title: Diagonally Scanned Light-Sheet Microscopy for Fast Volumetric Imaging of Adherent Cells.
abstract: In subcellular light-sheet fluorescence microscopy (LSFM) of adherent cells, glass substrates are advantageously rotated relative to the excitation and emission light paths to avoid glass-induced optical aberrations. Because cells are spread across the sample volume, three-dimensional imaging requires a light-sheet with a long propagation length, or rapid sample scanning. However, the former degrades axial resolution and/or optical sectioning, while the latter mechanically perturbs sensitive biological specimens on pliant biomimetic substrates (e.g., collagen and basement membrane). Here, we use aberration-free remote focusing to diagonally sweep a narrow light-sheet along the sample surface, enabling multicolor imaging with high spatiotemporal resolution. Further, we implement a dithered Gaussian lattice to minimize sample-induced illumination heterogeneities, significantly improving signal uniformity. Compared with mechanical sample scanning, we drastically reduce sample oscillations, allowing us to achieve volumetric imaging at speeds of up to 3.5 Hz for thousands of Z-stacks. We demonstrate the optical performance with live-cell imaging of microtubule and actin cytoskeletal dynamics, phosphoinositide signaling, clathrin-mediated endocytosis, polarized blebbing, and endocytic vesicle sorting. We achieve three-dimensional particle tracking of clathrin-associated structures with velocities up to 4.5 μm/s in a dense intracellular environment, and show that such dynamics cannot be recovered reliably at lower volumetric image acquisition rates using experimental data, numerical simulations, and theoretical modeling.
authors.affiliation: Lyda Hill Department of Bioinformatics, University of Texas Southwestern Medical Center, Dallas, Texas; Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas.
authors.forename: Kevin M
authors.initials: KM
authors.lastname: Dean
authors.affiliation: Lyda Hill Department of Bioinformatics, University of Texas Southwestern Medical Center, Dallas, Texas; Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas.
authors.forename: Philippe
authors.initials: P
authors.lastname: Roudot
authors.affiliation: Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas.
authors.forename: Carlos R
authors.initials: CR
authors.lastname: Reis
authors.affiliation: Lyda Hill Department of Bioinformatics, University of Texas Southwestern Medical Center, Dallas, Texas; Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas.
authors.forename: Erik S
authors.initials: ES
authors.lastname: Welf
authors.affiliation: Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas.
authors.forename: Marcel
authors.initials: M
authors.lastname: Mettlen
authors.affiliation: Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas. Electronic address: reto.fiolka@utsouthwestern.edu.
authors.forename: Reto
authors.initials: R
authors.lastname: Fiolka
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism; D002448:Cell Adhesion; D002460:Cell Line; D011992:Endosomes / Q000378:metabolism; D005110:Extracellular Space / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D021621:Imaging, Three-Dimensional / Q000379:methods*; D008853:Microscopy / Q000379:methods*; D015398:Signal Transduction
chemical_list: D049452:Green Fluorescent Proteins