecular targets, which has become increasingly essential for understanding complex biological processes and interactions. Vibrational microscopy has great potential owing to the sharp linewidth of vibrational spectra. In 2017, we demonstrated the coupling between electronic pre-resonant stimulated Raman scattering (epr-SRS) microscopy with a proposed library of 9-cyanopyronin-based dyes, named Manhattan Raman Scattering (MARS). Herein, we develop robust synthetic methodology to build MARS probes with different core atoms, expansion ring numbers, and stable isotope substitutions. We discover a predictive model to correlate their vibrational frequencies with structures, which guides rational design of MARS dyes with desirable Raman shifts. An expanded library of MARS probes with diverse functionalities is constructed. When coupled with epr-SRS microscopy, these MARS probes allow us to demonstrate not only many versatile labeling modalities but also increased multiplexing capacity. Hence, this work opens up next-generation vibrational imaging with greater utilities.

Super-resolution vibrational microscopy by stimulated Raman excited fluorescence.

Inspired by the revolutionary impact of super-resolution fluorescence microscopy, super-resolution Raman imaging has been long pursued because of its much higher chemical specificity than the fluorescence counterpart. However, vibrational contrasts are intrinsically less sensitive compared with fluorescence, resulting in only mild resolution enhancement beyond the diffraction limit even with strong laser excitation power. As such, it is still a great challenge to achieve biocompatible super-resolution vibrational imaging in the optical far-field. In 2019 Stimulated Raman Excited Fluorescence (SREF) was discovered as an ultrasensitive vibrational spectroscopy that combines the high chemical specificity of Raman scattering and the superb sensitivity of fluorescence detection. Herein we developed a novel super-resolution vibrational imaging method by harnessing SREF as the contrast mechanism. We first identified the undesired role of anti-Stokes fluorescence background in preventing direct adoption of super-resolution fluorescence technique. We then devised a frequency-modulation (FM) strategy to remove the broadband backgrounds and achieved high-contrast SREF imaging. Assisted by newly synthesized SREF dyes, we realized multicolor FM-SREF imaging with nanometer spectral resolution. Finally, by integrating stimulated emission depletion (STED) with background-free FM-SREF, we accomplished high-contrast super-resolution vibrational imaging with STED-FM-SREF whose spatial resolution is only determined by the signal-to-noise ratio. In our proof-of-principle demonstration, more than two times of resolution improvement is achieved in biological systems with moderate laser excitation power, which shall be further refined with optimized instrumentation and imaging probes. With its super resolution, high sensitivity, vibrational contrast, and mild laser excitation power, STED-FM-SREF microscopy is envisioned to aid a wide variety of applications.

Ultra-bright Raman dots for multiplexed optical imaging.

Imaging the spatial distribution of biomolecules is at the core of modern biology. The development of fluorescence techniques has enabled researchers to investigate subcellular structures with nanometer precision. However, multiplexed imaging, i.e. observing complex biological networks and interactions, is mainly limited by the fundamental 'spectral crowding' of fluorescent materials. Raman spectroscopy-based methods, on the other hand, have a much greater spectral resolution, but often lack the required sensitivity for practical imaging of biomarkers. Addressing the pressing need for new Raman probes, herein we present a series of Raman-active  nanoparticles (Rdots) that exhibit the combined advantages of ultra-brightness and compact sizes (~20 nm). When coupled with the emerging stimulated Raman scattering (SRS) microscopy, these Rdots are brighter than previously reported Raman-active organic probes by two to three orders of magnitude. We further obtain evidence supporting for SRS imaging of Rdots at single particle level. The compact size and ultra-brightness of Rdots allows immunostaining of specific protein targets (including cytoskeleton and low-abundant surface proteins) in mammalian cells and tissue slices with high imaging contrast. These Rdots thus offer a promising tool for a large range of studies on complex biological networks.

Stimulated Raman Excited Fluorescence Spectroscopy of Visible Dyes.

Fluorescence spectroscopy and Raman spectroscopy are two major classes of spectroscopy methods in physical chemistry. Very recently, stimulated Raman excited fluorescence (SREF) has been demonstrated ( Xiong, H.; et al. Nature Photonics , 2019 , 13 , 412 - 417 ) as a new hybrid spectroscopy that combines the vibrational specificity of Raman spectroscopy with the superb sensitivity of fluorescence spectroscopy (down to the single-molecule level). However, this proof-of-concept study was limited by both the tunability of the commercial laser source and the availability of the excitable molecules in the near-infrared. As a result, the generality of SREF spectroscopy remains unaddressed, and the understanding of the critical electronic preresonance condition is lacking. In this work, we built a modified excitation source to explore SREF spectroscopy in the visible region. Harnessing a large palette of red dyes, we have systematically studied SREF spectroscopy on a dozen different cases with a fine spectral interval of several nanometers. The results not only establish the generality of SREF spectroscopy for a wide range of molecules but also reveal a tight window of proper electronic preresonance for the stimulated Raman pumping process. Our theoretical modeling and further experiments on newly synthesized dyes also support the obtained insights, which would be valuable in designing and optimizing future SREF experiments for single-molecule vibrational spectroscopy and supermultiplex vibrational imaging.

Spectral tracing of deuterium for imaging glucose metabolism.

Cells and tissues often display pronounced spatial and dynamical metabolic heterogeneity. Common glucose-imaging techniques report glucose uptake or catabolism activity, yet do not trace the functional utilization of glucose-derived anabolic products. Here we report a microscopy technique for the optical imaging, via the spectral tracing of deuterium (STRIDE), of diverse macromolecules derived from glucose. Based on stimulated Raman-scattering imaging, STRIDE visualizes the metabolic dynamics of newly synthesized macromolecules, such as DNA, protein, lipids and glycogen, via the enrichment and distinct spectra of carbon-deuterium bonds transferred from the deuterated glucose precursor. STRIDE can also use spectral differences derived from different glucose isotopologues to visualize temporally separated glucose populations using a pulse-chase protocol. We also show that STRIDE can be used to image glucose metabolism in many mouse tissues, including tumours, brain, intestine and liver, at a detection limit of 10 mM of carbon-deuterium bonds. STRIDE provides a high-resolution and chemically informative assessment of glucose anabolic utilization.

Structure-activity-distribution relationship study of anti-cancer antimycin-type depsipeptides.

Small-molecule natural products have been an essential source of pharmaceuticals to treat human diseases, but very little is known about their behavior inside dynamic, live human cells. Here, we demonstrate the first structure-activity-distribution relationship (SADR) study of complex natural products, the anti-cancer antimycin-type depsipeptides, using the emerging bioorthogonal Stimulated Raman Scattering (SRS) Microscopy. Our results show that the intracellular enrichment and distribution of these compounds are driven by their potency and specific protein targets, as well as the lipophilic nature of compounds.

Operando and three-dimensional visualization of anion depletion and lithium growth by stimulated Raman scattering microscopy.

Visualization of ion transport in electrolytes provides fundamental understandings of electrolyte dynamics and electrolyte-electrode interactions. However, this is challenging because existing techniques are hard to capture low ionic concentrations and fast electrolyte dynamics. Here we show that stimulated Raman scattering microscopy offers required resolutions to address a long-lasting question: how does the lithium-ion concentration correlate to uneven lithium deposition? In this study, anions are used to represent lithium ions since their concentrations should not deviate for more than 0.1 mM, even near nanoelectrodes. A three-stage lithium deposition process is uncovered, corresponding to no depletion, partial depletion, and full depletion of lithium ions. Further analysis reveals a feedback mechanism between the lithium dendrite growth and heterogeneity of local ionic concentration, which can be suppressed by artificial solid electrolyte interphase. This study shows that stimulated Raman scattering microscopy is a powerful tool for the materials and energy field.