Fig 1.
(A-D) Scanning electron micrographs of Caco-2 cells at different magnifications. They are compared with atomic force micrographs of Caco-2 cells at the same magnification (E-H). The SEM micrographs display a cell surface completely covered with microvilli (A+B). At higher magnifications the clusters of microvilli appear linked at their tip (C+D). At high magnification the AFM visualizes highly ordered, equally distributed microvilli (G+H).
Fig 2.
AFM analysis of single microvilli.
(A) Atomic force microscopy of Caco-2 cells without and with green labels of the automated detection of microvilli at 5x5 μm2 and 2.5x2.5 μm2. The last row presents 3D reconstruction of the Caco-2 cell surface at 2.5x2.5 μm2 magnification with and without green labelled microvilli. (B) The diagram displays the mean volume ± SD, etc. and the mean height ± SD of one microvillus determined by automated quantification. (C) The minimal and maximal radius calculated by the inflection point and the curvature are presented by green circles in this draft. The SD is represented by the thickness of the circle (rmin) of 18 ± 2 nm and (rmax) of 63 ± 5 nm (n = 1574).
Fig 3.
AFM images of different lattice structures of microvilli—Contact mode.
The hexagonal (A) and the rhombic (E) lattice structure of the microvilli in the spatial domain imaged by atomic force microscopy. (B) and (F) show the elementary cell of the lattice structure. The red dots represent the microvilli and the lines the lattice planes. (C+G) show the Fourier transformation of (A+E) windowed by the Hann function. The Fourier representations are zoomed into the first Brillouin zones. (D+H) are the plotted profile lines of the red rectangles displayed in the Fourier representations (C+G).
Fig 4.
Analysis of different lattice structures of microvilli.
(A) Box plot of the median microvillar density for the rhombic and the hexagonal lattice structure. The rectangle represents the 1st and 3rd percentile while the whiskers represent the minimal and maximal measured value. (B) Box plot of the median lattice constant of the rhombic and the hexagonal lattice. The rectangle represents the 1st and 3rd percentile while the whiskers represent the minimal and maximal measured value.
Fig 5.
AFM images of the rhombic lattice arrangement of microvilli—AC mode.
Series of the same area of Caco-2 cells at different magnifications. (A) Overview of Caco-2 cells recorded with 1024 data point per scan line. At this image size the amplitude set point must be decreased to image the whole range in z of 1.5 μm. This leads to adhesion of the tip at some areas. (B) This resolution can detect highly and less ordered arrangements of microvilli. (C) With a lateral resolution of 6–7 nm/px the lattice arrangement can clearly be identified. Presented are 0 and 90 degree images of the same area (scan direction labelled with light grey arrow) and their Fourier transforms. For better comparison the left Fourier image is rotated as marked by the white arrow. (D) At the highest magnification of 2.9 nm/px the intermicrovillar links are visible in detail. A repeated scan is presented to demonstrate image stability. Furthermore the Fourier transform at this level yields to the presentation of harmonic components in the Fourier transformation.