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

Published: July 31, 2023

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. Add 10 mL of water plus 30 µL fluorescein (1 mg/mL stock) to the LLSM bath (~10 mL volume), press Image (H…

Representative 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 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) …

Declarações

The authors have nothing to disclose.

Materials

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

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Lattice Light Sheet Microscopy to Visualize Receptor-Ligand Interactions in Live Cells. J. Vis. Exp. (Pending Publication), e21457, doi: (2023).

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