Molecular probes of temperature (termed \"molecular thermometers\") have become broadly used for in situ temperature measurements. Here, we describe Boltzmann-edge vibrational thermometry (BET) detected by anti-Stokes fluorescence, where the relative population of vibrationally excited molecules acts as a calibration-free reporter of local temperature based on the Boltzmann distribution. We demonstrate that BET microscopy is readily compatible with biological samples and achieves single-molecule sensitivity. We then show that local environments can be characterized through the modulation of vibrational temperature by mid-infrared absorption, allowing for BET fingerprinting. This work provides a foundation for sensitive vibrational thermometry in biological imaging.
", "doi": "10.1021/acs.jpcb.5c04024", "pmcid": "PMC12396515", "issn": "1520-6106", "publisher": "American Chemical Society", "publication": "Journal of Physical Chemistry B", "publication_date": "2025-08-28", "series_number": "34", "volume": "129", "issue": "34", "pages": "8788-8797" }, { "id": "authors:zmfd4-erf28", "collection": "authors", "collection_id": "zmfd4-erf28", "cite_using_url": "https://authors.library.caltech.edu/records/zmfd4-erf28", "type": "article", "title": "Two-dimensional bond-selective fluorescence spectroscopy: violations of the resonance condition, vibrational cooling rate dispersion, and super-multiplex imaging", "author": [ { "family_name": "Kocheril", "given_name": "Philip A.", "orcid": "0000-0002-1265-5931", "clpid": "Kocheril-Philip-A" }, { "family_name": "Du", "given_name": "Jiajun", "orcid": "0000-0003-2693-834X", "clpid": "Du-Jiajun" }, { "family_name": "Wang", "given_name": "Haomin", "orcid": "0000-0001-7193-8651", "clpid": "Wang-Haomin" }, { "family_name": "Leighton", "given_name": "Ryan E.", "orcid": "0000-0003-0077-413X", "clpid": "Leighton-Ryan-E" }, { "family_name": "Lee", "given_name": "Dongkwan", "orcid": "0000-0001-6091-1349", "clpid": "Lee-Dongkwan" }, { "family_name": "Yang", "given_name": "Ziguang", "orcid": "0000-0003-3927-0393", "clpid": "Yang-Ziguang" }, { "family_name": "Naji", "given_name": "Noor", "orcid": "0009-0008-3401-6226", "clpid": "Naji-Noor" }, { "family_name": "Colazo", "given_name": "Adrian", "orcid": "0000-0002-0702-0851", "clpid": "Colazo-Adrian" }, { "family_name": "Wei", "given_name": "Lu", "orcid": "0000-0001-9170-2283", "clpid": "Wei-Lu" } ], "abstract": "Multidimensional spectroscopies have shaped our understanding of molecular phenomena, but they are often limited in sensitivity. In this work, we describe two-dimensional bond-selective fluorescence-detected infrared-excited (2D-BonFIRE) spectro-microscopy: an ultrasensitive two-dimensional spectroscopy and hyperspectral imaging technique. 2D-BonFIRE spectra are richly detailed, allowing for direct measurement of vibronic coupling and strong evidence of combination modes in congested spectral regions. Additionally, 2D-BonFIRE provides new insights into the nature of vibrational relaxation, including direct experimental observation of vibrational cooling rate dispersion, illuminating the inherent heterogeneity of vibrational decays in large molecules. Finally, we demonstrate that the high specificity of 2D-BonFIRE allows for single-shot 16-colour chemical imaging, with high promise for further palette expansion. 2D-BonFIRE holds significant potential as a tool for fundamental photophysics and a basis for super-multiplex bioimaging.
", "doi": "10.1039/d5sc02628h", "pmcid": "PMC12336853", "issn": "2041-6520", "publisher": "Royal Society of Chemistry", "publication": "Chemical Science", "publication_date": "2025-07-30", "series_number": "33", "volume": "16", "issue": "33", "pages": "14905-14918" }, { "id": "authors:7b4z7-qkq61", "collection": "authors", "collection_id": "7b4z7-qkq61", "cite_using_url": "https://authors.library.caltech.edu/records/7b4z7-qkq61", "type": "article", "title": "Wide-field bond-selective fluorescence imaging: from single-molecule to cellular imaging beyond video rate", "author": [ { "family_name": "Lee", "given_name": "Dongkwan", "orcid": "0000-0001-6091-1349", "clpid": "Lee-Dongkwan" }, { "family_name": "Wang", "given_name": "Haomin", "orcid": "0000-0001-7193-8651", "clpid": "Wang-Haomin" }, { "family_name": "Kocheril", "given_name": "Philip A.", "orcid": "0000-0002-1265-5931", "clpid": "Kocheril-Philip-A" }, { "family_name": "Bi", "given_name": "Xiaotian", "orcid": "0000-0003-4449-7488", "clpid": "Bi-Xiaotian" }, { "family_name": "Naji", "given_name": "Noor", "clpid": "Naji-Noor" }, { "family_name": "Wei", "given_name": "Lu", "orcid": "0000-0001-9170-2283", "clpid": "Wei-Lu" } ], "abstract": "Wide-field (WF) imaging is pivotal for observing dynamic biological events. While WF chemical microscopy offers high molecular specificity, it lacks the sensitivity for single-molecule detection. In contrast, WF fluorescence microscopy provides live-cell dynamic mapping but fails to leverage the rich chemical information necessary for functional interpretations. To address these limitations, we introduce wide-field bond-selective fluorescence-detected infrared-excited (WF-BonFIRE) spectro-microscopy. This technique combines rationally optimized imaging speed and field of view (FOV) to achieve single-molecule sensitivity with bond-selective contrast. We demonstrate WF-BonFIRE's capabilities in imaging single molecules, cells, astrocytes, and live neurons, capturing single FOVs up to 50\u00b5m\u00d750\u00b5m, with further expansion via multi-FOV mosaicking. Additionally, we have implemented a temporal-delay modulation scheme that allows real-time kilohertz WF-BonFIRE imaging with speeds up to 1500\u00a0Hz. We showcase the millisecond temporal resolution through monitoring the random motion of live Escherichia coli. Leveraging its ability to distinguish molecules through distinct narrow-band BonFIRE signals, we further demonstrate multicolor real-time E. coli tracking. WF-BonFIRE should significantly broaden the boundary for chemical imaging, enabling high-speed observations at unparalleled sensitivity levels.", "doi": "10.1364/optica.545195", "issn": "2334-2536", "publisher": "Optica Publishing Group", "publication": "Optica", "publication_date": "2025-02-20", "series_number": "2", "volume": "12", "issue": "2", "pages": "148" }, { "id": "authors:22794-a6f52", "collection": "authors", "collection_id": "22794-a6f52", "cite_using_url": "https://authors.library.caltech.edu/records/22794-a6f52", "type": "article", "title": "Nitrile Vibrational Lifetimes as Probes of Local Electric Fields", "author": [ { "family_name": "Kocheril", "given_name": "Philip A.", "orcid": "0000-0002-1265-5931", "clpid": "Kocheril-Philip-A" }, { "family_name": "Wang", "given_name": "Haomin", "orcid": "0000-0001-7193-8651", "clpid": "Wang-Haomin" }, { "family_name": "Lee", "given_name": "Dongkwan", "orcid": "0000-0001-6091-1349", "clpid": "Lee-Dongkwan" }, { "family_name": "Naji", "given_name": "Noor", "clpid": "Naji-Noor" }, { "family_name": "Wei", "given_name": "Lu", "orcid": "0000-0001-9170-2283", "clpid": "Wei-Lu" } ], "abstract": "Optical measurements of electric fields have wide-ranging applications in the fields of chemistry and biology. Previously, such measurements focused on shifts in intensity or frequency. Here, we show that nitrile vibrational lifetimes can report local electric fields through ultrasensitive picosecond mid-infrared–near-infrared double-resonance fluorescence spectro-microscopy on Rhodamine 800. Using a robust convolution fitting approach, we observe that the nitrile vibrational lifetimes are strongly linearly correlated (R2 = 0.841) with solvent reaction fields. Supported by density functional theory, we rationalize this trend through a doorway model of intramolecular vibrational energy redistribution. This work provides new fundamental insights into the nature of vibrational energy flow in large polyatomic molecular systems and establishes a theoretical basis for electric field sensing with vibrational lifetimes, offering a new experimental dimension for probing intracellular electrostatics.
", "doi": "10.1021/acs.jpclett.4c00597", "issn": "1948-7185", "publisher": "American Chemical Society", "publication": "Journal of Physical Chemistry Letters", "publication_date": "2024-05-16", "series_number": "9", "volume": "15", "issue": "9", "pages": "5306-5314" }, { "id": "authors:ckx23-fcn21", "collection": "authors", "collection_id": "ckx23-fcn21", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20230630-744789000.1", "type": "article", "title": "Bond-selective fluorescence imaging with single-molecule sensitivity", "author": [ { "family_name": "Wang", "given_name": "Haomin", "orcid": "0000-0001-7193-8651", "clpid": "Wang-Haomin" }, { "family_name": "Lee", "given_name": "Dongkwan", "orcid": "0000-0001-6091-1349", "clpid": "Lee-Dongkwan" }, { "family_name": "Cao", "given_name": "Yulu", "clpid": "Cao-Yulu" }, { "family_name": "Bi", "given_name": "Xiaotian", "orcid": "0000-0003-4449-7488", "clpid": "Bi-Xiaotian" }, { "family_name": "Du", "given_name": "Jiajun", "orcid": "0000-0003-2693-834X", "clpid": "Du-Jiajun" }, { "family_name": "Miao", "given_name": "Kun", "orcid": "0000-0001-6567-3650", "clpid": "Miao-Kun" }, { "family_name": "Wei", "given_name": "Lu", "orcid": "0000-0001-9170-2283", "clpid": "Wei-Lu" } ], "abstract": "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. The system utilizes tunable narrowband picosecond pulses to ensure high sensitivity, biocompatibility and robustness for bond-selective biological interrogations over a wide spectrum of reporter molecules. We demonstrate BonFIRE spectral imaging in both fingerprint and cell-silent spectroscopic windows with single-molecule sensitivity for common fluorescent dyes. We then demonstrate BonFIRE imaging on various intracellular targets in fixed and live cells, neurons and tissues, with promise for further vibrational multiplexing. For dynamic bioanalysis in living systems, we implement a high-frequency modulation scheme and demonstrate time-lapse BonFIRE microscopy of live HeLa cells. We expect BonFIRE to expand the bioimaging toolbox by providing a new level of bond-specific vibrational information and facilitate functional imaging and sensing for biological investigations.", "doi": "10.1038/s41566-023-01243-8", "pmcid": "PMC10756635", "issn": "1749-4885", "publisher": "Nature Publishing Group", "publication": "Nature Photonics", "publication_date": "2023-10", "series_number": "10", "volume": "17", "issue": "10", "pages": "846-855" }, { "id": "authors:x1cq9-y3414", "collection": "authors", "collection_id": "x1cq9-y3414", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20230630-537228000.5", "type": "article", "title": "Toward the Next Frontiers of Vibrational Bioimaging", "author": [ { "family_name": "Wang", "given_name": "Haomin", "orcid": "0000-0001-7193-8651", "clpid": "Wang-Haomin" }, { "family_name": "Lee", "given_name": "Dongkwan", "orcid": "0000-0001-6091-1349", "clpid": "Lee-Dongkwan" }, { "family_name": "Wei", "given_name": "Lu", "orcid": "0000-0001-9170-2283", "clpid": "Wei-Lu" } ], "abstract": "Chemical imaging based on vibrational contrasts can extract molecular information entangled in complex biological systems. To this end, nonlinear Raman scattering microscopy, mid-infrared photothermal (MIP) microscopy, and atomic force microscopy (AFM)-based force-detected photothermal microscopies are emerging with better chemical sensitivity, molecular specificity, and spatial resolution than conventional vibrational methods. Their utilization in bioimaging applications has provided biological knowledge in unprecedented detail. This Perspective outlines key methodological developments, bioimaging applications, and recent technical innovations of the three techniques. Representative biological demonstrations are also highlighted to exemplify the unique advantages of obtaining vibrational contrasts. With years of effort, these three methods compose an expanding vibrational bioimaging toolbox to tackle specific bioimaging needs, benefiting many biological investigations with rich information in both label-free and labeling manners. Each technique will be discussed and compared in the outlook, leading to possible future directions to accommodate growing needs in vibrational bioimaging.", "doi": "10.1021/cbmi.3c00004", "pmcid": "PMC10131268", "issn": "2832-3637", "publisher": "American Chemical Society", "publication": "Chemical and Biomedical Imaging", "publication_date": "2023-04-24", "series_number": "1", "volume": "1", "issue": "1", "pages": "3-17" }, { "id": "authors:91na4-n8n08", "collection": "authors", "collection_id": "91na4-n8n08", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210416-071920882", "type": "article", "title": "Toward photoswitchable electronic pre-resonance stimulated Raman probes", "author": [ { "family_name": "Lee", "given_name": "Dongkwan", "orcid": "0000-0001-6091-1349", "clpid": "Lee-Dongkwan" }, { "family_name": "Qian", "given_name": "Chenxi", "orcid": "0000-0003-4815-5565", "clpid": "Qian-Chenxi" }, { "family_name": "Wang", "given_name": "Haomin", "orcid": "0000-0001-7193-8651", "clpid": "Wang-Haomin" }, { "family_name": "Li", "given_name": "Lei", "orcid": "0000-0001-6164-2646", "clpid": "Li-Lei" }, { "family_name": "Miao", "given_name": "Kun", "orcid": "0000-0001-6567-3650", "clpid": "Miao-Kun" }, { "family_name": "Du", "given_name": "Jiajun", "orcid": "0000-0003-2693-834X", "clpid": "Du-Jiajun" }, { "family_name": "Shcherbakova", "given_name": "Daria M.", "orcid": "0000-0003-3384-6363", "clpid": "Shcherbakova-Daria-M" }, { "family_name": "Verkhusha", "given_name": "Vladislav V.", "orcid": "0000-0002-2083-8121", "clpid": "Verkhusha-Vladislav-V" }, { "family_name": "Wang", "given_name": "Lihong V.", "orcid": "0000-0001-9783-4383", "clpid": "Wang-Lihong-V" }, { "family_name": "Wei", "given_name": "Lu", "orcid": "0000-0001-9170-2283", "clpid": "Wei-Lu" } ], "abstract": "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 \u00b5M) 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.", "doi": "10.1063/5.0043791", "pmcid": "PMC8019356", "issn": "0021-9606", "publisher": "American Institute of Physics", "publication": "Journal of Chemical Physics", "publication_date": "2021-04-07", "series_number": "13", "volume": "154", "issue": "13", "pages": "Art. No. 135102" }, { "id": "authors:m20x1-9kw24", "collection": "authors", "collection_id": "m20x1-9kw24", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20200911-133137005", "type": "article", "title": "Visualizing Subcellular Enrichment of Glycogen in Live Cancer Cells by Stimulated Raman Scattering", "author": [ { "family_name": "Lee", "given_name": "Dongkwan", "clpid": "Lee-Dongkwan" }, { "family_name": "Du", "given_name": "Jiajun", "clpid": "Du-Jiajun" }, { "family_name": "Yu", "given_name": "Rona", "clpid": "Yu-Rona" }, { "family_name": "Su", "given_name": "Yapeng", "orcid": "0000-0002-6305-8467", "clpid": "Su-Yapeng" }, { "family_name": "Heath", "given_name": "James R.", "orcid": "0000-0001-5356-4385", "clpid": "Heath-J-R" }, { "family_name": "Wei", "given_name": "Lu", "orcid": "0000-0001-9170-2283", "clpid": "Wei-Lu" } ], "abstract": "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.", "doi": "10.1021/acs.analchem.0c02348", "pmcid": "PMC10676777", "issn": "0003-2700", "publisher": "American Chemical Society", "publication": "Analytical Chemistry", "publication_date": "2020-10-06", "series_number": "19", "volume": "92", "issue": "19", "pages": "13182-13191" }, { "id": "authors:n0npk-4qf04", "collection": "authors", "collection_id": "n0npk-4qf04", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20200924-122708002", "type": "article", "title": "Raman-guided subcellular pharmaco-metabolomics for metastatic melanoma cells", "author": [ { "family_name": "Du", "given_name": "Jiajun", "clpid": "Du-Jiajun" }, { "family_name": "Su", "given_name": "Yapeng", "orcid": "0000-0002-6305-8467", "clpid": "Su-Yapeng" }, { "family_name": "Qian", "given_name": "Chenxi", "orcid": "0000-0003-4815-5565", "clpid": "Qian-Chenxi" }, { "family_name": "Yuan", "given_name": "Dan", "clpid": "Yuan-Dan" }, { "family_name": "Miao", "given_name": "Kun", "orcid": "0000-0001-6567-3650", "clpid": "Miao-Kun" }, { "family_name": "Lee", "given_name": "Dongkwan", "clpid": "Lee-Dongkwan" }, { "family_name": "Ng", "given_name": "Alphonsus H. C.", "orcid": "0000-0003-0074-4598", "clpid": "Ng-Alphonsus-H-C" }, { "family_name": "Wijker", "given_name": "Reto S.", "orcid": "0000-0001-5104-9849", "clpid": "Wijker-Reto-S" }, { "family_name": "Ribas", "given_name": "Antoni", "orcid": "0000-0003-3669-8458", "clpid": "Ribas-Antoni" }, { "family_name": "Levine", "given_name": "Raphael D.", "clpid": "Levine-Raphael-D" }, { "family_name": "Heath", "given_name": "James R.", "orcid": "0000-0001-5356-4385", "clpid": "Heath-J-R" }, { "family_name": "Wei", "given_name": "Lu", "orcid": "0000-0001-9170-2283", "clpid": "Wei-Lu" } ], "abstract": "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.", "doi": "10.1038/s41467-020-18376-x", "pmcid": "PMC7518429", "issn": "2041-1723", "publisher": "Nature Publishing Group", "publication": "Nature Communications", "publication_date": "2020-09-24", "volume": "11", "pages": "Art. No. 4830" } ]