Illuminating life processes by vibrational probes

Abstract

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.

Fig. 1 |. Vibrational probes for bioimaging with high sensitivity, specificity and biocompatibility.

a, Small vibrational tags were reported in the cell-silent region for bioimaging, which has high specificity and optimal biocompatibility. Lists of chemical structures for metabolic imaging and drug imaging are included as examples, and the vibrational tags are highlighted in colors. M indicates small metabolites such as amino acids, fatty acids, glucose and other bioactive small molecules, such as drugs. b, Molecular vibrations as a contrast for sensitive bioimaging. Vibrational tags and dyes providing sensitive contrasts undergoing different processes of vibrational transition. Cross-sections are illustrated for each process with characteristic chemical structures of the corresponding vibrational reporters. Resonance enhancement can also be coupled into different processes to provide additional signal enhancement as indicated in the dashed box.

Fig. 2 |. Vibrational probes with multichromatic colors and diverse functions.

a, Vibrational palettes based on cyanine dyes are designed with distinct fingerprint signatures by introducing isotopes and modifying substituents on the periphery ring or the center Y-position of the conjugation system. Different vibrational colors’ resolution requires linear spectra decomposition across the entire window. b,c, The idea of super-multiplexing vibrational imaging is established in the cell-silent region with 20 or more vibrational dyes presenting well-resolved single-peak signatures in the narrow window. (b) Carbow palettes based on polyynes were developed by varying the conjugation length of the polyyne backbones, modifying the substituents at the end-capping phenyl ring, as well as isotope editing of the selected triple bonds. (c) MARS palettes based on xanthenes, were developed by changing the central atoms at the 10 position (O, C, Si), the number of extended rings substation on the periphery and isotope editing of the conjugated triple bonds at the 9 position^,. d–f, The general strategies to tune vibrational colors include (d) modifying substituents in conjugation, (e) isotope editing to generate distinct vibrational colors, which can be further combined with (f) varied intensities for barcoding to further expand the information capacity for target labeling and analysis. g–j, Vibrant vibrational colors can be engineered to render diverse functionality of biological systems. Biological structures can be imaged by (g) epitope labeling using vibrational dye-labeled antibodies and aptamers or (h) targeting organelles by functionalizing the vibrational reporters with chemical moieties for high-affinity interactions with specific organelles. Chemical structures of vibrational probes are listed for different organelles, including mitochondria (Mito), lysosomes (Lyso), plasma membrane (PM), ER and lipid droplets (LD). Biological activity can be probed by vibrational sensors or metabolic probes based on small vibrational tags. (i) Vibrational sensing can be achieved through a turn-on design (for example, a signal increase from pre-resonance enhancement upon the sensing reactions) or ratiometric readout (for example, frequency shifting upon sensing reactions). The sensing mechanisms of corresponding vibrational sensors are illustrated as examples. (j) Both general metabolic activity and metabolism of specific pathways can be monitored by vibrational imaging using listed metabolic probes labeled by small vibrational tags with high specificity and minimal perturbation of the biological systems.

Fig. 3 |. Metabolic imaging of biological systems using vibrational probes.

a, SRS imaging cholesterol trafficking probed by PhDY-Chol (structure shown in Fig. 1), with sensitivity to HPβCD (drug for NP-C disease) treatment in M12 cells. Scale bar, 10 µm. b, Simultaneous imaging of ODYA (left, structure shown in Fig. 1) and D₇-glucose by SRS microscopy reveals the correlation between the ratio of glucose-derived anabolism and fatty acid uptake and the level of cisplatin resistance in primary ovarian cancer cells (right). Scale bar, 20 µm. c, SRS imaging of unsaturated (top) and saturated (bottom) fatty acid metabolites (C–D SRS). d, SRS ratiometric image of 2,101/2,168 cm⁻¹ as a measure of conformational order for D-palmitate treated HeLa cells. e, Time-lapse SRS imaging of sucrose uptake probed by alkyne-labeled sucrose analog (structure shown in Fig. 1) in living BY2 cells. f, Simultaneous imaging of glucose uptake (3-OPG, structure shown in Fig. 1) and incorporation (D₇-glucose) by SRS microscopy reveals the different activity of glucose metabolism for in different cell types (co-cultured PC-3/RWPE-1 cells). Scale bar, 20 µm. g, SRS imaging of nucleic acid synthesis in HeLa cells measured by EU (for RNA, structure shown in Fig. 1). Scale bar, 10 µm. h, Protein synthesis and turnover visualized by vibrational imaging of D₅-glutamine targeting poly(Q) sequence of mutant Huntingtin (mHtt) exon1 proteins). i, Newly synthesized lipids (left), proteins (middle) and DNA (right) resolved from hyperspectral SRS imaging of deuterated water-labeled cells. j–u. Metabolic probes can also be extended to image the spatial metabolic heterogeneity inside organisms. Examples are (j) O-PTIR imaging of newly synthesized lipids labeled by azide-tagged palmitic acid (structure shown in Fig. 1) with immunofluorescence imaging showing the distribution of neurons (TUJ1) and astrocytes (GFAP, glial fibrillary acidic protein). Scale bar, 100 µm. (k) SRS image of 3D spheroid treated with 5 µM of lapatinib to visualize drug uptake and protein synthesis labeled by deuterated amino acids). Scale bar, 40 µm. (l) SRS imaging of D₉-choline incorporation in C. elegans larvae. Scale bar, 5 µm. (m) SRS imaging of deuterium incorporation for C. elegans nhr-25(ku217) mutant and wild types from deuterium-labeled bacteria diet. Scale bar, 50 µm. (n) SRS imaging of newly synthesized protein inside zebrafish labeled by deuterated amino acids. Scale bar, 10 µm. (o) SRS imaging of diet-regulated lipid metabolic activity in old flies labeled by D₂O (ref. 111). (p) SRS imaging of β-galactosidase activity in a Drosophila wing disc reported by 9CN-JCR-based sensors (mechanism illustrated in Fig. 2). Scale bar, 10 µm. (q) SRS imaging of lipid synthesis across the cerebrum of pulp mice labeled by D₇-glucose. (r) SRS imaging of protein synthesis in the pancreas collected from adult mice administered D₂O. (s) Metabolic volumetric SRS imaging of glioblastoma labeled by D₂O. Scale bar, 50 µm. (t) Discrete frequency mid-infrared (DFIR) imaging on D₂O-labeled biofilm by mapping absorbance at C–D 2,150 cm⁻¹. Scale bars, 40 µm. (u) Ratiometric SRS imaging (CD/CH) of the cross-sections of hypocotyl/root from adult Arabidopsis thaliana plants labeled by D₂O, revealing metabolic heterogeneity in the radial panels. Scale bar, 100 µm.

Fig. 4 |. Vibrational imaging of drugs and nanocarriers.

a, Live-cell Raman imaging of deuterated Mito-Q. b, Live-cell SRS imaging of diyne labeled ferrostatin (ferrostatin-2, structure shown in Fig. 1). Scale bar, 10 µm. c, SRS imaging of bisarylbutadiyne labeled anisomycin (BADY-ANS, structure shown in Fig. 1) in SKBR3 cells. Scale bar, 10 µm. d, SRS imaging of anticancer antimycintype depsipeptide in HeLa cells (PhDY-Ant, structure shown in Fig. 1). e, SRS imaging of methoxypyridazyl pyrimidyl butadiyne-labeled JQ1 (MPDY-JQ1, structure shown in Fig. 1) in living HeLa cells. Scale bar, 5 µm. f, Quantitative SERS imaging of alkyne-labeled cathepsin B inhibitor (Alt-AOMK, structure shown in Fig. 1) in living cells. g, SRS imaging of PBCA nanocarriers crossing the BBB of mouse brains. Scale bar, 5 µm. NP, nanoparticle; DAPI, 4′,6-diamidino-2-phenylindole. h, SRS imaging of deuterium- and alkyne-labeled PLGA nanoparticles in cells and tissues. Scale bar, 20 µm. i, Raman imaging of uptake of liposomal drug carrier systems labeled with deuterium in MCF-7 cells. j, SRS imaging of deuterated solid lipid nanoparticles (D-SLNs) inside macrophage cells. Scale bar, 5 µm. k, Multiplexed Raman imaging of SERS particles targeted in vivo cancer detection in a living mouse.

Fig. 5 |. Supermultiplexed vibrational imaging and cell profiling.

a, Multiplexing Raman mapping of ten different SERS particles injected in a nude mouse. b, SERS imaging of breast cancer to reveal the coexpression relationships among ten-panel immune checkpoints in breast cancer tissues biopsy to predict the optimal combination of antibody drugs for immunotherapy. Supermultiplexed SERS nanoprobes are prepared by incorporating a wide palette of small molecules with vibrational signatures in the cell-silent region. BF, bright field. c, Eight-color simultaneous imaging of DNA replication and protein synthesis in hippocampal neuronal cultures using MARS probes by epr-SRS and fluorescence microscopy. HPG labels proteins synthesized in the pulse period and AHA labels proteins synthesized in the chase period; NeuN, neurons; MBP, myelin basic protein. d, Ten-color simultaneous imaging of cellular organelles (mitochondria (Mito), lysosomes (Lyso), ER, PM, LD, Golgi, tubulin, actin and nucleus) in living cells using Carbow probes by epr-SRS and fluorescence microscopy. e, Supermultiplex time-lapse imaging of organelle interaction dynamics using Carbow probes by integrated stimulated Raman and fluorescence microscopy. FBS, fetal bovine serum; OA, oleic acid. f, Supermultiplexed volumetric imaging of 11 epitopes in a 1-mm-thick mouse cerebellum section by Raman dye imaging and tissue clearing (RADIANT) based on MARS probes. ConA, concanavalin A; LEL, Lycopersicon esculentum lectin; WGA, wheat germ agglutinin; GS-II, Griffonia simplicifolia lectin. g, MAP of single cells based on micro-Raman spectroscopy and deuterated metabolic probes. Workflow for high-throughput quantification of metabolic activity at single-cell level is illustrated with metabolic heterogeneity drevealed upon various drug treatments and different cancer subtypes. h, Single-cell metabolic analysis of microbial communities with D₂O using SRS-FISH. i, Supermultiplexed live-cell profiling with 14 Raman probes in the amide and cell-silent region with phenotypic profiling over multiple chemotherapy treatments shown on the top and single-cell live-cell spectra shown in the bottom. j, Spectral profiling for single-cell drug responses labeled by IR-active vibrational probes with workflow illustrated to map cell phenotypes after drug treatment. IC₅₀, half maximum inhibitory concentration; FM, fixed mirror; FT-IR, Fourier transform IR; MM, moving mirror.

Fig. 6 |. Functional sensing with vibrational probes.

a, Raman spectral of CD2 stretching from deuterated lipids with sensitivity to conformational order, which is helpful to reveal intracellular membrane phase separation (Fig. 3c). b, Rh800 with nitrile stretching sensitive to water environment. The linear response of nitrile peak frequency to bound water volume percentage (left) is utilized to reveal the intracellular water heterogeneity by SREF imaging (right). c, Local environment sensing by HDX of terminal alkynes (termed alkyne-HDX) monitored by Raman spectroscopy (left), which can be applied to detect DNA structural changes in situ by ratiometric SRS imaging (right). Scale bar, 20 µm. d, Raman imaging of pH-sensitive bisarylbutadiyne for ratiometric measurement of cellular pH. e, Enzymatic activity sensing and mapping by O-PTIR imaging of nitrile chameleons with both peak shift and signal enhancement upon enzymatic reactions. Activity distribution of phosphatase and caspase activities is shown in Dox-pretreated (1 µM) mouse cerebral cortex sections. Scale bar, 100 µm. f, Multiplexed sensing of intracellular labile Cu(I) and Cu(II) pools using SRS microscopy with activity-based copper Raman probes. g, Multiplexed enzymatic activity sensing and mapping by epr-SRS imaging of isotope-edited activatable 9CN-JCP (structures shown on the left), simultaneous detecting of the four enzyme activities in live cells by SRS imaging (multicolor imaging of A549 cells in the middle with corresponding SRS spectra shown on the right). Scale bar, 10 µm. h, Photo-activable Raman probes for subcellular imaging of organelles with high spatiotemporal controls using light-sensitive cyclopropenone caging. i, Super-resolution SRS imaging of HeLa cells labeled by photoswitchable diarylethene targeting mitochondria, with line profile shown along black dashed lines. Scale bar, 1 µm. j, Multiplexed SRS photo switches developed based on photosensitive polyynes with reversible light control.