Two-Photon Cell and Tissue Level Laser Ablation Methods to Study Morphogenetic Biomechanics

Abstract

Laser ablation is routinely performed to infer mechanical tension in cells and tissues. Here we describe our method of two-photon laser ablation at the cellular and tissue level in mouse embryos. The primary outcome of these experiments is initial retraction following ablation, which correlates with, and so can be taken as a measure of, the tensile stress that structure was under before ablation. Several experimental variables can affect interpretation of ablation tests.Pre-test factors include differences in physical properties such as viscoelasticity between experimental conditions. Factors relevant during the test include viability of the cells at the point of ablation, image acquisition rate and the potential for over-zealous ablations to cause air bubbles through heat dissipation. Post-test factors include intensity-biased image registration that can artificially produce apparent directionality. Applied to the closing portion of the mouse spinal neural tube, these methods have demonstrated long-range biomechanical coupling of the embryonic structure and have identified highly contractile cell populations involved in its closure process.

CFL1-dependent dynamicity of surface ectoderm filopodia-like protrusions enhances neurulation zippering speed in mice

“…For laser ablation, embryos were stained, positioned and ablated as previously described(72). After dissection, yolk sacs were kept for genotyping and live embryos were stained for 5 min in 1:500 Cell Mask (Thermo Fisher Scientific) in DMEM (without FBS).…”

“…Cell shape parameters (Figure 4 E – F, Supplementary Figure 2G, Supplementary Figure 6D) were measured using FIJI. Initial recoil of laser ablations (Supplementary Figure 6B – C) was calculated as the change in length of a line drawn between two reference points before and after the ablation(72).…”

The surface ectoderm exhibits spatially heterogenous tension that correlates with YAP localisation during spinal neural tube closure in mouse embryos

“…Laser ablation experiments were carried out as previously described ( Maniou et al, 2021 ; Marshall et al, 2022 ). Each decidua was transferred to an individual 1.5 mL Eppendorf in DMEM + FBS, gassed with 5 % CO 2 , 20 % O 2 and 75 % N 2 and kept on a hot block at 37 °C.…”

More than double the fun with two-photon excitation microscopy

“…In practice, we adjust and by first determining what type of photodamage is occurring within the sample. Linear photodamage from TPE is similar to photoablation, where the damaged area results in a small cavity, typically appearing as a dark non-fluorescent region 157 . Nonlinear photodamage is more complex to judge and may result in either darkening or intensifying the local signals due to the photochemical effects.…”