4.12. Modulated Polarization Microscopy (MPM)

Modulated polarization microscopy (MPM) visualizes cytoskeletal filaments based on their birefringence, but differs from standard polarization microscopy by exploiting the angle dependence of birefringence. MPM imaging as detailed by Kuhn and Poenie [61,62] was performed at a rate of one to two processed frames per second. Each image in the resulting MPM movie sequence was enhanced slightly using a 3 × 3 convolution kernel of [(−1/2 1/2 ½), (−1 1/2 ½), (−1/2 1/2 ½)], which added a small emboss effect to the image and improved visibility. Fluorescent images were acquired using a 12 bit CCD camera (Model DVC-1312M, DVC, Austin, TX, USA) on a Nikon Diaphot 200 fluorescence microscope. Image stacks were obtained using a MAC 2000 z-axis focus controller (Ludl Electronic Products, Hawthorne, NY, USA) and a custom image acquisition plugin written for ImageJ. The point-spread function (PSF) was measured under similar conditions using 100 nm fluorescent beads (L-5473, Molecular Probes) diluted in dH2O and dried on glass slides to give approximately one bead per field. Image stacks were deconvolved for 500 iterations using the expectation maximization algorithm in XCOSM [62]. 3D projections were calculated using the maximum value projection method in ImageJ, and stereo pairs were generated using images that differed in rotation by 10 degrees. Images were enhanced using a multiscale 3D line filter implemented as a custom plugin for ImageJ.