2025 Theses Doctoral

Advancing Chemical Specificity in Raman Microscopy: From Method Development to Applications

Qian, Naixin

Raman spectroscopy and microscopy, with signature of molecular vibration, intrinsically encode rich chemical specificity. Yet, to fully unleash this potential for solving real-world problems, particularly in complex biological and environmental systems, requires method development from multiple complementary angles, which I am very fortunate to have explored in the course of my PhD. The first approach centers on the development of vibrational probes that offer additional specificity for imaging biological systems. Vibrational tags, especially those with sharp resonances in the cell-silent region, enable bioorthogonal chemical imaging with minimal biological perturbation and high biocompatibility. Together with the advances in vibrational microscopy instrumentations such as pre-resonance stimulated Raman scattering and stimulated Raman excited fluorescence microscopy, these probes push the detection sensitivity of vibrational imaging down to the single-molecule level, paving the way for super-resolution imaging with vibrational microscopy. Furthermore, the intrinsically narrow linewidths of vibrational transitions, in contrast to the broad emission of fluorescence, open the opportunity for super-multiplexed vibrational imaging.

This capability holds special promise for imaging deep tissue and living cells. By coupling vibrational probe palettes with optical barcoding strategies, the development of vibrational barcodes can further drastically expand information encoding capacity for applications such as multiplexed virus detection.

Beyond probe-based labeling, the rich chemical information embedded in label-free vibrational spectra can be further exploited through data science. Using the development of a hyperspectral SRS microscopy platform for micro- and nanoplastics analysis as a case study, I show how tailored spectral matching algorithms and synthetic training data can enable high-throughput, chemically specific single-particle identification—even in highly heterogeneous real-world samples. These efforts illustrate how computational approaches, including emerging tools in artificial intelligence, offer a powerful new direction for advancing chemical specificity from the data side.

Vibrational microscopy also shows special utility in chemical sensing, using both label-free spectral signatures and functional vibrational probes. To demonstrate, I highlight two application areas from my work experience where vibrational imaging offers unique advantages over fluorescence microscopy: (1) the detection and spatial mapping of labile heavy metals such as Zn²⁺ and Cu⁺/²⁺ in living cells, and (2) the sensing of local water environments, including hydrogen-bonding and electrostatic interactions, through the vibrational Stark effect and OH stretching analysis.

Finally, I highlight a particularly elusive and broadly impactful scientific question: the charge and chemistry at the water–hydrophobe interface. In the course of investigation, spectroscopy efforts in understanding the fundamental interfacial water structure and properties have inspired a real-world application. By connecting the insights accumulated from the fundamental spectroscopy study of water and technique development in appreciation of the rising concern on micro-nano plastic contamination and exposure, I connect the dots by revealing the spontaneous electrostatic and chemical activity at the surface of microplastics in aqueous environments. The fundamental understanding of the structure-activity relationship at water-hydrophobe interface also has real-life implications on the active interactions of microplastics with the biological systems.

In summary, I showcased with my own experience how the chemical specificity of Raman microscopy can be systematically advanced through multidimensional strategies. By integrating interdisciplinary advances in probe development, sensing, instrumentation, and data science, Raman microscopy can be tailored in a problem-driven way into a practical analytical platform with impactful applications for chemistry, biology, and environmental science.