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

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.

Silicon (Si)-based material is a promising anode material for next-generation lithium-ion batteries (LIBs). Herein, we report the fabrication of a silicon oxide-carbon (SiO/C) nanocomposite through the reaction between silicon particles with fresh surface and HO in a mild hydrothermal condition, as well as conducting carbon coating synchronously. We found that controllable oxidation could be realized for Si particles to produce uniform SiO after the removal of the native passivation layer. The uniform oxidation and conductive coating offered the as-fabricated SiO/C composite good stability at both particle and electrode level over electrochemical cycling. The as-fabricated SiO/C composite delivered a high reversible capacity of 1133 mAh g at 0.5 A g with 89.1% capacity retention after 200 cycles. With 15 wt % SiO/C composite, graphite-SiO/C hybrid electrode displayed a high reversible specific capacity of 496 mAh g and stable electrochemical cycling with a capacity retention of 90.1% for 100 cycles.

Manipulating Oxidation of Silicon with Fresh Surface Enabling Stable Battery Anode.

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.

Urea, an agricultural fertilizer, nourishes humanity. The century-old Bosch-Meiser process provides the world's urea. It is multi-step, consumes enormous amounts of non-renewable energy, and has a large CO footprint. Thus, developing an eco-friendly synthesis for urea is a priority. Herein we report a single-step Pd/LTA-3A catalyzed synthesis of urea from CO and NH under ambient conditions powered solely by solar energy. Pd nanoparticles serve the dual function of catalyzing the dissociation of NH and providing the photothermal driving force for urea formation, while the absorption capacity of LTA-3A removes by-product H O to shift the equilibrium towards urea production. The solar urea conversion rate from NH and CO is 87 μmol g  h . This advance represents a first step towards the use of solar energy in urea production. It provides insights into green fertilizer production, and inspires the vision of sustainable, modular plants for distributed production of urea on farms.

Chenxi Qian

Division of Chemistry and Chemical Engineering

California Institute of Technology

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

Chenxi Qian

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Division of Chemistry and Chemical Engineering

California Institute of Technology

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

Division of Chemistry and Chemical Engineering