eoe sub articles

EoE Sub Articles

1. Washing the coverslips
NOTE: Wash (24 mm x 60 mm, #1.5) coverslips according to Smith et al., 2013.
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	<li>Arrange coverslips in a plastic slide mailer container.</li>
	<li>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. 
	<stro...

tirf microscopy to visualize actin and microtubule coupling dynamics

Source: Henty-Ridilla, J.L., <a href="https://app.jove.com/v/64074">Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence (TIRF) Microscopy.</a> J. Vis. Exp. (2022)
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.

<img alt="Figure 1" src="/files/ftp_upload/21456/21456_Figure1.jpg" /> 
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...

TIRF Microscopy to Visualize Actin and Microtubule Coupling Dynamics

Source: Henty-Ridilla, J.L., Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence (TIRF) Microscopy. J. ...

Actin monomers polymerize in a head-to-tail manner to form thin fibers, called microfilaments. Similarly, tubulin dimers polymerize into longer hollow cylindrical microtubules. These cytoskeleton components maintain cell shape and facilitate cellular motility.
To visualize the actin-microtubule coupling dynamics by total internal reflection microscopy, TIRF, take a customized imaging assembly.
The imaging assembly consists of two coverslips: a top m-PEG silane-coated coverslip and a bottom biotin-PEG silane-coated coverslip. The two coverslips are spaced longitudinally with double-sided adhesive tape and sealed at both ends to create a flow channel.
Pipette BSA into the flow channel to prime the chamber. Add streptavidin solution, and incubate. The streptavidin molecules bind to the biotin molecules attached to the bottom coverslip&#39;s PEG-silane.
Flow TIRF buffer into the channel. Add a cytoskeleton mix solution containing unlabeled and green fluorophore-labeled tubulin dimers, along with unlabeled, biotin-labeled, and purple fluorophore-tagged actin monomers.
Next, add a reaction solution containing ATP &#8212; actin polymerization-promoting agent, and GTP &#8212; tubulin polymerization-promoting agent, and actin-microtubule coupling protein. Incubate.
The tubulin dimers bind to the GTP molecules and polymerize, while the actin monomers bind to the ATP molecules and polymerize to form microtubules and microfilaments, respectively.
Place the imaging assembly under a TIRF microscope. Excite the fluorophores with lasers of appropriate wavelengths.
The excitation light reflects internally, and creates a light field that selectively illuminates the fluorophores in a limited region near the glass-water interface, where there are two media with different refractive indexes. In this region, the microfilaments appear purple, and the microtubules appear green.