Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy

Summary

The goal of this protocol is to show how to use Lattice Light-Sheet Microscopy to four-dimensionally visualize surface receptor dynamics in live cells. Here T cell receptors on CD4⁺ primary T cells are shown.

Abstract

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the structure-function relationship of these receptors in situ, we need to visualize and track them on the live cell surface with enough spatiotemporal resolution. Here we show how to use recently developed Lattice Light-Sheet Microscopy (LLSM) to image T-cell receptors (TCRs) four-dimensionally (4D, space and time) at the live cell membrane. T cells are one of the main effector cells of the adaptive immune system, and here we used T cells as an example to show that the signaling and function of these cells are driven by the dynamics and interactions of the TCRs. LLSM allows for 4D imaging with unprecedented spatiotemporal resolution. This microscopy technique therefore can be generally applied to a wide array of surface or intracellular molecules of different cells in biology.

Introduction

The precise dynamics of molecules trafficking and diffusing on the three-dimensional cell surface in real time have been an enigma to solve. Microscopy has always been a balance of speed, sensitivity, and resolution; if any one or two are maximized, the third is minimized. Therefore, due to the small size and immense speed with which surface receptors move, tracking their dynamics has remained a major technological challenge to the field of cell biology. For example, many studies have been conducted using total internal reflection fluorescence (TIRF) microscopy^1,2,3, which ha....

Protocol

5C.C7 TCR-transgenic RAG2 knockout mice in B10.A background were used in this study according to a protocol approved by the Institutional Animal Care and Use Committee of the University of Chicago.

1. Harvest and Activate T Cells

NOTE: This part of the protocol is based on previous protocols. See citations for further detail^20,21.

1. Euthanize a 10-12 week-old 5C.C7 transgenic mouse of either sex (~20-25 g) according to the approved IACUC protocol (i.e., CO₂ chamber followed by cervical dislocation).
2. Spray mouse carcass thoro....

Results

Here, we describe the isolation, preparation, and imaging of primary mouse 5C.C7 T cells using a lattice light-sheet microscope. During section 3, it is imperative to align the microscope correctly, and to collect PSF daily with which to deconvolve the data after collection. In Figure 2, we show the correct alignment images that will be seen when aligning the microscope. Figure 2A and Figure 2B show.......

Discussion

The presented protocol was optimized for the usage of CD4⁺ T cells isolated from 5C.C7 transgenic mice on the LLSM instrument used, and therefore other cell systems and LLSMs may need to be optimized differently. However, this protocol shows the power of 4D imaging, as it can be used to quantify the dynamics of a surface receptor on an entire cell with the least distortion in physiological conditions. Therefore, there are many possible future applications of this technique.

A critic.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1 mL Syringe	BD	309659	For T cell harvest
2-Mercaptoethanol	Sigma-Aldrich	M3148-25ML	For T cell culture
5 mm round coverslips	World Precision Instruments	502040	For Imaging
70um Sterile Cell Strainer	Corning	7201431	For T cell harvest
Alexa Fluor 488 anti-mouse TCR β chain Antibody	BioLegend	109215	For Imaging
Fetal Bovine Serum (FBS)	X&Y Cell Culture	FBS-500	For T cell culture
Ficoll	GE Healthcare	17-1440-02	Denisty gradient reagent for T cell harvest
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
L-Glutamine	Thermo Fisher Scientific	25030-081	For T cell culture
LLSpy	Janelia Research Campus	N/A	LLSpy was used under license from Howard Hughes Medical Institute, Janelia Research Campus. Contact innovation@janelia.hhmi.org for access. Other deconvolution and deksewing methods are available in image processing softwares such as Fiji, Slidebook, Amira, and others. https://llspy.readthedocs.io/en/latest/
Moth Cytochrome C (MCC), sequence ANERADLIAYLKQATK	Elimbio	Custom Synthesis	For T cell harvest
Penacillin/Streptamycin	Life Technologies	15140122_3683884612	For T cell culture
Poly-L-Lysine	Phenix Research Products	P8920-100ML	For Imaging
RBC Lysis Buffer	eBioscience	00-4300-54	For T cell harvest
Recombinant mouse IL-2	Sigma-Aldrich	I0523	For T cell culture
RPMI 1640 Medium	Corning	MT10040CV	For T cell culture
Slidebook	3i	N/A	LLSM imaging software
Surgical Dissection Tools	Nova-Tech International	DSET10	For T cell harvest
T-25 Flasks	Eppendorf	2231710126	For T cell culture
Thermo Scientific Pierce Fab Micro Preparation Kits	Thermo Fisher Scientific	44685	For preparing Fab