A comparison of methods for optical sectioning using structured illumination microscopy

摘要

Structured Illumination Microscopy is a simple and effective method to remove out-of-focus light in widefield fluorescence microscopy. Neil et al. originally proposed a simple square-law method for calculating the optically sectioned image from the three raw images with the structured illumination pattern super-imposed. However, the Neil method does not make the most efficient use of the three raw images. The three structured illumination images can also be used to separate three copies of the image covering shifted regions of frequency space in a similar manner to that developed by Gustafsson et al. These can then be combined using a generalized Wiener filter to create an image with a well-behaved optical transfer function in which the missing cone has been filled in, providing optical sectioning. Here, we compare the Neil and Gustafsson methods and show that the Gustafsson method provides an image with higher fidelity and a better Signal to Noise Ratio (SNR) at low photon counts. We apply the two methods to images of fluorescent beads and GFP labeled septins in Aspergillus nidulans.