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

Abstract

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 alpha-galactosylceramide (alphaGalCer)-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 approximately 25 to approximately 160 nm, with the smaller domains corresponding to a single CD1d molecule.

Nanoscale monitoring of drug actions on cell membrane using atomic force microscopy

“…When performing force spectroscopy on living cells, the AFM probe can be contaminated after recording only a few force curves [110] . Therefore, current AFM methods (such as simultaneous topography and recognition imaging [112,113] ) are mainly adapted for chemically fixed cells, which allow us to obtain data with a higher signal-noise ratio. Studies that convincingly demonstrate the reliability of force spectroscopy experiments on living mammalian cells are still needed.…”

Molecular imaging of membrane proteins and microfilaments using atomic force microscopy

“…This method was also applied to mapping of interactions between calcitonin and calcitonin receptor in osteocalst cells using AFM tips modified with calcitonin (Lehenkari et al, 2000). In addition, it was reported that the combined imaging of fluorescence, topography and recognition was applied to detect density, distribution, and localization of YFP-labeled CD1d molecules on α-galactosylceramide-loaded THP1 cells (Duman et al, 2010).…”

“…To overcome this limitation, a combination of AFM and confocal microscopy has been recognized as an alternative method for the investigation of biological materials with high resolution (Franz and Muller, 2005;Sharma et al, 2005;Duman et al, 2010). It would be both convenient and useful to directly install AFM on a confocal microscope, but there may be compatibility issues between the two.…”

Mapping CD20 molecules on the lymphoma cell surface using atomic force microscopy

“…AFM achieves nanometer resolution and can work in various environments, including the liquids necessary for living cells. By tethering antibodies or ligands onto the AFM tip, specific molecules (receptors or antigens) on the cell surface can be located by using either binding force mapping [10] or dynamic force recognition mapping [11]. In this work, we combined the AFM lift scan method and the binding force mapping method to locate CD20 molecules on the surface of lymphoma Raji cells.…”

“…Living cells have a soft and dynamic surface, which means that the scanning AFM tip can deform the cell surface [15]. Therefore, to avoid the deformation of the cell membrane, fixing agents are often used [11]. AFM images of the local cell surface obtained in the normal tapping mode revealed that the cell surface was rough (Figure 2(b)-(d)).…”