Single-molecule (SM) optical spectroscopy and imaging approaches have proven transformative, allowing for deep insights into molecular dynamics and behaviour. Despite their intrinsically weaker signals, vibrational spectro-microscopies have advanced significantly within the past decade, allowing for far-field vibrational spectroscopy and imaging at the SM level under ambient conditions. In this Perspective, we first discuss the critical insights and advancements that allowed for SM vibrational spectroscopy and imaging to be realized, highlighting the technical developments that have allowed vibrational spectro-microscopies to break the SM barrier with far-field optics. We then discuss the unique and exciting opportunities of these SM vibrational methods.
\nMolecular 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" } ]