Extended depth from focus reconstruction using NIH ImageJ plugins: quality and resolution of elevation maps

In this work, NIH ImageJ plugins for extended depth-from-focus reconstructions (EDFR) based on spatial domain operations were compared and tested for usage optimization. Also, some preprocessing solutions for light microscopy image stacks were evaluated, suggesting a general routine for the ImageJ user to get reliable elevation maps from grayscale image stacks. Two reflected light microscope image stacks were used to test the EDFR plugins: one bright-field image stack for the fracture of carbon-epoxy composite and its darkfield corresponding stack at same (x,y,z) spatial coordinates. Image quality analysis consisted of the comparison of signal-to-noise ratio and resolution parameters with the consistence of elevation maps, based on roughness and fractal measurements. Darkfield illumination contributed to enhance the homogeneity of images in stack and resulting height maps, reducing the influence of digital image processing choices on the dispersion of topographic measurements. The subtract background filter, as a preprocessing tool, contributed to produce sharper focused images. In general, the increasing of kernel size for EDFR spatial domain-based solutions will produce smooth height maps. Finally, this work has the main objective to establish suitable guidelines to generate elevation maps by light microscopy.