Fast reconstruction and optical-sectioning three-dimensional structure illumination microscopy

Public summary•3D structured illumination microscopy is enhanced integrating spatial reconstruction and optical sectioning.•Reconstruction is speed up by nearly three orders of magnitude and fidelity has been greatly improved.•We achieve high-throughput and near-real-time 3D super-resolution imaging with high speed and fidelity.AbstractThree-dimensional structure 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 z 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 z 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 z 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 xyz super-resolution, multi-color, long-term, and polarization imaging with less photodamage, removed defocused backgrounds, and reduced reconstruction time.Graphical abstract