Super-resolution stimulated Raman Scattering microscopy with A-PoD

Abstract

Unlike traditionally-mapped Raman imaging, stimulated Raman scattering (SRS) imaging achieved the capability of imaging metabolic dynamics and a greatly improved signal-noise-ratio. However, its spatial resolution is still limited by the numerical aperture or scattering cross section. To achieve super-resolved SRS imaging, we developed a new deconvolution algorithm Adam optimization-based Pointillism Deconvolution (A-PoD) for SRS imaging, and demonstrated a spatial resolution of 52 nm on polystyrene beads. By changing the genetic algorithm to A-PoD, the image deconvolution process was shortened by more than 3 orders of magnitude, from a few hours to a few seconds. By applying A-PoD to spatially correlated multi-photon fluorescence (MPF) imaging and deuterium oxide (D2O)-probed SRS (DO-SRS) imaging data from diverse samples, we compared nanoscopic distributions of proteins and lipids in cells and subcellular organelles. We successfully differentiated newly synthesized lipids in lipid droplets using A-PoD coupled with DO-SRS. The A-PoD-enhanced DO-SRS imaging method was also applied to reveal the metabolic change in brain samples from Drosophila on different diets. This new approach allows us to quantitatively measure the nanoscopic co-localization of biomolecules and metabolic dynamics in organelles. We expect that the A-PoD algorithm will have a wide range of applications, from nano-scale measurements of biomolecules to processing astronomical images.

Subcellular resolution DO-SRS and 2PEF imaging of metabolic dynamics regulated by L-methionine in amyotrophic lateral sclerosis

“…This multimodal imaging scheme empowers us to visualize the spatial distributions of different molecules at a nanoscopic scale for a better understanding and treatment for ALS. Future study utilizing super-resolution deconvolution algorithms can significantly enhance spatial resolution of imaging in every single channel (from ~300 nm to ~80 nm) [20,21]. Multimodal images can also be collected in a hyperspectral manner, and a recently developed molecular detection program based on penalized reference matching method can also be applied for multiplex hyperspectral imaging of various biomolecules [22].…”

Optical Metabolic Imaging Uncovers Sex- and Diet-dependent Lipid Changes in Aging Drosophila Brain

“…3D Drosophila brain images were taken by SRS imaging system at 2850 cm -1 . To enhance the detection precision, the pixel numbers were increased by 2 times along the lateral direction and 4 times along the axial direction and then A-PoD 89 was used to convert the preprocessed 3D images to the super-resolved images. From the super-resolved image, the numbers and sizes of LDs were measured with the 3D objects counter plugin 90 of ImageJ.…”

Bioorthogonal Imaging Uncovers Sex- and Diet-dependent Lipid Metabolic Changes in Drosophila Brain during Aging

“…LD analysis 3D Drosophila brain images were taken by SRS imaging system at 2850 cm -1 . To enhance the detection precision, the pixel numbers were increased by 2 times along the lateral direction and 4 times along the axial direction and then A-PoD (Jang et al, 2022) was used to convert the preprocessed 3D images to the super-resolved images. From the super-resolved image, the numbers and sizes of LDs were measured with the 3D objects counter plugin (Bolte and Cordelières, 2006) of ImageJ.…”