Format

Send to

Choose Destination
J Vis Exp. 2014 Mar 24;(85). doi: 10.3791/51100.

Visualization of the immunological synapse by dual color time-gated stimulated emission depletion (STED) nanoscopy.

Author information

1
Center for Human Immunobiology, Texas Children's Hospital and Baylor College of Medicine; mace@bcm.edu.
2
Center for Human Immunobiology, Texas Children's Hospital and Baylor College of Medicine.

Abstract

Natural killer cells form tightly regulated, finely tuned immunological synapses (IS) in order to lyse virally infected or tumorigenic cells. Dynamic actin reorganization is critical to the function of NK cells and the formation of the IS. Imaging of F-actin at the synapse has traditionally utilized confocal microscopy, however the diffraction limit of light restricts resolution of fluorescence microscopy, including confocal, to approximately 200 nm. Recent advances in imaging technology have enabled the development of subdiffraction limited super-resolution imaging. In order to visualize F-actin architecture at the IS we recapitulate the NK cell cytotoxic synapse by adhering NK cells to activating receptor on glass. We then image proteins of interest using two-color stimulated emission depletion microscopy (STED). This results in <80 nm resolution at the synapse. Herein we describe the steps of sample preparation and the acquisition of images using dual color STED nanoscopy to visualize F-actin at the NK IS. We also illustrate optimization of sample acquisition using Leica SP8 software and time-gated STED. Finally, we utilize Huygens software for post-processing deconvolution of images.

PMID:
24686478
PMCID:
PMC4157735
DOI:
10.3791/51100
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for MyJove Corporation Icon for PubMed Central
Loading ...
Support Center