Simultaneous imaging of the surface and the submembraneous cytoskeleton in living cells by tapping mode atomic force microscopy (original) (raw)
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Cytoskeleton of living, unstained cells imaged by scanning force microscopy
Biophysical Journal, 1993
Subsurface cytoskeletal structure can be visualized in either fixed or living mammalian cells in aqueous medium with -50 nm resolution using the Scanning Force Microscope (SFM). In living cells, changes in cell topography, or subsurface cytoskeleton caused by the introduction of drugs (colchicine) or cross-linking of surface receptors (by antibodies against IgE bound to the IgE receptor) can be followed in time. Contrast in SFM images of cell surfaces result from both topographic features of the cell and from variations in cell surface "stiffness". The SFM is therefore capable of measuring local compliance and stress in living cells, and so should make it possible to map the cytoskeletal forces used to generate cell motions and changes in cell shape. ment the cells were stimulated by injecting 1.0 gg/ml DNP-BSA in 0006 3495/93/04
Ultramicroscopy, 2000
Di$culties in the proper adjustment of the scanning parameters are often encountered when using tapping-mode atomic force microscopy (TMAFM) for imaging thick and soft material, and particularly living cells, in aqueous bu!er. A simple procedure that drastically enhances the successfull imaging of the surface of intact cells by TMAFM is described. It is based on the observation, in liquid, of a de#ection signal, concomitant with the damping of the amplitude that can be followed by amplitude}distance curves. For intact cells, the evolution of the de#ection signal, steeper than the amplitude damping allows a precise adjustment of the feedback value. Besides its use in "nding the appropriate tapping conditions, the de#ection signal provides images of living cells that essentially reveal the organization of the membrane cytoskeleton. This allows to show that changes in the membrane surface topography are associated with a reorganization of the membrane skeleton. Studies on the relationships between the cell surface topography and membrane skeleton organization in living cells open a new "eld of applications for the atomic force microscope.
Imaging of the Surface of Living Cells by Low-Force Contact-Mode Atomic Force Microscopy
Biophysical Journal, 1998
The membrane surface of living CV-1 kidney cells in culture was imaged by contact-mode atomic force microscopy using scanning forces in the piconewton range. A simple procedure was developed for imaging of the cell surface with forces as low as 20 -50 pN, i.e., two orders of magnitude below those commonly used for cell imaging. Under these conditions, the indentation of the cells by the tip could be reduced to less than l0 nm, even at the cell center, which gave access to the topographic image of the cell surface. This surface appeared heterogeneous with very few villosities and revealed, only in distinct areas, the submembrane cytoskeleton. At intermediate magnifications, corresponding to 20 -5 m scan sizes, the surface topography likely reflected the organization of submembrane and intracellular structures on which the plasma membrane lay. By decreasing the scan size, a lateral resolution better than 20 nm was routinely obtained for the cell surface, and a lateral resolution better than 10 nm was obtained occasionally. The cell surface appeared granular, with packed particles, likely corresponding to proteins or protein-lipid complexes, between ϳ5 and 30 nm xy size.
Imaging of the Membrane Surface of MDCK Cells by Atomic Force Microscopy
The membrane surface of polarized renal epithelial cells (MDCK cells) grown as a monolayer was imaged with the atomic force microscope. The surface topography of dried cells determined by this approach was consistent with electron microscopy images previously reported. Fixed and living cells in aqueous medium gave more fuzzy images, likely because of the presence of the cell glycocalix. Treatment of living cells with neuraminidase, an enzyme that partly degrades the glycocalix, allowed sub-micrometer imaging. Protruding particles, 10 to 60 nm xy size, occupy most of the membrane surface. Protease treatment markedly reduced the size of these particles, indicating that they corresponded to proteins. Tip structure effects were probably involved in the exaggerated size of imaged membrane proteins. Although further improvements in the imaging conditions , including tip sharpness, are required, atomic force microscope already offers the unique possibility to image proteins at the membrane surface of living cells.
Atomic force microscopy combined with confocal laser scanning microscopy: A new look at cells
Bioimaging, 1993
A stand-a lone atomic fo rce microscope (AFM) has been developed, which features a la rge sca n area a nd which allows opera tion under liquid. This system was combined with a co nfoca l laser sca nning microscope (C LSM). Information about cell structures, obtained by CLSM, can be complemented with images of the cell surface obtained with the AFM. This is illustra ted by stud ying the pseudopodia of cells from a human cell line (K562-cells, predecessor of eryth ro bl as ts) and the cytoskeleton of monk ey kidney cells (in air and under liquid), both stained with F-actin-specific Auo rescent pro bes. Im ages of the cytoskeleton during the cytotox ic interaction betwee n a na tu ral killer and a K562 target cell are presented. O ur results show that co mbination of these techniques ca n provid e new information about cells and cellular structu res. Key words: a to mic fo rce microscopy, co nfoca l lase r sca nnin g microscopy, cy toskeleto n, cy totox ic. interacti o n.
Atomic Force Microscopy as a Tool for the Investigation of Living Cells
Medicina, 2013
Atomic force microscopy is a valuable and useful tool for the imaging and investigation of living cells in their natural environment at high resolution. Procedures applied to living cell preparation before measurements should be adapted individually for different kinds of cells and for the desired measurement technique. Different ways of cell immobilization, such as chemical fixation on the surface, entrapment in the pores of a membrane, or growing them directly on glass cover slips or on plastic substrates, result in the distortion or appearance of artifacts in atomic force microscopy images. Cell fixation allows the multiple use of samples and storage for a prolonged period; it also increases the resolution of imaging. Different atomic force microscopy modes are used for the imaging and analysis of living cells. The contact mode is the best for cell imaging because of high resolution, but it is usually based on the following: (i) image formation at low interaction force, (ii) low ...