Fiber laser based stimulated Raman photothermal microscopy towards a high-performance and user-friendly chemical imaging platform

Xiaowei Ge; Yifan Zhu; Dingcheng Sun; Hongli Ni; Yueming Li; Chinmayee V. Prabhu Dessai; Ji-Xin Cheng

doi:10.1186/s43074-025-00196-1

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Xiaowei Ge, Yifan Zhu, Dingcheng Sun, Hongli Ni, Yueming Li, Chinmayee V. Prabhu Dessai, Ji-Xin Cheng. Fiber laser based stimulated Raman photothermal microscopy towards a high-performance and user-friendly chemical imaging platform[J]. PhotoniX. doi: 10.1186/s43074-025-00196-1

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Fiber laser based stimulated Raman photothermal microscopy towards a high-performance and user-friendly chemical imaging platform

doi: 10.1186/s43074-025-00196-1

1.
Department of Electrical & Computer Engineering, Boston University, Boston, MA, USA
2.
Department of Biomedical Engineering, Boston University, Boston, MA, USA
3.
Department of Chemistry, Boston University, Boston, MA, USA
4.
Photonics Center, Boston University, Boston, MA, USA

Funds: This work is supported by NIH grants R35GM136223, R01EB032391, R01EB035429 to JXC.

Received Date: 2025-06-04
Accepted Date: 2025-09-06
Rev Recd Date: 2025-08-26

Available Online: 2025-09-29

Abstract

Abstract

Stimulated Raman scattering (SRS) microscopy is a highly sensitive chemical imaging technique. However, the SRS imaging performance hinges on two key factors: the reliance on low-noise but bulky solid-state laser sources and stringent sample requirements necessitated by high numerical aperture (NA) optics. Here, we present a fiber laser based stimulated Raman photothermal (SRP) microscope that addresses these limitations. While appreciating the portability and compactness of a noisy source, fiber laser SRP enables a two-order-of-magnitude improvement in signal to noise ratio over fiber laser SRS without balance detection. Furthermore, with the use of low NA, long working distance optics for signal collection, SRP expands the allowed sample space from millimeters to centimeters, which diversifies the sample formats to multi-well plates and thick tissues. The sensitivity and imaging depth are further amplified by using urea for both thermal enhancement and tissue clearance. Together, fiber laser SRP microscopy provides a robust, user-friendly platform for diverse applications.
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- Stimulated Raman')">" data-track="click" data-track-action="view keyword" data-track-label="link">Stimulated Raman,
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- Photothermal microscopy')">" data-track="click" data-track-action="view keyword" data-track-label="link">Photothermal microscopy,
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References(51)

References

[1]	Cheng J-X, Yuan Y, Ni H, Ao J, Xia Q, Bolarinho R, et al. Advanced vibrational microscopes for life science. Nat Methods. 2025;22:912.
[2]	Cheng J-X, Xie XS. Vibrational spectroscopic imaging of living systems: an emerging platform for biology and medicine. Science. 2015. https://doi.org/10.1126/science.aaa8870.
[3]	Gao X, Li X, Min W. Absolute stimulated raman cross sections of molecules. J Phys Chem Lett. 2023. https://doi.org/10.1021/acs.jpclett.3c01064.
[4]	Freudiger CW, Min W, Saar BG, Lu S, Holtom GR, He C, et al. Label-free biomedical imaging with high sensitivity by stimulated raman scattering microscopy. Science. 2008;322:1857.
[5]	Ji M, et al. Detection of human brain tumor infiltration with quantitative stimulated Raman scattering microscopy. Sci Transl Med. 2015;7:309ra163.
[6]	Oh S, et al. Protein and lipid mass concentration measurement in tissues by stimulated raman scattering microscopy. Proc Natl Acad Sci USA. 2022;119:e2117938119.
[7]	Shen Y, Xu F, Wei L, Hu F, Min W. Live-cell quantitative imaging of proteome degradation by stimulated raman scattering. Angew Chem Int Ed. 2014;53:5596.
[8]	Yue S, et al. Cholesteryl ester accumulation induced by PTEN loss and PI3K/AKT activation underlies human prostate cancer aggressiveness. Cell Metab. 2014;19:393.
[9]	Shi L, et al. Optical imaging of metabolic dynamics in animals. Nat Commun. 2018. https://doi.org/10.1038/s41467-018-05401-3.
[10]	Zhao G, et al. Ovarian cancer cell fate regulation by the dynamics between saturated and unsaturated fatty acids. Proc Natl Acad Sci U S A. 2022;119:e2203480119.
[11]	Li Y, et al. Microglial lipid droplet accumulation in tauopathy brain is regulated by neuronal AMPK. Cell Metabolism. 2024;36:1351.
[12]	Hong W, Karanja CW, Abutaleb NS, Younis W, Zhang X, Seleem MN, et al. Antibiotic susceptibility determination within one cell cycle at single-bacterium level by stimulated raman metabolic imaging. Anal Chem. 2018;90:3737.
[13]	Zhang M, Hong W, Abutaleb NS, Li J, Dong P-T, Zong C, et al. Rapid determination of antimicrobial susceptibility by stimulated raman scattering imaging of D2O metabolic incorporation in a single bacterium. Adv Sci. 2020;7:2001452.
[14]	Ge X, Pereira FC, Mitteregger M, Berry D, Zhang M, Hausmann B, et al. SRS-FISH: a high-throughput platform linking microbiome metabolism to identity at the single-cell level. Proc Natl Acad Sci U S A. 2022;119:e2203519119.
[15]	Pereira FC, et al. The Parkinson’s drug entacapone disrupts gut microbiome homeostasis via iron sequestration, bioRxiv. 2023;11;12–566429.
[16]	Wei L, Chen Z, Shi L, Long R, Anzalone AV, Zhang L, et al. Super-multiplex vibrational imaging. Nature. 2017;544:465.
[17]	Shi L, Wei M, Miao Y, Qian N, Shi L, Singer RA, Benninger RKP, Min W. Highly-multiplexed volumetric mapping with Raman dye imaging and tissue clearing. Nat Biotechnol. 2022;40:364.
[18]	Prince RC, Frontiera RR, Potma EO. Stimulated raman scattering: from bulk to nano. Chem Rev. 2017;117:5070.
[19]	Xu C, Wise FW. Recent advances in fibre lasers for nonlinear microscopy. Nat Photon. 2013;7:875.
[20]	Freudiger CW, Yang W, Holtom GR, Peyghambarian N, Xie XS, Kieu KQ. Stimulated raman scattering microscopy with a robust fibre laser source. Nat Photon. 2014. https://doi.org/10.1038/nphoton.2013.360.
[21]	Nose K, Ozeki Y, Kishi T, Sumimura K, Nishizawa N, Fukui K, et al. Sensitivity enhancement of fiber-laser-based stimulated Raman scattering microscopy by collinear balanced detection technique. Opt Express. 2012;20:13958.
[22]	Ni H, Lin P, Zhu Y, Zhang M, Tan Y, Zhan Y, et al. Multiwindow SRS imaging using a rapid widely tunable fiber laser. Anal Chem. 2021;93:15703.
[23]	de Andrade RB, Kerdoncuff H, Berg-Sørensen K, Gehring T, Lassen M, Andersen UL. Quantum-enhanced continuous-wave stimulated Raman scattering spectroscopy. Optica, OPTICA. 2020;7:470.
[24]	Casacio CA, Madsen LS, Terrasson A, Waleed M, Barnscheidt K, Hage B, et al. Quantum-enhanced nonlinear microscopy. Nature. 2021. https://doi.org/10.1038/s41586-021-03528-w.
[25]	Xu Z, Oguchi K, Taguchi Y, Takahashi S, Sano Y, Mizuguchi T, et al. Quantum-enhanced stimulated Raman scattering microscopy in a high-power regime. Opt Lett. 2022;47:5829.
[26]	Bertoncini A, Laptenok SP, Genchi L, Rajamanickam VP, Liberale C. 3D-printed high-NA catadioptric thin lens for suppression of XPM background in stimulated Raman scattering microscopy. J Biophotonics. 2021;14:e202000219.
[27]	Tsikritsis D, Legge EJ, Belsey NA. Practical considerations for quantitative and reproducible measurements with stimulated Raman scattering microscopy. Analyst. 2022;147:4642.
[28]	Zhang J, Lin H, Xu J, Zhang M, Ge X, Zhang C, Huang WE, Cheng JX. High-throughput single-cell sorting by stimulated Raman-activated cell ejection, bioRxiv. 2023;10:16–562526.
[29]	Suzuki Y, et al. Label-free chemical imaging flow cytometry by high-speed multicolor stimulated Raman scattering. Proc Natl Acad Sci U S A. 2019;116:15842.
[30]	Yu Y, Mutlu AS, Liu H, Wang MC. High-throughput screens using photo-highlighting discover BMP signaling in mitochondrial lipid oxidation. Nat Commun. 2017. https://doi.org/10.1038/s41467-017-00944-3.
[31]	Zhu Y, et al. Stimulated raman photothermal microscopy toward ultrasensitive chemical imaging. Sci Adv. 2023;9:eadi2181.
[32]	Gaiduk A, Yorulmaz M, Ruijgrok PV, Orrit M. Room-temperature detection of a single molecule’s absorption by photothermal contrast. Science. 2010;330:353.
[33]	Gaiduk A, Ruijgrok PV, Yorulmaz M, Orrit M. Detection limits in photothermal microscopy. Chem Sci. 2010;1:343.
[34]	Wei M, Shi L, Shen Y, Zhao Z, Guzman A, Kaufman LJ, et al. Volumetric chemical imaging by clearing-enhanced stimulated raman scattering microscopy. Proc Natl Acad Sci U S A. 2019;116:6608.
[35]	Ueda HR, Ertürk A, Chung K, Gradinaru V, Chédotal A, Tomancak P, et al. Tissue clearing and its applications in neuroscience. Nat Rev Neurosci. 2020;21:61.
[36]	Brinkmann M, et al. Portable all-fiber dual-output widely tunable light source for coherent raman imaging. Biomed Opt Express. 2019;10:4437.
[37]	Adhikari S, Spaeth P, Kar A, Baaske MD, Khatua S, Orrit M. Photothermal microscopy: imaging the optical absorption of single nanoparticles and single molecules. ACS Nano. 2020;14:16414.
[38]	Thorn K. A quick guide to light microscopy in cell biology. MBoC. 2016;27:219.
[39]	Zhang Y, Gross H. Systematic design of microscope objectives. Part I: System review and analysis. Adv Opt Technol. 2019;8:313.
[40]	Lin H, et al. Microsecond fingerprint stimulated Raman spectroscopic imaging by ultrafast tuning and spatial-spectral learning. Nat Commun. 2021. https://doi.org/10.1038/s41467-021-23202-z.
[41]	Zhang C, Li J, Lan L, Cheng J-X. Quantification of lipid metabolism in living cells through the dynamics of lipid droplets measured by stimulated raman scattering imaging. Anal Chem. 2017;89:4502.
[42]	Chen T, Yavuz A, Wang MC. Dissecting lipid droplet biology with coherent Raman scattering microscopy. J Cell Sci. 2021;135:jcs252353.
[43]	Shou J, Ozeki Y. Dual-polarization hyperspectral stimulated raman scattering microscopy. Appl Phys Lett. 2018;113:033701.
[44]	Tuck M, et al. Multimodal imaging based on vibrational spectroscopies and mass spectrometry imaging applied to biological tissue: a multiscale and multiomics review. Anal Chem. 2021;93:445.
[45]	Vanna R, De la Cadena A, Talone B, Manzoni C, Marangoni M, Polli D, et al. Vibrational imaging for label-free cancer diagnosis and classification. Riv Nuovo Cim. 2022;45:107.
[46]	Shen Y, Hu F, Min W. Raman imaging of small biomolecules. Annu Rev Biophys. 2019;48:347.
[47]	Zhang W, et al. Multi-molecular hyperspectral PRM-SRS microscopy. Nat Commun. 2024;15:1599.
[48]	Durst ME, Mertz J. Multiphoton Photothermal Imaging in Scattering Samples, in Optics in the Life Sciences (OSA, Monterey, California, 2011), p. NMD6.
[49]	Wang L, Lin H, Zhu Y, Ge X, Li M, Liu J, et al. Overtone photothermal microscopy for high-resolution and high-sensitivity vibrational imaging. Nat Commun. 2024;15:5374.
[50]	Ni H, Yuan Y, Li M, Zhu Y, Ge X, Yin J, et al. Millimetre-deep micrometre-resolution vibrational imaging by shortwave infrared photothermal microscopy. Nat Photon. 2024;18:944.
[51]	Zhang D, Slipchenko MN, Leaird DE, Weiner AM, Cheng J-X. Spectrally modulated stimulated Raman scattering imaging with an angle-to-wavelength pulse shaper. Opt Express. 2013;21:13864.