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Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy
Published on: January 30, 2020
Posterior approach to correct for focal plane offsets in lattice light-sheet structured illumination microscopy
Yu Shi1, Tim A Daugird2, Wesley R Legant1,2
1University of North Carolina at Chapel Hill and North Carolina State University, Joint Department of Biomedical Engineering, Chapel Hill, North Carolina, United States.
Significance:
Lattice light-sheet structured illumination microscopy (latticeSIM) has proven highly effective in producing three-dimensional images with super resolution rapidly and with minimal photobleaching. However, due to the use of two separate objectives, sample-induced aberrations can result in an offset between the planes of excitation and detection, causing artifacts in the reconstructed images.
Aim:
We introduce a posterior approach to detect and correct the axial offset between the excitation and detection focal planes in latticeSIM and provide a method to minimize artifacts in the reconstructed images.
Approach:
We utilized the residual phase information within the overlap regions of the laterally shifted structured illumination microscopy information components in frequency space to retrieve the axial offset between the excitation and the detection focal planes in latticeSIM.
Results:
We validated our technique through simulations and experiments, encompassing a range of samples from fluorescent beads to subcellular structures of adherent cells. We also show that using transfer functions with the same axial offset as the one present during data acquisition results in reconstructed images with minimal artifacts and salvages otherwise unusable data.
Conclusion:
We envision that our method will be a valuable addition to restore image quality in latticeSIM datasets even for those acquired under non-ideal experimental conditions.
