Metabolic Imaging at Video Rates Using Raman with Airy Light-Sheet Illumination and Sparse Photon Detection
Authors:
Haokun Luo1* ([email protected]), Ramachandran Kasu2, Demetrios N. Christodoulides1, Andreas E. Vasdekis2 (PI)
Institutions:
1University of Southern California; 2University of Idaho
Abstract
Nowadays, Raman imaging represents only a modest fraction of all research and clinical microscopy to date even though it exhibits great potential. This limited adoption is primarily attributed to the ultralow Raman scattering cross-sections of most biomolecules, resulting in low-light or photon-sparse conditions. Imaging biological samples under such conditions is suboptimal, leading to either extremely low frame rates or the need for higher levels of irradiance. In this study, researchers address this tradeoff by introducing Raman imaging capable of operating at both video rates and with irradiance levels 1,000 times lower than existing methods. To achieve this, the team utilized a carefully designed Airy light-sheet microscope, which efficiently images large specimen areas (Dunn et al. 2023). The Airy beam, known for its unique diffraction-free properties such as self-healing and refocusing, has been employed in lightsheet microscopy (LSI) (Subedi et al. 2020; Subedi et al. 2021) and selective plane illumination imaging schemes. The team investigated the diffraction-free behavior of Airy beams as a function of cubic phase modulation ‘α’, both theoretically and experimentally.
Additionally, group members implemented subphoton per pixel image acquisition and reconstruction techniques to address challenges arising from sparse photon availability during short integration times. This project demonstrates the versatility of this approach through successful imaging of various samples, including the three-dimensional metabolic activity of individual microbial cells and their associated cell-to-cell variability. Moreover, to visualize small-scale targets, the group leveraged photon sparsity and photon superlocalization to increase magnification without sacrificing field-of-view, thereby overcoming another significant limitation in modern light-sheet microscopy.
References
Dunn, L. et al. 2023. “Video-Rate Raman-Based Metabolic Imaging by Airy Light-Sheet Illumination and Photon-Sparse Detection,” Proceedings of the National Academies of Sciences 120(9), e2210037120. DOI:10.1073/ pnas.2210037120.
Subedi, N. R., et al. 2020. “Integrative Quantitative- Phase and Airy Light-Sheet Imaging,” Scientific Reports 10(1), 20150. DOI:10.1038/s41598-020-76730-x.
Subedi, N. R., et al. 2021. “Airy Light-sheet Raman Imaging,” Optics Express 29(20), 31941–51. DOI:10.1364/ OE.435293