Fast reconstruction and optical-sectioning three-dimensional structured illumination microscopy.

Three-dimensional structured illumination microscopy (3DSIM) is a popular method for observing subcellular/cellular structures or animal/plant tissues with gentle phototoxicity and 3D super-resolution. However, its time-consuming reconstruction process poses challenges for high-throughput imaging and real-time observation. Moreover, traditional 3DSIM typically requires more than six layers for successful reconstruction and is susceptible to defocused backgrounds. This poses a great gap between single-layer 2DSIM and 6-layer 3DSIM, and limits the observation of thicker samples. To address these limitations, we developed FO-3DSIM, a novel method that integrates spatial-domain reconstruction with optical-sectioning SIM. FO-3DSIM enhances reconstruction speed by up to 855.7 times with superior performance with limited layers and under high defocused backgrounds. It retains the high-fidelity, low-photon reconstruction capabilities of our previously proposed Open-3DSIM. Utilizing fast reconstruction and optical sectioning, we achieved large field-of-view (FOV) 3D super-resolution imaging of mouse kidney actin, covering a region of 0.453 mm × 0.453 mm × 2.75 μm within 23 min of acquisition and 13 min of reconstruction. Near real-time performance was demonstrated in live actin imaging with FO-3DSIM. Our approach reduces photodamage through limited layer reconstruction, allowing the observation of ER tubes with just three layers. We anticipate that FO-3DSIM will pave the way for near real-time, large FOV 6D imaging, encompassing super-resolution, multi-color, long-term, and polarization imaging with less photodamage, removed defocused backgrounds, and reduced reconstruction time.