Raman microscopy unmixed: A comprehensive guide to the molecular imaging technology reshaping biology
Bioengineers at the University of California San Diego bridge the gap between spectroscopy developers and biologists through a systematic guide explaining modern Raman imaging technologies, probes, and applications
The University of California San Diego researchers have published a comprehensive review on Raman microscopy and its applications in the life sciences, aiming to bridge a long-standing communication gap between physicists who develop the instrumentation and the biologists who stand to benefit most from it.
Raman microscopy generates chemical images of biological samples by using the interaction between light and molecular vibrations, without requiring stains, dyes, or labels. Different chemical bonds in proteins, lipids, DNA, and metabolites scatter light at characteristic frequencies, creating molecular fingerprints that reveal cellular composition and how chemical contents change in real time.
The review was published in Photonix Life and authored by Erick Alvarado, Zhi Li, Yajuan Li, and corresponding author Lingyan Shi from the Shu Chien-Gene Lay Department of Bioengineering at the University of California San Diego.
"Over the past decade, Raman microscopy has advanced tremendously, but there is still a clear disconnect between what the technology can currently do and what biologists know is available," said Shi, a tenured associate professor of bioengineering at UC San Diego and a pioneer of the DO-SRS and SuMMIT-SRS metabolic imaging platforms. "We wrote this review to provide the kind of resource we wish we had when we first started working in this interdisciplinary field."
"Looking ahead, Raman microscopy is at a critical inflection point as it moves from the lab toward clinical and industrial translation," Shi added. "With the convergence of quantum-enhanced imaging, AI-assisted diagnosis, and miniaturized probe technologies, this label-free chemical imaging approach will truly become an everyday tool for biologists and clinicians, advancing precision medicine into a new era of single-molecule, real-time, and multidimensional analysis."
Structured framework for comparing technologies
One notable strength of the review is its systematic comparison of technical advances along four performance axes: signal sensitivity, imaging speed, volumetric (3D) imaging capability, and spatial resolution. This structured framework is applied consistently across both spontaneous and coherent Raman modalities, enabling readers to directly assess which technologies and trade-offs are most relevant to their specific experimental needs.
From cells to the clinic
The review highlights how Raman microscopy is being applied across biological scales. Recent examples featured in the article include tracking metabolic changes during aging and neurodegenerative disease using heavy water as a tracer; detecting amyloid plaques in Alzheimer's disease brain tissue without any labels; achieving diagnostic-quality virtual tissue staining with machine learning in just 3 minutes, compared with more than 36 hours using conventional preparation methods; and imaging drug penetration in tissue in real time.
Pushing fundamental limits
The article documents several major technical milestones, including tip-enhanced methods that have pushed spatial resolution to 2.5 nanometers—sufficient to distinguish individual protein complexes on cell membranes. In terms of sensitivity, quantum-enhanced approaches using squeezed light have surpassed the classical noise limit, boosting signal detection by more than 50% while enabling faster live-cell imaging. Computational methods, including the A-PoD super-resolution algorithm developed in the Shi laboratory, have achieved sub-59-nanometer resolution through software-based post-processing.
Emerging frontiers
Looking ahead, the review points to several transformative directions: miniature fiber-optic Raman probes that have achieved more than 98% accuracy for cancer diagnosis during endoscopy; quantum coherent effects that can selectively amplify specific molecular signals; ultrafast time-resolved Raman spectroscopy for tracking molecular dynamics on the femtosecond timescale; and multimodal platforms that combine Raman imaging with mass spectrometry and clinical MRI for comprehensive tissue characterization.
"The field is at a turning point," said Alvarado, a PhD student in the Shi laboratory and first author of the review. "The technology is now mature enough to address real biological and clinical questions, but realizing that potential requires physicists and biologists to speak the same language. That is exactly what we are trying to build with this review."
See the article:
Raman microscopy unmixed: Technical advances, bio-orthogonal tags, and applications in life sciences
https://doi.org/10.3724/PXLIFE.2025-0016
Method of Research
Literature review
Subject of Research
Not applicable
Article Title
Raman microscopy unmixed: Technical advances, bio-orthogonal tags, and applications in life sciences
The protein microcrystallography beamline (BL18U1) at the Shanghai Synchrotron Radiation Facility
BL18U1 supports high-quality diffraction data collection and structure determination for diverse crystallographic samples at SSRF
image:
This figure shows the complete experimental endstation of BL18U1, including the MD2 microdiffractometer, Pilatus 3 6M detector, automated sample changer, and cryogenic cooling system. Together, these key instruments transform the beamline’s optical performance into a practical experimental platform for high-precision crystal positioning, high-throughput sample handling, and rapid diffraction data acquisition.
view moreCredit: Wen-Ming Qin
An advanced platform for structural biology
The BL18U1 microcrystallography beamline at SSRF is one of the five beamlines operated by the National Facility for Protein Science in Shanghai and the first microcrystallography beamline at a third-generation synchrotron light source in China. Designed for microcrystals and other challenging samples, BL18U1 has become an important platform for structural biology and crystallographic research.
High-quality beam, flexible energy, integrated instrumentation
BL18U1 combines advanced optical design with stable microbeam performance. At the sample position, it delivers a beam of about 9.8 μm horizontally and 4.6 μm vertically, providing suitable conditions for diffraction studies of protein microcrystals, membrane protein crystals, small-molecule crystals, and other difficult samples. With a tunable energy range of 5–18 keV, the beamline can meet different experimental needs and support absorption-edge measurements as well as anomalous diffraction experiments.
The experimental station is equipped with an MD2 microdiffractometer, a Pilatus 3 6M detector, a Rigaku ACTOR robotic sample changer, and a cryogenic cooling system. Together with the MXCuBE3 control system, these components enable precise sample alignment, efficient automated operation, rapid data acquisition, and streamlined data processing.
From routine diffraction to sulfur-SAD phasing
The study shows that BL18U1 can routinely provide high-quality diffraction data. In lysozyme experiments, the beamline achieved diffraction data to 1.28 Å resolution with excellent processing statistics. The beamline also demonstrated long-wavelength anomalous diffraction capability at 2.02 Å, enhanced anomalous signals from endogenous sulfur atoms enabled sulfur-SAD phasing, reliable electron density map calculation, and atomic model building.
Strong support for high-quality structure determination
By the end of 2024, data collected at BL18U1 had contributed to 1,687 macromolecular structures deposited in the Protein Data Bank, with most structures in the 1.5–2.5 Å high-resolution range. These results show that BL18U1 has consistently supported the determination of numerous high-quality crystal structures and plays an important role in structural biology and structure-based research at SSRF.
The complete study is via by DOI: https://doi.org/10.1007/s41365-026-01991-6
Journal
Nuclear Science and Techniques
Method of Research
Experimental study
Subject of Research
Not applicable
Article Title
The protein microcrystallography beamline (BL18U1) at the Shanghai Synchrotron Radiation Facility
Article Publication Date
18-Jun-2026
This figure highlights the integrated MXCuBE3 control framework implemented at BL18U1. Through the coordinated control of the diffractometer, sample changer, detector, and database system, the beamline has achieved a highly automated workflow that unifies experiment control, data display, storage, and processing, greatly improving user efficiency and operational intelligence
This figure demonstrates BL18U1’s capability for routine high-quality diffraction data collection and automated processing. The beamline delivered a dataset at 1.28 Å resolution with an Rmerge of only 0.065, clearly showing that BL18U1 can reliably support high-resolution macromolecular structure determination.
This figure highlights the obvious anomalous signal obtained at 2.02 Å, especially from sulfur atoms. The successful recovery of reliable electron density maps and atomic models demonstrates that BL18U1 has progressed beyond routine crystallography and is capable of supporting demanding long-wavelength native-SAD phasing experiments.
This figure summarizes the resolution distribution of 1,687 PDB structures supported by BL18U1 as of the end of 2024. The results show that most structures fall within the high-resolution range of 1.5–2.5 Å, highlighting BL18U1’s long-term and stable capability to produce high-quality structural data, as well as its important supporting role in structural biology and structure-based drug discovery.
Credit
Wen-Ming Qin
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