Example of fluorescence confocal image used to determine substratum deformation.

<p>The three panels at the top of the figure (<i>a–c</i>) show the first horizontal slice of a –pixel stack, which is focused at the free surface of the substratum (). <i>(</i><b><i>a</i></b><i>)</i>, Tracer beads fluorescence in undeformed conditions used as reference for traction force microscopy. The scale bar is 10 microns long. The axes indicate the reference system for both the substratum deformation and the traction stresses. <i>(</i><b><i>b</i></b><i>)</i>, Tracer beads fluorescence when the substratum is deformed by a migrating cell, whose outline is indicated by the green contour. <i>(</i><b><i>c</i></b><i>)</i>, Image obtained by merging the fluorescence from tracer beads in undeformed (<i>a</i>) and deformed (<i>b</i>) conditions, which reveals the deformation of the substratum. White speckles indicate perfect match between undeformed and deformed conditions and thus zero local deformation. Blue and orange speckles indicate mismatch between undeformed and deformed conditions and thus non-zero local deformation. Regions of locally large deformation are indicated with yellow arrows. The dashed green line indicates the location of the vertical section shown in panels <i>d–g</i>. <i>(</i><b><i>d</i></b><i>)</i>, Three-dimensional illustration of the relative positions of the horizontal plane in panel (<i>x</i>–<i>y</i>, yellow axes) and the vertical plane in panel (<i>s</i>–<i>z</i>, red axes). The black axes indicate the three-dimensional reference system used to express both the substratum deformation and the traction stresses. The three panels at the bottom right corner of the figure (<i>e–g</i>) show vertical slices of the same –stack passing through the dashed green line in panel at . <i>(</i><b><i>e</i></b><i>)</i>, Image obtained by merging the fluorescence from tracer beads in undeformed (<i>f</i>) and deformed (<i>g</i>) conditions. Speckle patterns with orange top and blue bottom are found in locations where the cell is pulling up on the substratum. Conversely, speckle patterns with blue top and orange bottom are found the when cell is pushing down on the substratum. <i>(</i><b><i>f</i></b><i>)</i>, Tracer beads fluorescence in deformed condition. <i>(</i><b><i>g</i></b><i>)</i>, Tracer beads fluorescence in underformed condition.