Diagonally Scanned Light-Sheet Microscopy for Fast Volumetric Imaging of Adherent Cells

Kevin M. Dean, Philippe Roudot, Carlos R. Reis, Erik S. Welf, Marcel Mettlen, Reto Fiolka

Research output: Contribution to journalArticle

17 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)1456-1465
Number of pages10
JournalBiophysical Journal
Volume110
Issue number6
DOIs
StatePublished - Mar 29 2016

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Microscopy
Light
Clathrin
Glass
Transport Vesicles
Biomimetics
Three-Dimensional Imaging
Blister
Phosphatidylinositols
Endocytosis
Lighting
Fluorescence Microscopy
Basement Membrane
Microtubules
Actins
Collagen

ASJC Scopus subject areas

  • Biophysics

Cite this

Diagonally Scanned Light-Sheet Microscopy for Fast Volumetric Imaging of Adherent Cells. / Dean, Kevin M.; Roudot, Philippe; Reis, Carlos R.; Welf, Erik S.; Mettlen, Marcel; Fiolka, Reto.

In: Biophysical Journal, Vol. 110, No. 6, 29.03.2016, p. 1456-1465.

Research output: Contribution to journalArticle

Dean, Kevin M. ; Roudot, Philippe ; Reis, Carlos R. ; Welf, Erik S. ; Mettlen, Marcel ; Fiolka, Reto. / Diagonally Scanned Light-Sheet Microscopy for Fast Volumetric Imaging of Adherent Cells. In: Biophysical Journal. 2016 ; Vol. 110, No. 6. pp. 1456-1465.
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