Two fundamental parameters of the highly dynamic, ultrathin lamellipodia of migrating fibroblasts have been determined-its thickness in living cells (176 +/- 14 nm), by standing-wave fluorescence microscopy, and its F-actin density (1580 +/- 613 microm of F-actin/microm(3)), via image-based photometry. In combination with data from previous studies, we have computed the density of growing actin filament ends at the lamellipodium margin (241 +/- 100/microm) and the maximum force (1.86 +/- 0.83 nN/microm) and pressure (10.5 +/- 4.8 kPa) obtainable via actin assembly. We have used cell deformability measurements (. J. Cell Sci. 44:187-200;. Proc. Natl. Acad. Sci. USA. 79:5327-5331) and an estimate of the force required to stall the polymerization of a single filament (. Proc. Natl. Acad. Sci. USA. 78:5613-5617;. Biophys. J. 65:316-324) to argue that actin assembly alone could drive lamellipodial extension directly.

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We report some recent algorithmic refinements and the resulting simulated and real image reconstructions of fluorescence micrographs by using a blind-deconvolution algorithm based on maximum likelihood estimation. Blind-deconvolution methods encompass those that do not require either calibrated or theoretical predetermination of the point-spread function (PSF). Instead, a blind deconvolution reconstructs the PSF concurrently with deblurring of the image data. Two-dimensional computer simulations give some definitive evidence of the integrity of the reconstructions of both the fluorescence concentration and the PSF. A reconstructed image and a reconstructed PSF from a two-dimensional fluorescent data set show that the blind version of the algorithm produces images that are comparable with those previously produced by a precursory nonblind version of the algorithm. They furthermore show a remarkable similarity, albeit not perfectly identical, with a PSF measurement taken for the same data set, provided by Agard and colleagues. A reconstructed image of a three-dimensional confocal data set shows a substantial axial smear removal. There is currently an existing trade-off in using the blind deconvolution in that it converges at a slightly slower rate than the nonblind approach. Future research, of course, will address this present limitation.

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