An orientation‐independent DIC microscope allows high resolution imaging of epithelial cell migration and wound healing in a cnidarian model

Abstract

Epithelial cell dynamics can be difficult to study in intact animals or tissues. Here we use the medusa form of the hydrozoan Clytia hemisphaerica, which is covered with a monolayer of epithelial cells, to test the efficacy of an orientation-independent differential interference contrast (OI-DIC) microscope for in vivo imaging of wound healing. OI-DIC provides an unprecedented resolution phase image of epithelial cells closing a wound in a live, non-transgenic animal model. In particular, the OI-DIC microscope equipped with a 40×/0.75NA objective lens and using the illumination light with wavelength 546 nm demonstrated a resolution of 460 nm. The repair of individual cells, the adhesion of cells to close a gap, and the concomitant contraction of these cells during closure is clearly visualized.

Live-cell imaging of subcellular structures for quantitative evaluation of pluripotent stem cells

“…Non-invasive visualization of subcellular structures has been enabled by recent development of differential interference contrast (DIC) microscope combined with retardation modulation 19,20 and two switchable orthogonal shear directions 21–23 such as an orientation-independent differential interference contrast (OI-DIC) microscopy 24–28 . These microscopes allow quantitative measurement of subcellular structures, providing information about not only morphology but also the density and dynamics of subcellular structures.…”

Past, present and future of Clytia hemisphaerica as a laboratory jellyfish

“…Building on a body of historical work (Neppi et al 1979;Schmid, 1974;Schmid et al, 1976;Schmid & Tardent, 1971), recent cellular and molecular analyses have shed a new light on regeneration processes in Clytia. Studies of wound healing exploited the optical clarity of the exumbrella monolayered epithelium for high resolution imaging and revealed the extreme rapidity and simplicity of this process, which occurs much faster than for cultured cells or bilaterian embryos (Kamran et al, 2017;Malamy & Shribak, 2018). Both purse-string-like supra-cellular actomyosin contractions and lamellipodia-dependent cell crawling contribute to wound healing (Kamran et al, 2017;Malamy & Shribak, 2018).…”

“…Studies of wound healing exploited the optical clarity of the exumbrella monolayered epithelium for high resolution imaging and revealed the extreme rapidity and simplicity of this process, which occurs much faster than for cultured cells or bilaterian embryos (Kamran et al, 2017;Malamy & Shribak, 2018). Both purse-string-like supra-cellular actomyosin contractions and lamellipodia-dependent cell crawling contribute to wound healing (Kamran et al, 2017;Malamy & Shribak, 2018). The balance between these mechanisms depends on the depth of the wound and the extent of damage to the basement membrane / ECM.…”

Single‐shot quantitative phase imaging as an extension of differential interference contrast microscopy

“…In addition, phase‐shifted DIC (PS‐DIC) used a rotating analyzer with DIC microscopy and demonstrated quantification of a bovine cell line (King et al, 2008). Orientation‐independent DIC (OI‐DIC) incorporated two beam‐shearing assemblies in DIC microscopy (Shribak et al., 2017) and was applied to quantitative chromatin biology (Imai et al., 2017) and imaging of epithelial cell dynamics (Malamy & Shribak, 2018). Recently, gradient light interference microscopy (GLIM) applied a phase‐based spatial light modulator (SLM) to DIC microscopy and demonstrated the visualization of thick structures such as embryos and cell sheets (Fanous et al., 2019; Nguyen et al, 2017).…”