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Improved localization of cellular membrane receptors using combined fluorescence microscopy and simultaneous topography and recognition imaging

2010; IOP Publishing; Volume: 21; Issue: 11 Linguagem: Inglês

10.1088/0957-4484/21/11/115504

1361-6528

Memed Duman, Michael Pfleger, Rong Zhu, Christian Rankl, Lilia A. Chtcheglova, Isabel Neundlinger, Bianca L. Bozna, Barbara Mayer, Mariolina Salio, Dawn Shepherd, Paolo Polzella, Manuel Moertelmaier, Gerald Kada, Andreas Ebner, Michael Dieudonné, Gerhard J. Schütz, Vincenzo Cerundolo, Ferry Kienberger, Peter Hinterdorfer,

Nanofabrication and Lithography Techniques

The combination of fluorescence microscopy and atomic force microscopy has a great potential in single-molecule-detection applications, overcoming many of the limitations coming from each individual technique. Here we present a new platform of combined fluorescence and simultaneous topography and recognition imaging (TREC) for improved localization of cellular receptors. Green fluorescent protein (GFP) labeled human sodium-glucose cotransporter (hSGLT1) expressed Chinese Hamster Ovary (CHO) cells and endothelial cells (MyEnd) from mouse myocardium stained with phalloidin-rhodamine were used as cell systems to study AFM topography and fluorescence microscopy on the same surface area. Topographical AFM images revealed membrane features such as lamellipodia, cytoskeleton fibers, F-actin filaments and small globular structures with heights ranging from 20 to 30 nm. Combined fluorescence and TREC imaging was applied to detect density, distribution and localization of YFP-labeled CD1d molecules on α-galactosylceramide (αGalCer)-loaded THP1 cells. While the expression level, distribution and localization of CD1d molecules on THP1 cells were detected with fluorescence microscopy, the nanoscale distribution of binding sites was investigated with molecular recognition imaging by using a chemically modified AFM tip. Using TREC on the inverted light microscope, the recognition sites of cell receptors were detected in recognition images with domain sizes ranging from ∼ 25 to ∼ 160 nm, with the smaller domains corresponding to a single CD1d molecule.