label-free-super-resolution-imaging-enabled-vibrational-imaging

Label-free Super-resolution Imaging Enabled by Vibrational Imaging of Swelled Tissue and Analysis

The universal utilization of fluorescence microscopy, especially super-resolution microscopy, has greatly advanced knowledge about modern biology. ...

California Institute of Technology

Heterotrimeric G proteins mediate cell growth and differentiation by coupling cell surface receptors to intracellular effector enzymes. The G-protein alpha subunit, Galpha(16), and its murine homologue Galpha(15), are expressed specifically in hematopoietic cells and their expression is highly regulated during differentiation of normal and leukemic cells. In this study, we examined the phosphorylation of Galpha(15)/Galpha(16) and its role in receptor and effector coupling. We observed a PMA-stimulated intact cell phosphorylation of Galpha(15) in COS7 cells transfected with Galpha(15) and protein kinase Calpha (PKCalpha), and phosphorylation of endogenous Galpha(16) in HL60 cells. We also showed that peptides derived from the two G-proteins were phosphorylated in vitro using purified brain PKC. Furthermore, we identified the putative phosphorylation site and showed that mutation or deletion of this PKC phosphorylation site inhibited phospholipase C (PLC) activation. The behavior of double mutants with the constitutively active G-protein mutation (QL-mutant) and mutation in the putative phosphorylation site suggests that the phosphorylation site of Galpha(15/16) is essential for receptor-coupled activation of PLC, but not for direct interaction of the G-protein with PLC-beta.

Regulation of hematopoietic-specific G-protein Galpha15 and Galpha16 by protein kinase C.

Several photoactive platinum alpha-diimine intercalators have been prepared to develop new probes of DNA oxidation and reduction chemistry. Five water-soluble bis(mes')Pt(II) complexes (mes' = N,N,N,3,5-pentamethylaniline) with various aromatic alpha-diimine ligands (dppz = dipyridophenazine, np = naphtha[2,3-f][1,omega]phenanthroline, CN-np = naphtho[2,3-f][1,10]phenanthroline-9-carbonitrile, CN(2)-np = naphtho[2,3-f][1,10]phenanthroline-9,14-dicarbonitrile, and bp = benzo-[f][1,10]phenanthroline) were synthesized. The complex [(np)Pt(mes')(2)]Cl(2) was also characterized by X-ray crystallography, and the crystal structure shows that the ortho-methyl groups of the mes' ligands conveniently block substitution at the vacant sites of platinum without overlapping with the intercalating alpha-diimine ligand. The Pt(II) complexes were found to have excited-state oxidation and reduction potentials of -0.6 to -1.0 and 1.0 to 1.5 V versus NHE, respectively, making them potent photoreductants as well as photooxidants. Many of the complexes are found to promote the photooxidation of N(2)-cyclopropyldeoxyguanosine (d(Cp)G). Photoexcited [(dppz)Pt(mes')(2)](2+) is found to be most efficient in this photooxidation, as well as in the photoreduction of N(4)-cyclopropylcytidine ((Cp)C); these modified nucleosides rapidly decompose in a ring-opening reaction upon oxidation or reduction. Photoexcited [(dppz)Pt(mes')(2)]Cl(2), upon intercalation into the DNA pi stack, is found, in addition, to promote reductive and oxidative damage within the DNA duplex, as is also probed using the kinetically fast electron and hole traps, (Cp)C and (Cp)G. These Pt complexes may therefore offer useful reactive tools to compare and contrast directly reductive and oxidative chemistry in double helical DNA.

Reductive and oxidative DNA damage by photoactive platinum(II) intercalators.

Heteroleptic cyclometalated complexes of Ir(III) containing the dipyridophenazine ligand are synthesized through the direct introduction of a functionalized dipyridophenazine ligand onto a bis(dichloro)-bridged Ir(III) precusor and characterized by 1H NMR, mass spectrometry, as well as spectroscopic and electrochemical properties. The excited state of the Ir(III) complexes have sufficient driving force to oxidize purines and to reduce pyrimidine nucleobases. Luminescence and EPR measurements of the Ir(III) complex with an unmodified dppz bound to DNA show the formation of a guanine radical upon irradiation, resulting from an oxidative photoinduced electron-transfer process. Evidence is also obtained indirectly for reductive photoinduced electron transfer from the excited complex to the thymine base in DNA. We have also utilized cyclopropylamine-substituted nucleosides as ultrafast kinetic traps to report transient charge occupancy in oligonucleotides when DNA is irradiated in the presence of noncovalently bound complexes. These experiments establish that the derivatized Ir(III) complexes, with photoactivation, can trigger the oxidation of guanine and the reduction of cytosine.

Synthesis and characterization of iridium(III) cyclometalated complexes with oligonucleotides: insights into redox reactions with DNA.

Three-dimensional visualization of tissue structures using optical microscopy facilitates the understanding of biological functions. However, optical microscopy is limited in tissue penetration due to severe light scattering. Recently, a series of tissue-clearing techniques have emerged to allow significant depth-extension for fluorescence imaging. Inspired by these advances, we develop a volumetric chemical imaging technique that couples Raman-tailored tissue-clearing with stimulated Raman scattering (SRS) microscopy. Compared with the standard SRS, the clearing-enhanced SRS achieves greater than 10-times depth increase. Based on the extracted spatial distribution of proteins and lipids, our method reveals intricate 3D organizations of tumor spheroids, mouse brain tissues, and tumor xenografts. We further develop volumetric phasor analysis of multispectral SRS images for chemically specific clustering and segmentation in 3D. Moreover, going beyond the conventional label-free paradigm, we demonstrate metabolic volumetric chemical imaging, which allows us to simultaneously map out metabolic activities of protein and lipid synthesis in glioblastoma. Together, these results support volumetric chemical imaging as a valuable tool for elucidating comprehensive 3D structures, compositions, and functions in diverse biological contexts, complementing the prevailing volumetric fluorescence microscopy.

Volumetric chemical imaging by clearing-enhanced stimulated Raman scattering microscopy.

Polyglutamine (polyQ) diseases are a group of neurodegenerative disorders, involving the deposition of aggregation-prone proteins with long polyQ expansions. However, the cytotoxic roles of these aggregates remain highly controversial, largely due to a lack of proper tools for quantitative and nonperturbative interrogations. Common methods including  biochemical, spectroscopic assays, and live-cell fluorescence imaging all suffer from certain limitations. Here, we propose coupling stimulated Raman scattering microscopy with deuterium-labeled glutamine for live-cell imaging, quantification, and spectral analysis of native polyQ aggregates with subcellular resolution. First, through the enrichment of deuterated glutamine in the polyQ sequence of mutant Huntingtin (mHtt) exon1 proteins for Huntington's disease, we achieved sensitive and specific stimulated Raman scattering (SRS) imaging of carbon-deuterium bonds (C-D) from aggregates without GFP labeling, which is commonly employed in fluorescence microscopy. We revealed that these aggregates became 1.8-fold denser compared to those with GFP. Second, we performed ratiometric quantifications, which indicate a surprising dependence of protein compositions on aggregation sizes. Our further calculations, for the first time, reported the absolute concentrations for sequestered mHtt and non-mHtt proteins within the same aggregates. Third, we adopted hyperspectral SRS for Raman spectroscopic studies of aggregate structures. By inducing a cellular heat shock response, a potential therapeutic approach for inhibiting aggregate formation, we found a possible aggregate intermediate state with changed solvation microenvironments. Our method may hence readily unveil new features and mechanistic insight of polyQ aggregates and pave the way for comprehensive  investigations.

Live-Cell Imaging and Quantification of PolyQ Aggregates by Stimulated Raman Scattering of Selective Deuterium Labeling.

Glycogen, a branched glucose polymer, helps regulate glucose homeostasis through immediate storage and release of glucose. Reprogramming of glycogen metabolism has recently been suggested to play an emerging role in cancer progression and tumorigenesis. However, regulation of metabolic rewiring for glycogen synthesis and breakdown in cancer cells remains less understood. Despite the availability of various glycogen detection methods, selective visualization of glycogen in living cells with high spatial resolution has proven to be highly challenging. Here, we present an optical imaging strategy to visualize glycogen in live cancer cells with minimal perturbation by combining stimulated Raman scattering microscopy with metabolic incorporation of deuterium-labeled glucose. We revealed the subcellular enrichment of glycogen in live cancer cells and achieved specific glycogen mapping through distinct spectral identification. Using this method, different glycogen metabolic phenotypes were characterized in a series of patient-derived BRAF mutant melanoma cell lines. Our results indicate that cell lines manifesting high glycogen storage level showed increased tolerance to glucose deficiency among the studied melanoma phenotypes. This method opens up the possibility for noninvasive study of complex glycogen metabolism at subcellular resolution and may help reveal new features of glycogen regulation in cancer systems.

Visualizing Subcellular Enrichment of Glycogen in Live Cancer Cells by Stimulated Raman Scattering.

Non-invasively probing metabolites within single live cells is highly desired but challenging. Here we utilize Raman spectro-microscopy for spatial mapping of metabolites within single cells, with the specific goal of identifying druggable metabolic susceptibilities from a series of patient-derived melanoma cell lines. Each cell line represents a different characteristic level of cancer cell de-differentiation. First, with Raman spectroscopy, followed by stimulated Raman scattering (SRS) microscopy and transcriptomics analysis, we identify the fatty acid synthesis pathway as a druggable susceptibility for differentiated melanocytic cells. We then utilize hyperspectral-SRS imaging of intracellular lipid droplets to identify a previously unknown susceptibility of lipid mono-unsaturation within de-differentiated mesenchymal cells with innate resistance to BRAF inhibition. Drugging this target leads to cellular apoptosis accompanied by the formation of phase-separated intracellular membrane domains. The integration of subcellular Raman spectro-microscopy with lipidomics and transcriptomics suggests possible lipid regulatory mechanisms underlying this pharmacological treatment. Our method should provide a general approach in spatially-resolved single cell metabolomics studies.

Raman-guided subcellular pharmaco-metabolomics for metastatic melanoma cells.

Reversibly photoswitchable probes allow for a wide variety of optical imaging applications. In particular, photoswitchable fluorescent probes have significantly facilitated the development of super-resolution microscopy. Recently, stimulated Raman scattering (SRS) imaging, a sensitive and chemical-specific optical microscopy, has proven to be a powerful live-cell imaging strategy. Driven by the advances of newly developed Raman probes, in particular the pre-resonance enhanced narrow-band vibrational probes, electronic pre-resonance SRS (epr-SRS) has achieved super-multiplex imaging with sensitivity down to 250 nM and multiplexity up to 24 colors. However, despite the high demand, photoswitchable Raman probes have yet to be developed. Here, we propose a general strategy for devising photoswitchable epr-SRS probes. Toward this goal, we exploit the molecular electronic and vibrational coupling, in which we switch the electronic states of the molecules to four different states to turn their ground-state epr-SRS signals on and off. First, we showed that inducing transitions to both the electronic excited state and triplet state can effectively diminish the SRS peaks. Second, we revealed that the epr-SRS signals can be effectively switched off in red-absorbing organic molecules through light-facilitated transitions to a reduced state. Third, we identified that photoswitchable proteins with near-infrared photoswitchable absorbance, whose states are modulable with their electronic resonances detunable toward and away from the pump photon energy, can function as the photoswitchable epr-SRS probes with desirable sensitivity (<1 µM) and low photofatigue (>40 cycles). These photophysical characterizations and proof-of-concept demonstrations should advance the development of novel photoswitchable Raman probes and open up the unexplored Raman imaging capabilities.

Toward photoswitchable electronic pre-resonance stimulated Raman probes.

Amyloid aggregation, formed by aberrant proteins, is a pathological hallmark for neurodegenerative diseases, including Alzheimer's disease and Huntington's disease. High-resolution holistic mapping of the fine structures from these aggregates should facilitate our understanding of their pathological roles. Here, we achieved label-free high-resolution imaging of the polyQ and the amyloid-beta (Aβ) aggregates in cells and tissues utilizing a sample-expansion stimulated Raman strategy. We further focused on characterizing the Aβ plaques in 5XFAD mouse brain tissues. 3D volumetric imaging enabled visualization of the whole plaques, resolving both the fine protein filaments and the surrounding components. Coupling our expanded label-free Raman imaging with machine learning, we obtained specific segmentation of aggregate cores, peripheral filaments together with cell nuclei and blood vessels by pre-trained convolutional neural network models. Combining with 2-channel fluorescence imaging, we achieved a 6-color holistic view of the same sample. This ability for precise and multiplex high-resolution imaging of the protein aggregates and their micro-environment without the requirement of labeling would open new biomedical applications.

High spatial-resolution imaging of label-free  protein aggregates by VISTA.

Innovations in high-resolution optical imaging have allowed visualization of nanoscale biological structures and connections. However, super-resolution fluorescence techniques, including both optics-oriented and sample-expansion based, are limited in quantification and throughput especially in tissues from photobleaching or quenching of the fluorophores, and low-efficiency or non-uniform delivery of the probes. Here, we report a general sample-expansion vibrational imaging strategy, termed VISTA, for scalable label-free high-resolution interrogations of protein-rich biological structures with resolution down to 78 nm. VISTA achieves decent three-dimensional image quality through optimal retention of endogenous proteins, isotropic sample expansion, and deprivation of scattering lipids. Free from probe-labeling associated issues, VISTA offers unbiased and high-throughput tissue investigations. With correlative VISTA and immunofluorescence, we further validated the imaging specificity of VISTA and trained an image-segmentation model for label-free multi-component and volumetric prediction of nucleus, blood vessels, neuronal cells and dendrites in complex mouse brain tissues. VISTA could hence open new avenues for versatile biomedical studies.

Super-resolution label-free volumetric vibrational imaging.

Photoactivatable probes, with high-precision spatial and temporal control, have largely advanced bioimaging applications, particularly for fluorescence microscopy. While emerging Raman probes have recently pushed the frontiers of Raman microscopy for noninvasive small-molecule imaging and supermultiplex optical imaging with superb sensitivity and specificity, photoactivatable Raman probes remain less explored. Here, we report the first general design of multicolor photoactivatable alkyne Raman probes based on cyclopropenone caging for live-cell imaging and tracking. The fast photochemically generated alkynes from cyclopropenones enable background-free Raman imaging with desired photocontrollable features. We first synthesized and spectroscopically characterized a series of model cyclopropenones and identified the suitable light-activating scaffold. We further engineered the scaffold for enhanced chemical stability in a live-cell environment and improved Raman sensitivity. Organelle-targeting probes were then generated to achieve targeted imaging of mitochondria, lipid droplets, endoplasmic reticulum, and lysosomes. Multiplexed photoactivated imaging and tracking at both subcellular and single-cell levels was next demonstrated to monitor the dynamic migration and interactions of the cellular contents. We envision that this general design of multicolor photoactivatable Raman probes would open up new ways for spatial-temporal controlled profiling and interrogations in complex biological systems with high information throughput.

Multicolor Photoactivatable Raman Probes for Subcellular Imaging and Tracking by Cyclopropenone Caging.

Alkyne-tagged Raman probes have shown high promise for noninvasive and sensitive visualization of small biomolecules to understand their functional roles in live cells. However, the potential for alkynes to sense cellular environments that goes beyond imaging remains to be further explored. Here, we report a general strategy for Raman imaging-based local environment sensing by hydrogen-deuterium exchange (HDX) of terminal alkynes (termed alkyne-HDX). We first demonstrate, in multiple Raman probes, that deuterations of the alkynyl hydrogens lead to remarkable shifts of alkyne Raman peaks for about 130 cm, providing resolvable signals suited for imaging-based analysis with high specificity. Both our analytical derivation and experimental characterizations subsequently establish that HDX kinetics are linearly proportional to both alkyne ps and environmental pDs. After validating the quantitative nature of this strategy, we apply alkyne-HDX to sensing local chemical and cellular environments. We establish that alkyne-HDX exhibits high sensitivity to various DNA structures and demonstrates the capacity to detect DNA structural changes  from UV-induced damage. We further show that this strategy is also applicable to resolve subtle pD variations in live cells. Altogether, our work lays the foundation for utilizing alkyne-HDX strategy to quantitatively sense the local environments for a broad spectrum of applications in complex biological systems.

Lu Wei

Division of Chemistry and Chemical Engineering

California Institute of Technology

Label-free Super-resolution Imaging Enabled by Vibrational Imaging of Swelled Tissue and Analysis