An aperture-shifting light-microscopic method for rapidly quantifying positions of cells in 3D matrices.

Abstract

Rapid measurements of large numbers of cells in 3D are often required for measurements of cell migration. A method is presented for quantifying the position of cells in three-dimensional gels using brightfield microscopy. Images were recorded using transmitted light with a closed condenser aperture diaphragm positioned off axis to produce oblique illumination. Two or more images, each at the same focal plane, were obtained with the aperture shifted equally to either side of the optical axis. All in-focus objects were at the same position in the two images, but out-of-focus objects were displaced parallel to the aperture movement. The method was tested using gels containing 12-microm-diameter glass beads or cells that had migrated several hundred micrometers into the gel. The position in the image plane varied linearly with axial position, being reversed for objects above and below the focal plane. Beads and cells could be visualized up to a depth of >1 mm in gels. The method facilitates measurements of positions of cells in 3D matrices by eliminating the need to perform optical sectioning and can be used with any brightfield microscope. Supplementary material for this article can be found at http://www.interscience.wiley.com/jpages/0196-4763/suppmat/54_2/v54.125.html