Highly sensitive volumetric single-molecule imaging.

Volumetric subcellular imaging has long been essential for studying structures and dynamics in cells and tissues. However, due to limited imaging speed and depth of field, it has been challenging to perform live-cell imaging and single-particle tracking. Here we report a 2.5D fluorescence microscopy combined with highly inclined illumination beams, which significantly reduce not only the image acquisition time but also the out-of-focus background by ∼2-fold compared to epi-illumination. Instead of sequential z -scanning, our method projects a certain depth of volumetric information onto a 2D plane in a single shot using multi-layered glass for incoherent wavefront splitting, enabling high photon detection efficiency. We apply our method to multi-color immunofluorescence imaging and volumetric super-resolution imaging, covering ∼3-4 µm thickness of samples without z -scanning. Additionally, we demonstrate that our approach can substantially extend the observation time of single-particle tracking in living cells.