A subscription to JoVE is required to view this content. Sign in or start your free trial.
TIRF Microscopy to Visualize Actin and Microtubule Coupling Dynamics
Overview
This video describes TIRF, a total internal reflection microscopy-based technique to visualize actin and microtubule polymerization dynamics. The method allows the high-resolution imaging of actin and microtubule coupling dynamics in real-time, which is essential for understanding cellular crosstalks.
Protocol
1. Washing the coverslips
NOTE: Wash (24 mm x 60 mm, #1.5) coverslips according to Smith et al., 2013.
- Arrange coverslips in a plastic slide mailer container.
- Submerge coverslips sequentially in the following solutions and sonicate for 30-60 min, rinsing with ddH2O 10 times in between each solution: ddH2O with one drop of dish soap; 0.1 M KOH. Store coverslips in 100% ethanol for up to 6 months.
Representative Results
Figure 1. Experimental schematics: flow chamber assembly to image acquisition. (A) Imaging chamber assembly. Top to bottom: IBIDI imaging chambers are taped along perfusion wells (denoted by arrow); the second (white) layer of tape backing (left on in the image shown to better orient users) is removed and Epoxy is applied at the edge of the perfusion chamber (arrow). Note: To more easily orient users where to pl.......
This article has been published
Video Coming Soon
Source: Henty-Ridilla, J.L., Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence (TIRF) Microscopy. J. Vis. Exp. (2022)
ABOUT JoVE
Copyright © 2025 MyJoVE Corporation. All rights reserved