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).