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

Abstract

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

Protocol

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.

1. Add 10 mL of water plus 30 µL fluorescein (1 mg/mL stock) to the LLSM bath (~10 mL volume), press Image (Home) to move the objective to the image position, and look at a single Bessel laser beam pattern. Align the laser beam using the guides and pre-set re....

Results

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 th.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
5 mm round coverslips	World Precision Instruments	502040	For Imaging
Alexa Fluor 488 anti-mouse TCR β chain Antibody	BioLegend	109215	For Imaging
Fluorescein sodium salt	Sigma-Aldrich	F6377	For microscope alignment
FluoSpheres Carboxylate-Modified Microspheres	Thermo Fisher Scientific	F8810	For microscope alignment
Imaris	Bitplane	N/A	Tracking Software; Other options for tracking software include Amira or Trackmate (Fiji).
Lattice Light-Sheet Microscope	3i	N/A	Microscope Used
Leibovitz's L-15 Medium, no phenol red	Thermo Fisher Scientific	21083027	For Imaging
Slidebook	3i	N/A	LLSM imaging software
Poly-L-Lysine 	Phenix Research Products 	P8920-100ML 	For Imaging