Shu Chien - Gene Lay Department of Bioengineering, University of California San Diego, La Jolla, CA, USA.
School of Medicine, University of California, Irvine, CA, USA.
Nat Methods. 2023 Mar;20(3):448-458. doi: 10.1038/s41592-023-01779-1. Epub 2023 Feb 16.
Stimulated Raman scattering (SRS) offers the ability to image metabolic dynamics with high signal-to-noise ratio. However, its spatial resolution is limited by the numerical aperture of the imaging objective and the scattering cross-section of molecules. To achieve super-resolved SRS imaging, we developed a deconvolution algorithm, adaptive moment estimation (Adam) optimization-based pointillism deconvolution (A-PoD) and demonstrated a spatial resolution of lower than 59 nm on the membrane of a single lipid droplet (LD). We applied A-PoD to spatially correlated multiphoton fluorescence imaging and deuterium oxide (DO)-probed SRS (DO-SRS) imaging from diverse samples to compare nanoscopic distributions of proteins and lipids in cells and subcellular organelles. We successfully differentiated newly synthesized lipids in LDs using A-PoD-coupled DO-SRS. The A-PoD-enhanced DO-SRS imaging method was also applied to reveal metabolic changes in brain samples from Drosophila on different diets. This new approach allows us to quantitatively measure the nanoscopic colocalization of biomolecules and metabolic dynamics in organelles.
受激拉曼散射（SRS）能够以高信噪比成像代谢动力学。然而，其空间分辨率受到成像物镜的数值孔径和分子的散射截面的限制。为了实现超分辨 SRS 成像，我们开发了一种去卷积算法，基于自适应矩估计（Adam）优化的点画去卷积（A-PoD），并在单个脂质滴（LD）的膜上证明了低于 59nm 的空间分辨率。我们将 A-PoD 应用于空间相关的多光子荧光成像和氘代水（DO）探测的受激拉曼散射（DO-SRS）成像，以比较细胞和亚细胞器中蛋白质和脂质的纳米级分布。我们成功地使用 A-PoD 耦合的 DO-SRS 区分了 LD 中合成的新脂质。该 A-PoD 增强的 DO-SRS 成像方法还应用于揭示不同饮食的果蝇脑组织样本中的代谢变化。这种新方法使我们能够定量测量细胞器中生物分子和代谢动力学的纳米级共定位。
