Related Experiment Video
Updated: May 9, 2026
A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins
Published on: March 22, 2012
Nanomapping of CD1d-glycolipid complexes on THP1 cells by using simultaneous topography and recognition imaging
Memed Duman1, Lilia A Chtcheglova, Rong Zhu
1Institute for Biophysics, University of Linz, Altenbergerstrasse 69, A-4040, Linz, Austria.
Insights
Simultaneous topography and recognition imaging (TREC) reveals single CD1d molecule distribution on THP1 cells. This method visualizes glycolipid binding sites, showing different cluster sizes for various glycolipids with single-molecule resolution.
Area of Science:
- Immunology
- Cell Biology
- Biophysics
Background:
- CD1d molecules present glycolipids to invariant natural killer T (iNKT) cells, crucial for immunity and autoimmunity.
- Understanding CD1d-glycolipid complex distribution is key to iNKT cell function.
Purpose of the Study:
- To develop and apply simultaneous topography and recognition imaging (TREC) for visualizing single CD1d molecules.
- To detect the density, distribution, and localization of CD1d molecules on THP1 cells loaded with different glycolipids.
Main Methods:
- Simultaneous topography and recognition imaging (TREC) using atomic force microscopy.
- Functionalization of AFM tips with biotinylated iNKT cell receptors (TCR).
- Analysis of THP1 cells pulsed with three distinct glycolipids (α-GalCer, C20, OCH12).
Main Results:
- TREC successfully mapped single CD1d-glycolipid binding sites with nanoscale resolution.
- CD1d-α-GalCer and CD1d-C20:2 formed smaller microdomains (~10,000 nm²).
- OCH12 loaded CD1d complexes formed larger clusters (~30,000 nm²).
- Recognition spots as small as 25 nm indicated single CD1d binding sites.
Conclusions:
- TREC provides high-resolution insights into CD1d-glycolipid complex organization on cell surfaces.
- Glycolipid identity influences the formation and size of CD1d microdomains.
- This technique advances the study of immune recognition at the single-molecule level.
Abstract:
CD1d molecule, a monomorphic major histocompatibility complex class I-like molecule, presents different types of glycolipids to invariant natural killer T (iNKT) cells that play an important role in immunity to infection and tumors, as well as in regulating autoimmunity. Here, we present simultaneous topography and recognition imaging (TREC) analysis to detect density, distribution and localization of single CD1d molecules on THP1 cells that were loaded with different glycolipids. TREC was conducted using magnetically coated atomic force microscopy tips functionalized with a biotinylated iNKT cell receptor (TCR). The recognition map revealed binding sites visible as dark spots, resulting from oscillation amplitude reduction during specific binding between iNKT TCR and the CD1d-glycolipid complex. THP1 cells were pulsed with three different glycolipids (α-GalCer, C20 and OCH12) for 4 and 16 hr. Whereas CD1d-α-GalCer and CD1d-C20:2 complexes on cellular membrane formed smaller microdomains up to ~10 000 nm(2) (dimension area), OCH12 loaded CD1d complexes presented larger clusters with a dimension up to ~30 000 nm(2). Moreover, the smallest size of recognition spots was about 25 nm, corresponding to a single CD1d binding site. TREC successfully revealed the distribution and localization of CD1d-glycolipid complexes on THP1 cell with single molecule resolution under physiological conditions.
