Figure 1
Comparison of optical sectioning strength. A tilted layer of fluorescent dye was processed with the HiLo ImageJ plugin and our MATLAB script. (a) Shows the experimental setup depicting the fluorescent layer tilt in the inset. The tilt is exaggerated to make clear that only a narrow strip in the centre was in focus. An example of the HiLo processed layer is shown for (b) the ImageJ plugin and (c) the MATLAB script with the optical sectioning parameter σ set to 1 for both processing modalities which corresponded to approximately the same optical section thickness. The thickness as a function of σ (d) was obtained by processing the same data of the tilted fluorescent layer with the plugin and our MATLAB script and calculating the average full-width-half-maximum (FWHM) of Gaussian fits to five horizontal intensity line plots through the processed images. (1) Shows an example of such a horizontal line plot, the line is made thicker to improve visualisation but was only one pixel thick for the measurement. The two processing modalities reached a minimum section thickness of 6.6 ± 0.3 μm (plugin) and 6.8 ± 0.2 μm (MATLAB) at the lowest setting for σ. The graininess is an example of artefacts that can arise when speckle structure translated through to the final image. This could be avoided in thicker samples or by setting σ to higher values, i.e. thicker optical sections.
