Dual-Bessel-BeamStimulated Emission Depletion Microscopyfor Super-resolution Volumetric Projection Imaging of Lipid DropletDynamics

Volumetric imaging efficiently captures comprehensive spatial structures and dynamic function information on organisms in biomedical research. However, optical diffraction limit restricts the visualization of fine structure details at nanoscale. To address this limitation, we developed dual-Bessel-beam stimulated emission depletion (DB-STED) microscopy to enhance the information throughput and lateral resolution. This technique combines a zeroth-order Bessel beam for excitation with a first-order hollow Bessel beam for depletion, aligned both spatially and temporally to achieve super-resolution volumetric projection imaging. We validated this approach using fluorescent beads embedded in agarose, achieving a resolution of 69 nm over a depth of 10 μm with a numerical aperture of 1.4. The high-throughput and super-resolution capability enables detailed observation of lipid droplet motion within entire cells, providing valuable insights into lipid dynamics.