Bioimaging harnessing optical contrasts and chemical specificity is of vital importance in probing complex biology. Vibrational spectroscopy based on mid-infrared excitation can reveal rich chemical information about molecular distributions. However, its full potential for bioimaging is hindered by the achievable sensitivity. Here we report bond-selective fluorescence-detected infrared-excited (BonFIRE) spectro-microscopy. BonFIRE employs two-photon excitation in the mid- and near-infrared to upconvert vibrational excitations to electronic states for fluorescence detection, thus encoding vibrational information into fluorescence. We demonstrate BonFIRE spectral imaging in large spectroscopic windows on samples ranging from single molecules to biological specimens. We then extend BonFIRE imaging from point-scanning to wide-field configuration, enabling ultrafast imaging with a large field of view. We expect BonFIRE to expand the bioimaging toolbox by providing a new level of bond-specific vibrational information and facilitate biological investigations.
KEYWORDS: Raman spectroscopy, Environmental sensing, Chemical analysis, Visualization, In situ remote sensing, Hydrogen, Complex systems, Biological and chemical sensing
Alkyne-tagged Raman probes have shown high promise for noninvasive and sensitive visualization of small biomolecules in live cells. Here, we report a general strategy for Raman imaging-based local environment sensing by hydrogen-deuterium exchange (HDX) of terminal alkynes. We first demonstrate, in multiple Raman probes, that deuterations of the alkynyl hydrogens lead to remarkable shifts of alkyne Raman peaks, 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 pKas and environmental pDs. We establish that alkyne-HDX exhibits high sensitivity to various DNA structures and subtle pD variations in live cells. After validating the quantitative nature of this strategy, our work lays the foundation for utilizing alkyne-HDX strategy to quantitatively sense the local chemical and cellular environments.
Innovations in optical spectroscopy and microscopy have revolutionized our understanding in live biological systems at the sub-cellular levels. In this talk, I will present our recent advancements in developing and applying stimulated Raman scattering (SRS) imaging coupled with novel vibrational tags for specific and highly sensitive investigations of complex biological (i.e. cancer- and neuronal-) systems.
High-resolution optical imaging techniques allowed the visualization of nanoscale biological structures and molecules. Current fluorescence-based super-resolution techniques are limited in throughput and quantification. We developed a high-resolution versatile imaging modality, called Vibrational Imaging of Swelled Tissues and Analysis (VISTA), based on a sample-expansion vibrational imaging strategy. We achieve label-free super-resolution imaging, and successfully used a U-net convolutional network to predict specific cellular features. We further applied VISTA to investigate protein aggregations in biological samples, including aggregates formed in Huntington’s disease and Alzheimer’s disease. This precise and multiplex high-resolution imaging modality opens new avenues for versatile biomedical studies.
We utilized Raman spectro-microscopy to non-invasively probe metabomics within single live cells, aiming to identify druggable metabolic susceptibilities from a series of patient-derived BRAF mutant melanoma cell lines. Each cell line represents a phenotype with different characteristic level of de-differentiation and BRAFi (BRAF inhibitor) resistance. First, with single-cell Raman spectroscopy and stimulated Raman scattering (SRS) microscopy, followed by transcriptomics analysis, we identified lipid processes as major metabolic functional difference between different phenotypes. We then utilized hyperspectral-SRS imaging on intracellular single organelles to identify a previously unknown susceptibility of lipid desaturation within de-differentiated cell lines. Drugging this target leads to cellular apoptosis accompanied by phase separated intracellular domains. The integration of subcellular Raman spectro-microscopy with lipidomics and transcriptomics suggests highly heterogenous metabolic responses and possible lipid regulatory mechanisms underlying this pharmacological treatment. Our method should provide a general approach in spatially-resolved single cell metabolomics studies.
KEYWORDS: Raman scattering, Proteins, Spectroscopy, Raman spectroscopy, Microscopy, Luminescence, Green fluorescent protein, Spectral resolution, In vivo imaging, In vitro testing
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 controversial, largely due to a lack of proper tools for quantitative and nonperturbative interrogations. Common methods including in vitro 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 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 an aggregation intermediate state. Vibrational line shapes of the polyQ aggregates suggested that they experience a hyper-hydrated environment during the intermediate state. Our method should fill the gap and serve as a suitable tool to study native polyQ aggregates. It may unveil new features of polyQ aggregates and pave the way for comprehensive in vivo investigations.
Innovations in optical spectroscopy and microscopy have revolutionized our understanding in
biological systems at sub-cellular levels. In this talk, I will discuss about our recent development by coupling stimulated Raman scattering (SRS) microscopy with chemical probes that could allow new subcellular bioanalysis in live cells. The introduced tags offer additional SRS contrast channel for quantification of biological contents that were previously difficult. Both physical and chemical principles underlying the optical microscopy will be presented, as well as our efforts in biomedical applications including cancer- and neuronal- metabolism.
Innovations in novel probes have significantly push the development of new optical spectroscopy and microscopy methods for revealing new information in biological systems. In this talk, I will discuss our recent
development by introducing chemical probes to stimulated Raman scattering (SRS) microscopy
that could allow multi-functional imaging at sub-cellular level. Both physical and chemical principles underlying the
investigation and design of new probes when coupled to the Raman imaging modalities will be presented, as well as our efforts in biomedical applications including cancer- and neuronal- metabolism
Innovations in optical spectroscopy and microscopy have revolutionized our understanding in
biological systems. In this talk, I will discuss our recent development by coupling stimulated Raman scattering (SRS) microscopy with chemical probes that could allow high-sensitivity bio-analysis with fast speed at the sub-cellular level. Both physical and chemical principles underlying the optical microscopy will be presented, as well as our efforts in biomedical applications including cancer- and neuronal- metabolism.
Glucose is consumed as an energy source by virtually all living organisms, from bacteria to humans. Its uptake activity closely reflects the cellular metabolic status in various pathophysiological transformations, such as diabetes and cancer. Extensive efforts such as positron emission tomography, magnetic resonance imaging and fluorescence microscopy have been made to specifically image glucose uptake activity but all with technical limitations. Here, we report a new platform to visualize glucose uptake activity in live cells and tissues with subcellular resolution and minimal perturbation. A novel glucose analogue with a small alkyne tag (carbon-carbon triple bond) is developed to mimic natural glucose for cellular uptake, which can be imaged with high sensitivity and specificity by targeting the strong and characteristic alkyne vibration on stimulated Raman scattering (SRS) microscope to generate a quantitative three dimensional concentration map. Cancer cells with differing metabolic characteristics can be distinguished. Heterogeneous uptake patterns are observed in tumor xenograft tissues, neuronal culture and mouse brain tissues with clear cell-cell variations. Therefore, by offering the distinct advantage of optical resolution but without the undesirable influence of bulky fluorophores, our method of coupling SRS with alkyne labeled glucose will be an attractive tool to study energy demands of living systems at the single cell level.
Two-photon excited fluorescence microscopy (TPFM) offers the highest penetration depth with subcellular resolution in light microscopy, due to its unique advantage of nonlinear excitation. However, a fundamental imaging-depth limit, accompanied by a vanishing signal-to-background contrast, still exists for TPFM when imaging deep into scattering samples. Formally, the focusing depth, at which the in-focus signal and the out-of-focus background are equal to each other, is defined as the fundamental imaging-depth limit. To go beyond this imaging-depth limit of TPFM, we report a new class of super-nonlinear fluorescence microscopy for high-contrast deep tissue imaging, including multiphoton activation and imaging (MPAI) harnessing novel photo-activatable fluorophores, stimulated emission reduced fluorescence (SERF) microscopy by adding a weak laser beam for stimulated emission, and two-photon induced focal saturation imaging with preferential depletion of ground-state fluorophores at focus. The resulting image contrasts all exhibit a higher-order (third- or fourth- order) nonlinear signal dependence on laser intensity than that in the standard TPFM. Both the physical principles and the imaging demonstrations will be provided for each super-nonlinear microscopy. In all these techniques, the created super-nonlinearity significantly enhances the imaging contrast and concurrently extends the imaging depth-limit of TPFM. Conceptually different from conventional multiphoton processes mediated by virtual states, our strategy constitutes a new class of fluorescence microscopy where high-order nonlinearity is mediated by real population transfer.
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