Low-photobleaching line-scanning confocal microscopy using dual inclined beams

Confocal microscopy is an indispensable tool for biological imaging due to its high resolution and optical sectioning capability. However, its slow imaging speed and severe photobleaching have largely prevented further applications. Here, we present dual inclined beam line-scanning (LS) confocal microscopy. The reduced excitation intensity of our imaging method enabled a 2-fold longer observation time of fluorescence compared to traditional LS microscopy while maintaining a good sectioning capability and single-molecule sensitivity. We characterized the performance of our method and applied it to subcellular imaging and three-dimensional single-molecule RNA imaging in mammalian cells.