Figure 5.

Multidimensional atomic force microscopy (AFM). (A) Principles of the AFM technology. Piezoelectric actuator moves the microscope stage with ångström (Å) precision while very fine tip (probe) of the cantilever (100 nm) is moving (contact mode) or oscillates up and down (tapping mode) along the surface of the cell. The deflection of the tip is measured using laser beam reflected from the surface of the cantilever. Reflected light is subsequently projected onto the position sensitive photodiode array. From the output signals data processing electronics and visualization software creates 3D virtualization of the cell surface characteristics. Note that the tip of the cantilever (red) has subcellular dimensions [Inset photograph, courtesy of: Drs Alexandre Berquand (Veeco Instruments GmbH, Mannhaim, Germany), Ian Schroeder (Leica Microsystems) and Frank Lafont (Pasteur Institute, Lille, France)]. (B) Advanced visualization of AFM data showing improved recognition of unique morphometric features (indicated with white arrows). Standard 2D AFM height image (upper), pseudo (non-realistic) 3D AFM height image (middle) and photo-realistically rendered 3D height image (lower). Note that live realistic 3D volume rendering of AFM data allows even very small cell surface structures to be identified with great precision. (C) Hybrid images of a cell showing 3D representation of confocal fluorescence, height and deflection AFM modes (upper panel). Reconstruction of 3D cell surface visualized together with the fluorescence confocal data (green texture layer). Note volume measuring tool that provides unique morphometric capabilities (lower). Multiple surface visualization of atomic force microscopy and fluorescence data were performed using Surface3D PRO engine (ScienceGL Inc.,) (data used with permission from ScienceGL Inc., Attleboro, MA, USA).