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

Abstract

The combination of fluorescence microscopy and atomic force microscopy has a greatpotential in single-molecule-detection applications, overcoming many of the limitationscoming from each individual technique. Here we present a new platform of combinedfluorescence and simultaneous topography and recognition imaging (TREC) for improvedlocalization of cellular receptors. Green fluorescent protein (GFP) labeled humansodium-glucose cotransporter (hSGLT1) expressed Chinese Hamster Ovary (CHO) cellsand endothelial cells (MyEnd) from mouse myocardium stained with phalloidin-rhodaminewere used as cell systems to study AFM topography and fluorescence microscopy on thesame surface area. Topographical AFM images revealed membrane features such aslamellipodia, cytoskeleton fibers, F-actin filaments and small globular structures withheights ranging from 20 to 30 nm. Combined fluorescence and TREC imaging was appliedto detect density, distribution and localization of YFP-labeled CD1d molecules onα-galactosylceramide(αGalCer)-loaded THP1 cells. While the expression level, distribution and localization of CD1dmolecules on THP1 cells were detected with fluorescence microscopy, the nanoscaledistribution of binding sites was investigated with molecular recognition imaging by using achemically modified AFM tip. Using TREC on the inverted light microscope, the recognitionsites 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.