Quantitative dynamic footprinting microscopy

Quantitative dynamic footprinting (qDF) allows visualization of the footprints of live leukocytes rolling on a selectin-coated cover glass. qDF works on the principle of total internal reflection fluorescence, which involves fluorescence excitation in a thin slice (∼200 nm) of the cell proximal to the cover glass while the rest of the cell remains dark. Dual color qDF (DqDF) is an advancement of qDF, which enables simultaneous visualization of two fluorochromes in the footprints of rolling leukocytes. When the fluorochrome is localized either in the cell cytoplasm or plasma membrane, the two-dimensional qDF image is used to create a three-dimensional rendition of the footprint topography. DqDF is a useful tool to study leukocyte adhesion under flow, and has recently been used to reveal mechanisms that enable neutrophils to roll at high shear stresses that prevail in venules during inflammation.