highly-multiplexed-tissue-imaging-with-raman-dyes

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. ...

multi-modal-spatial-metabolomics-with-mid-infrared-microscope-guided

Multi-Modal Spatial Metabolomics with Mid-Infrared Microscope Guided Mass Spectrometry Imaging

Spatial metabolomics is a rapidly evolving field to map the distribution of metabolites within tissues, organs, and even single cells. This approach ...

Columbia University

Columbia College

Fudan University

Zhongshan-Xuhui Hospital

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.

Super-multiplex vibrational imaging.

Recently we have reported electronic pre-resonance stimulated Raman scattering (epr-SRS) microscopy as a powerful technique for super-multiplex imaging ( Wei, L. ; Nature 2017 , 544 , 465 - 470 ). However, under rigorous electronic resonance, background signal, which mainly originates from pump-probe process, overwhelms the desired vibrational signature of the chromophores. Here we demonstrate electronic resonant stimulated Raman scattering (er-SRS) microspectroscopy and imaging through suppression of electronic background and subsequent retrieval of vibrational peaks. We observed a change of the vibrational band shapes from normal Lorentzian, through dispersive shapes, to inverted Lorentzian as the electronic resonance was approached, in agreement with theoretical prediction. In addition, resonant Raman cross sections have been determined after power-dependence study as well as Raman excitation profile calculation. As large as 10 cm of resonance Raman cross section is estimated in er-SRS, which is about 100 times higher than previously reported in epr-SRS. These results of er-SRS microspectroscopy pave the way for the single-molecule Raman detection and ultrasensitive biological imaging.

Electronic Resonant Stimulated Raman Scattering Micro-Spectroscopy.

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.

Probe design for super-multiplexed vibrational imaging.

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.

Biological imaging of chemical bonds by stimulated Raman scattering 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.

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

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, C-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). 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.

Mid-infrared metabolic imaging with vibrational probes.

Powerful optical tools have revolutionized science and technology. The prevalent fluorescence detection offers superb sensitivity down to single molecules but lacks sufficient chemical information. In contrast, Raman-based vibrational spectroscopy provides exquisite chemical specificity about molecular structure, dynamics and coupling, but is notoriously insensitive. 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.

Stimulated Raman Excited Fluorescence Spectroscopy and 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.

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

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.

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

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 (D O) metabolic labeling to a scope of mouse organs and tissues. The premise is that, as D 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 charaztereized intratumoral metabolic heterogeneity of glioblastoma. Altogether, the integrated platform paves a way to map the metabolic tissue atlas for complex mammalian systems.

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

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.

VIBRANT: spectral profiling for single-cell drug responses.

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.

Probing the structure of water in individual living cells.

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.

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

Vibrational spectroscopy and fluorescence spectroscopy have historically been two established but separate fields of molecular spectroscopy. While vibrational spectroscopy provides exquisite chemical information, fluorescence spectroscopy often offers orders of magnitude higher detection sensitivity. However, they each lack the advantages of each other. In recent years, a series of novel nonlinear optical spectroscopy studies have been developed that merge both spectroscopies into a single double-resonance process. These techniques combine the chemical specificity of Raman or infrared (IR) spectroscopy with the superb detection sensitivity and spatial resolution of fluorescence microscopy. Many facets have been explored, including Raman transition versus IR transition, time domain versus frequency domain, and spectroscopy versus microscopy. Notably, single-molecule vibrational spectroscopy has been achieved at room temperature without the need for plasmonics. Even superresolution vibrational imaging beyond the diffraction limit was demonstrated. This review summarizes the growing field of vibrational-encoded fluorescence microscopy, including key technical developments, emerging applications, and future prospects.

Merging Vibrational Spectroscopy with Fluorescence Microscopy: Combining the Best of Two Worlds.

Vibration of chemical bonds can serve as imaging contrast. Vibrational probes, synergized with major advances in chemical bond imaging instruments, have recently flourished and proven valuable in illuminating life processes. Here, we review how the development of vibrational probes with optimal biocompatibility, enhanced sensitivity, multichromatic colors and diverse functionality has extended chemical bond imaging beyond the prevalent label-free paradigm into various novel applications such as imaging metabolites, metabolic imaging, drug imaging, super-multiplex imaging, vibrational profiling and vibrational sensing. These advancements in vibrational probes have greatly facilitated understanding living systems, a new field of vibrational chemical biology.

ArticleTotal : 2	Year
Publication title Cited by 5	2022
Publication title Cited by 2	2025

Article	Year
Super-multiplex vibrational imaging. Nature\| PubMed ID: 28424513	2017
Electronic Resonant Stimulated Raman Scattering Micro-Spectroscopy. The journal of physical chemistry. B\| PubMed ID: 30208710	2018
Probe design for super-multiplexed vibrational imaging. Physical biology\| PubMed ID: 30870829	2019
Biological imaging of chemical bonds by stimulated Raman scattering microscopy. Nature methods\| PubMed ID: 31471618	2019
Optical mapping of biological water in single live cells by stimulated Raman excited fluorescence microscopy. Nature communications\| PubMed ID: 31628307	2019
Mid-infrared metabolic imaging with vibrational probes. Nature methods\| PubMed ID: 32601425	2020
Stimulated Raman Excited Fluorescence Spectroscopy and Imaging. Nature photonics\| PubMed ID: 32607124	2019
9-Cyanopyronin probe palette for super-multiplexed vibrational imaging. Nature communications\| PubMed ID: 34312393	2021
Highly-multiplexed volumetric mapping with Raman dye imaging and tissue clearing. Nature biotechnology\| PubMed ID: 34608326	2022
Towards Mapping Mouse Metabolic Tissue Atlas by Mid-Infrared Imaging with Heavy Water Labeling. Advanced science (Weinheim, Baden-Wurttemberg, Germany)\| PubMed ID: 35319171	2022
VIBRANT: spectral profiling for single-cell drug responses. Nature methods\| PubMed ID: 38374266	2024
Probing the structure of water in individual living cells. Nature communications\| PubMed ID: 38902250	2024
Viewing 3D spatial biology with highly-multiplexed Raman imaging: from spectroscopy to biotechnology. Chemical communications (Cambridge, England)\| PubMed ID: 39041798	2024
Merging Vibrational Spectroscopy with Fluorescence Microscopy: Combining the Best of Two Worlds. Annual review of physical chemistry\| PubMed ID: 39899841	2025
Illuminating life processes by vibrational probes. Nature methods\| PubMed ID: 40360917	2025

Lixue Shi

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