Multicolor 2.5D HILO microscopy for fast volumetric single-molecule imaging with high sensitivity

Three-dimensional (3D) subcellular imaging has long been essential for biological studies. However, the imaging speed is limited by the scanning process of a bulky piezo stage along the z-axis. Here we report a 2.5D fluorescence microscopy technique that largely reduces the image acquisition time while maintaining high resolution and high photon detection efficiency. Our approach enables us to perform single-shot immunofluorescence imaging covering ~5 um thickness while minimizing degradation of spatial resolution. Furthermore, our approach is capable of extending the observation time of single-particle tracking due to the extended depth of field. Finally, we show fast volumetric super-resolution imaging.