VIBRANT: spectral profiling for single-cell drug responses.

High-content cell profiling has proven invaluable for single-cell phenotyping in response to chemical perturbations. However, methods with improved throughput, information content and affordability are still needed. We present a new high-content spectral profiling method named vibrational painting (VIBRANT), integrating mid-infrared vibrational imaging, multiplexed vibrational probes and an optimized data analysis pipeline for measuring single-cell drug responses. Three infrared-active vibrational probes were designed to measure distinct essential metabolic activities in human cancer cells. More than 20,000 single-cell drug responses were collected, corresponding to 23 drug treatments. The resulting spectral profile is highly sensitive to phenotypic changes under drug perturbation. Using this property, we built a machine learning classifier to accurately predict drug mechanism of action at single-cell level with minimal batch effects. We further designed an algorithm to discover drug candidates with new mechanisms of action and evaluate drug combinations. Overall, VIBRANT has demonstrated great potential across multiple areas of phenotypic screening.

Mid-infrared metabolic imaging with vibrational probes.

Understanding metabolism is indispensable in unraveling the mechanistic basis of many physiological and pathological processes. However, in situ metabolic imaging tools are still lacking. Here we introduce a framework for mid-infrared (MIR) metabolic imaging by coupling the emerging high-information-throughput MIR microscopy with specifically designed IR-active vibrational probes. We present three categories of small vibrational tags including azide bond, 13C-edited carbonyl bond and deuterium-labeled probes to interrogate various metabolic activities in cells, small organisms and mice. Two MIR imaging platforms are implemented including broadband Fourier transform infrared microscopy and discrete frequency infrared microscopy with a newly incorporated spectral region (2,000-2,300 cm-1). Our technique is uniquely suited to metabolic imaging with high information throughput. In particular, we performed single-cell metabolic profiling including heterogeneity characterization, and large-area metabolic imaging at tissue or organ level with rich spectral information.

Super-multiplex vibrational imaging.

The ability to visualize directly a large number of distinct molecular species inside cells is increasingly essential for understanding complex systems and processes. Even though existing methods have successfully been used to explore structure-function relationships in nervous systems, to profile RNA in situ, to reveal the heterogeneity of tumour microenvironments and to study dynamic macromolecular assembly, it remains challenging to image many species with high selectivity and sensitivity under biological conditions. For instance, fluorescence microscopy faces a 'colour barrier', owing to the intrinsically broad (about 1,500 inverse centimetres) and featureless nature of fluorescence spectra that limits the number of resolvable colours to two to five (or seven to nine if using complicated instrumentation and analysis). Spontaneous Raman microscopy probes vibrational transitions with much narrower resonances (peak width of about 10 inverse centimetres) and so does not suffer from this problem, but weak signals make many bio-imaging applications impossible. Although surface-enhanced Raman scattering offers high sensitivity and multiplicity, it cannot be readily used to image specific molecular targets quantitatively inside live cells. Here we use stimulated Raman scattering under electronic pre-resonance conditions to image target molecules inside living cells with very high vibrational selectivity and sensitivity (down to 250 nanomolar with a time constant of 1 millisecond). We create a palette of triple-bond-conjugated near-infrared dyes that each displays a single peak in the cell-silent Raman spectral window; when combined with available fluorescent probes, this palette provides 24 resolvable colours, with the potential for further expansion. Proof-of-principle experiments on neuronal co-cultures and brain tissues reveal cell-type-dependent heterogeneities in DNA and protein metabolism under physiological and pathological conditions, underscoring the potential of this 24-colour (super-multiplex) optical imaging approach for elucidating intricate interactions in complex biological systems.

Biological imaging of chemical bonds by stimulated Raman scattering microscopy.

All molecules consist of chemical bonds, and much can be learned from mapping the spatiotemporal dynamics of these bonds. Since its invention a decade ago, stimulated Raman scattering (SRS) microscopy has become a powerful modality for imaging chemical bonds with high sensitivity, resolution, speed and specificity. We introduce the fundamentals of SRS microscopy and review innovations in SRS microscopes and imaging probes. We highlight examples of exciting biological applications, and share our vision for potential future breakthroughs for this technology.

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.

Novel loci fsd6.1 and Csgl3 regulate ultra-high fruit spine density in cucumber.

KEY MESSAGE: Quantitative Trait Loci (QTL) analysis of multiple populations in multiple environments revealed that the fsd6.2 locus, which includes the candidate gene Csgl3, controls high fruit spine density in natural cucumbers. GWAS identified a novel locus fsd6.1, which regulates ultra-high fruit spine density in combination with Csgl3, and evolved during cucumber domestication. Fruit spine density, a domestication trait, largely influences the commercial value of cucumbers. However, the molecular basis of fruit spine density in cucumber remains unclear. In this study, four populations were derived from five materials, which included three with low fruit spine density, one with high fruit spine density, and one with ultra-high fruit spine density. Fruit spine densities were measured in 15 environments over a span of 6 years. The distributions were bimodal suggesting that fruit spine density is controlled by a major-effect QTL. QTL analysis determined that the same major-effect QTL, fsd6.2, is present in four populations. Fine mapping indicated that Csgl3 is the candidate gene at the fsd6.2 locus. Phylogenetic and geographical distribution analyses revealed that Csgl3 originated from China, which has the highest genetic diversity for fruit spine density. One novel minor-effect QTL, fsd6.1, was detected in the HR and HP populations derived from the cross between 65G and 02245. In addition, GWAS identified a novel locus that colocates with fsd6.1. Inspection of a candidate region of about 18 kb in size using pairwise LD correlations, combined with genetic diversity and phylogenetic analysis of fsd6.1 in natural populations, indicated that Csa6G421750 is the candidate gene responsible for ultra-high fruit spine density in cucumber. This study provides new insights into the origin of fruit spine density and the evolution of high/ultra-high fruit spine density during cucumber domestication.

Combined fine mapping, genetic diversity, and transcriptome profiling reveals that the auxin transporter gene ns plays an important role in cucumber fruit spine development.

KEY MESSAGE: Map-based cloning was used to identify the ns gene, which was involved in the formation of cucumber numerous fruit spines together with other genes under regulation by plant hormone signal transduction. The cucumber (Cucumis sativus) fruit spine density has an important impact on the commercial value. However, little is known about the regulatory mechanism for the fruit spine formation. Here, we identified NUMEROUS SPINES (NS), which regulate fruit spine development by modulating the Auxin signaling pathway. We fine-mapped the ns using a 2513 F2 population derived from NCG122 (numerous fruit spines line) and NCG121 (few fruit spines line), and showed that NS encoded auxin transporter-like protein 3. Genetic diversity analysis of the NS gene in natural populations revealed that one SNP and one InDel in the coding region of ns are co-segregated with the fruit spine density. The NS protein sequence was highly conserved among plants, but its regulation of fruit spine development in cucumber seems to be a novel function. Transcriptome profiling indicated that the plant hormone signal transduction-related genes were highly enriched in the up-regulated genes in NCG122 versus NCG121. Moreover, expression pattern analysis of the auxin signal pathway-related genes in NCG122 versus NCG121 showed that upstream genes of the pathway (like ns candidate gene Csa2M264590) are down-regulated, while the downstream genes are up-regulated. Quantitative reverse transcription PCR confirmed the differential expression during the fruit spine development. Therefore, reduced expression of ns may promote the fruit spine formation. Our findings provide a valuable framework for dissecting the regulatory mechanism for the fruit spine development.

Viewing 3D spatial biology with highly-multiplexed Raman imaging: from spectroscopy to biotechnology.

Understansding complex biological systems requires the simultaneous characterization of a large number of interacting components in their native 3D environment with high spatial resolution. Highly-multiplexed Raman imaging is an emerging general strategy for detecting biomarkers with scalable multiplexity and ultra-sensitivity based on a series of stimulated Raman scattering (SRS) techniques. Here we review recent advances in highly-multiplexed Raman imaging and how they contribute to the technological revolution in 3D spatial biology, focusing on the developmental pathway from spectroscopy study to biotechnology invention. We envision highly-multiplexed Raman imaging is taking off, which will greatly facilitate our understanding in biological and medical research fields.

Probing the structure of water in individual living cells.

Water regulates or even governs a wide range of biological processes. Despite its fundamental importance, surprisingly little is known about the structure of intracellular water. Herein we employ a Raman micro-spectroscopy technique to uncover the composition, abundance and vibrational spectra of intracellular water in individual living cells. In three different cell types, we show a small but consistent population (~3%) of non-bulk-like water. It exhibits a weakened hydrogen-bonded network and a more disordered tetrahedral structure. We attribute this population to biointerfacial water located in the vicinity of biomolecules. Moreover, our whole-cell modeling suggests that all soluble (globular) proteins inside cells are surrounded by, on average, one full molecular layer (about 2.6 Angstrom) of biointerfacial water. Furthermore, relative invariance of biointerfacial water is observed among different single cells. Overall, our study not only opens up experimental possibilities of interrogating water structure in vivo but also provides insights into water in life.

Vibrational Solvatochromism Study of the C-H···O Improper Hydrogen Bond.

The improper C-H···O hydrogen bond is an important weak interaction, with broad implications for protein and nucleic acid structure, molecular recognition, enzyme catalysis, and drug interaction. Despite its wide identification in crystal structures, the general existence of C-H···O hydrogen bonds remains elusive especially for natural C-H groups in bulk aqueous solutions at room temperature. Vibrational spectroscopy is a promising methodology to tackle this challenge, as formation of C-H···O hydrogen bonds usually causes shifts of the C-H stretch frequency. Yet, prior observations are inconclusive, as they are all based on a simple blue-shift in aqueous solution and cannot distinguish if it is an effect caused by solvent reorganization or a specific hydrogen-bonding interaction. In this work, we used vibrational solvatochromism as a calibration of the solvent reorganization effect and identified a specific H-bonding interaction. We performed vibrational solvatochromism study of C-H(D) of multiple alcohol molecules including the CH mode of CD3CH(OH)CD3 and the CD3 modes of CD3OH, CD3CH2OH, and CD3CH(OH)CD3 in a series of solvents. We found an abnormal blue-shift of the Raman frequency of the C-H and C-D bonds at both the Cα and Cß positions of alcohols in water, which lies in an opposite direction to the expected trend due to vibrational solvatochromism. This experimental evidence supports that the improper C-H···O hydrogen bonds might generally exist between nonpolarized C-H and water in liquid solutions at room temperature.

Highly-Multiplexed Tissue Imaging with Raman Dyes.

Visualizing a vast scope of specific biomarkers in tissues plays a vital role in exploring the intricate organizations of complex biological systems. Hence, highly multiplexed imaging technologies have been increasingly appreciated. Here, we describe an emerging platform of highly-multiplexed vibrational imaging of specific proteins with comparable sensitivity to standard immunofluorescence via electronic pre-resonance stimulated Raman scattering (epr-SRS) imaging of rainbow-like Raman dyes. This method circumvents the limit of spectrally-resolvable channels in conventional immunofluorescence and provides a one-shot optical approach to interrogate multiple markers in tissues with subcellular resolution. It is generally compatible with standard tissue preparations, including paraformaldehyde-fixed tissues, frozen tissues, and formalin-fixed paraffin-embedded (FFPE) human tissues. We envisage this platform will provide a more comprehensive picture of protein interactions of biological specimens, particularly for thick intact tissues. This protocol provides the workflow from antibody preparation to tissue sample staining, to SRS microscope assembly, to epr-SRS tissue imaging.

Towards Mapping Mouse Metabolic Tissue Atlas by Mid-Infrared Imaging with Heavy Water Labeling.

Understanding metabolism is of great significance to decipher various physiological and pathogenic processes. While great progress has been made to profile gene expression, how to capture organ-, tissue-, and cell-type-specific metabolic profile (i.e., metabolic tissue atlas) in complex mammalian systems is lagging behind, largely owing to the lack of metabolic imaging tools with high resolution and high throughput. Here, the authors applied mid-infrared imaging coupled with heavy water (D2 O) metabolic labeling to a scope of mouse organs and tissues. The premise is that, as D2 O participates in the biosynthesis of various macromolecules, the resulting broad C-D vibrational spectrum should interrogate a wide range of metabolic pathways. Applying multivariate analysis to the C-D spectrum, the authors successfully identified both inter-organ and intra-tissue metabolic signatures of mice. A large-scale metabolic atlas map between different organs from the same mice is thus generated. Moreover, leveraging the power of unsupervised clustering methods, spatially-resolved metabolic signatures of brain tissues are discovered, revealing tissue and cell-type specific metabolic profile in situ. As a demonstration of this technique, the authors captured metabolic changes during brain development and characterized intratumoral metabolic heterogeneity of glioblastoma. Altogether, the integrated platform paves a way to map the metabolic tissue atlas for complex mammalian systems.

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.

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.

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.

Proteomics analysis of host cells infected with infectious bursal disease virus.

The effect of infectious bursal disease virus (IBDV) infection on cellular protein expression is essential for viral pathogenesis. To characterize the cellular response to IBDV infection, the differential proteomes of chicken embryo fibroblasts, with and without IBDV infection, were analyzed at different time points with two-dimensional gel electrophoresis (2-DE) followed by MALDI-TOF/TOF identification. Comparative analysis of multiple 2-DE gels revealed that the majority of protein expression changes appeared at 48 and 96 h after IBDV infection. Mass spectrometry identified 51 altered cellular proteins, including 13 up-regulated proteins and 38 down-regulated proteins 12-96 h after infection. Notably 2-DE analysis revealed that IBDV infection induced the increased expression of polyubiquitin, apolipoprotein A-I, heat shock 27-kDa protein 1, actins, tubulins, eukaryotic translation initiation factor 4A isoform 2, acidic ribosomal phosphoprotein, and ribosomal protein SA isoform 2. In addition, IBDV infection considerably suppressed those cellular proteins involved in ubiquitin-mediated protein degradation, energy metabolism, intermediate filaments, host translational apparatus, and signal transduction. Moreover 38 corresponding genes of the differentially expressed proteins were quantitated by real time RT-PCR to examine the transcriptional profiles between infected and uninfected chicken embryo fibroblasts. Western blot further confirmed the inhibition of Rho protein GDP dissociation inhibitor expression and the induction of polyubiquitin during IBDV infection. Subcellular distribution analysis of the cytoskeletal proteins vimentin and beta-tubulin clearly demonstrated that IBDV infection induced the disruption of the vimentin network and microtubules late in IBDV infection. Thus, this work effectively provides useful dynamic protein-related information to facilitate further investigation of the underlying mechanism of IBDV infection and pathogenesis.

Optimized sample preparation for two-dimensional gel electrophoresis of soluble proteins from chicken bursa of Fabricius.

BACKGROUND: Two-dimensional gel electrophoresis (2-DE) is a powerful method to study protein expression and function in living organisms and diseases. This technique, however, has not been applied to avian bursa of Fabricius (BF), a central immune organ. Here, optimized 2-DE sample preparation methodologies were constructed for the chicken BF tissue. Using the optimized protocol, we performed further 2-DE analysis on a soluble protein extract from the BF of chickens infected with virulent avibirnavirus. To demonstrate the quality of the extracted proteins, several differentially expressed protein spots selected were cut from 2-DE gels and identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). RESULTS: An extraction buffer containing 7 M urea, 2 M thiourea, 2% (w/v) 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), 50 mM dithiothreitol (DTT), 0.2% Bio-Lyte 3/10, 1 mM phenylmethylsulfonyl fluoride (PMSF), 20 U/ml Deoxyribonuclease I (DNase I), and 0.25 mg/ml Ribonuclease A (RNase A), combined with sonication and vortex, yielded the best 2-DE data. Relative to non-frozen immobilized pH gradient (IPG) strips, frozen IPG strips did not result in significant changes in the 2-DE patterns after isoelectric focusing (IEF). When the optimized protocol was used to analyze the spleen and thymus, as well as avibirnavirus-infected bursa, high quality 2-DE protein expression profiles were obtained. 2-DE maps of BF of chickens infected with virulent avibirnavirus were visibly different and many differentially expressed proteins were found. CONCLUSION: These results showed that method C, in concert extraction buffer IV, was the most favorable for preparing samples for IEF and subsequent protein separation and yielded the best quality 2-DE patterns. The optimized protocol is a useful sample preparation method for comparative proteomics analysis of chicken BF tissues.

Competitive replication of different genotypes of infectious bursal disease virus on chicken embryo fibroblasts.

Infectious bursal disease virus (IBDV) causes immunosuppression in chickens. We investigated the molecular changes in chicken embryo fibroblasts (CEF) adapted IBDV by genomic sequencing. IBDV were serially passaged in CEF and chickens were infected with the IBDV obtained after different numbers of passages in CEF. Chicken infections showed that 16th, 20th, and 21st passage viruses were pathogenic, while 26th and 36th passage viruses were non-pathogenic. Sequencing demonstrated that the initial changes during the serial passage comprised of a single-nucleotide deletion in the 3' non-coding region of segment B of the virus after 19th passage, followed by changes in the VP1 gene after the 20th passage of the virus and changes in VP2, VP5 after the 21st passage of the virus. These data suggested that the attenuation of very virulent IBDV was due to multigenic mutations and there are in vitro and in vivo competitive replications in IBDV quasispecies.

Optical mapping of biological water in single live cells by stimulated Raman excited fluorescence microscopy.

Water is arguably the most common and yet least understood material on Earth. Indeed, the biophysical behavior of water in crowded intracellular milieu is a long-debated issue. Understanding of the spatial and compositional heterogeneity of water inside cells remains elusive, largely due to a lack of proper water-sensing tools with high sensitivity and spatial resolution. Recently, stimulated Raman excited fluorescence (SREF) microscopy was reported as the most sensitive vibrational imaging in the optical far field. Herein we develop SREF into a water-sensing tool by coupling it with vibrational solvatochromism. This technique allows us to directly visualize spatially-resolved distribution of water states inside single mammalian cells. Qualitatively, our result supports the concept of biological water and reveals intracellular water heterogeneity between nucleus and cytoplasm. Quantitatively, we unveil a compositional map of the water pool inside living cells. Hence we hope SREF will be a promising tool to study intracellular water and its relationship with cellular activities.

Stimulated Raman Excited Fluorescence Spectroscopy and Imaging.

Powerful optical tools have revolutionized science and technology. The prevalent fluorescence detection offers superb sensitivity down to single molecules but lacks sufficient chemical information1-3. In contrast, Raman-based vibrational spectroscopy provides exquisite chemical specificity about molecular structure, dynamics and coupling, but is notoriously insensitive3-5. Here we report a hybrid technique of Stimulated Raman Excited Fluorescence (SREF) that integrates superb detection sensitivity and fine chemical specificity. Through stimulated Raman pumping to an intermediate vibrational eigenstate followed by an upconversion to an electronic fluorescent state, SREF encodes vibrational resonance into the excitation spectrum of fluorescence emission. By harnessing narrow vibrational linewidth, we demonstrated multiplexed SREF imaging in cells, breaking the "color barrier" of fluorescence. By leveraging superb sensitivity of SREF, we achieved all-far-field single-molecule Raman spectroscopy and imaging without plasmonic enhancement, a long-sought-after goal in photonics. Thus, through merging Raman and fluorescence spectroscopy, SERF would be a valuable tool for chemistry and biology.