eoe sub articles

EoE Sub Articles

1. Conducting LLSM Daily Alignment
NOTE: (Important) This alignment protocol is based on the LLSM instrument used (see the Table of Materials). Each LLSM may be different and require different alignment strategies, especially those that are home-built. Carry out the appropriate routine alignment and continue to section 2.
<ol>
	<li>Add 10 mL of water plus 30 &#181;L fluorescein (1 mg/mL stock) to the LLSM bath (~10 mL volume), press Image (H...

lattice light sheet microscopy to visualize receptor-ligand interactions in live cells

Source: Rosenberg, J., et al. <a href="https://app.jove.com/v/59914">Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy.</a> J. Vis. Exp. (2020).
In this video, we describe the lattice light-sheet microscopy technique to visualize interactions between live T-cells and antigen-presenting cells. The method involves focusing an ultrathin lattice light sheet on an interacting cell pair to obtain a series of two-dimensional images, which are combined to obtain high-resolution four-dimensional images that reveal the spatiotemporal dynamics of the cell-cell interaction.

<img alt="Figure 1" src="/files/ftp_upload/21457/21457_Figure1.jpg" /> 
Figure 1: LLSM alignment. (A) Desired beam pattern for LLSM imaging experiment. (B) Screenshot of the beam alignment process; on the left is the focus window showing the narrowed, focused beam; at the top right is a graph showing that the beam is centered within the window; at the bottom right is the finder camera, which should also be a thin, focused beam. (C) ...

Lattice Light Sheet Microscopy to Visualize Receptor-Ligand Interactions in Live Cells

Source: Rosenberg, J., et al. Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy. J. Vis. Exp. (2020).
...

Lattice light-sheet microscopy, LLSM, enables the visualization of cell-surface receptor-ligand interactions in live cells with high spatiotemporal resolution.
Take a coverslip containing transduced ligand-expressing cells emitting red fluorescence. Attach the coverslip cell-side-up onto a greased sample holder. Fix the holder on the piezo of a microscope stage to allow precise sample positioning and scanning during imaging. Adjust the software settings to focus on a single ligand-expressing cell.
Pipette the receptor-expressing cell suspension onto the coverslip. The receptors are tagged with green fluorophore-labeled antibodies. Image the interacting cell pairs.
The microscope&#39;s excitation laser emits a circular, linearly-polarized laser beam that passes through cylindrical lenses to create a light sheet. The light sheet is projected onto a modulator that removes excess diffraction and transforms it into an ultrathin lattice pattern.
The lattice light sheet illuminates an ultrathin section of the interacting cell pair, and the fluorescence emitted by the interacting cells is captured perpendicular to the incident beam. Image the interacting cell pair in multiple Z-planes, and capture two-dimensional images in each focal plane with a high-speed camera.
Using suitable software, combine these images to form a z-stack &#8212; a high-resolution four-dimensional image demonstrating the spatiotemporal dynamics of the cell-surface receptor-ligand interaction.

lattice-light-sheet-microscopy-to-visualize-receptor-ligand

Research

JoVE Encyclopedia of Experiments

Biological Techniques

Microscopy Techniques

In this video, we describe the lattice light-sheet microscopy technique to visualize interactions between live T-cells and antigen-presenting cells. The method involves focusing an ultrathin lattice light sheet on an interacting cell pair to obtain a series of two-dimensional images, which are combined to obtain high-resolution four-dimensional images that reveal the spatiotemporal dynamics of the cell-cell interaction.

Overview

Protocol

<img title="figure-representative results-25" alt="figure-representative results-25" src="/files/ftp_upload/21457/21457_Figure1.jpg" /> 
Figure 1: LLSM alignment. (A) Desired beam pattern for LLSM imaging experiment. (B) Screenshot of the beam alignment process; on the left is the focus window showing the narrowed, focused beam; at the top right is a graph showing that the beam is centered within the window; at the bottom right is the finder camera, which should also be a thin, focused beam. (C) ...

Lattice Light Sheet Microscopy to Visualize Receptor-Ligand Interactions in Live Cells

In This Article

Overview

Protocol

Representative Results

Reprints and Permissions