Abstract
The use of structured illumination in fluorescence microscopy allows the suppression of out of focus light and an increase in effective spatial resolution. In this paper we consider different approaches for reconstructing 2D structured illumination images in order to combine these two attributes, to allow fast, optically sectioned, superresolution imaging. We present a linear reconstruction method that maximizes the axial frequency extent of the combined 2D structured illumination passband along with an empirically optimized approximation to this scheme. These reconstruction methods are compared to other schemes using structured illumination images of fluorescent samples. For sinusoidal excitation at half the incoherent cutoff frequency we find that removing information in the zero order passband except for a small region close to the excitation frequency, where it replaces the complementary information from the displaced first order passband, enables optimal reconstruction of optically sectioned images with enhanced spatial resolution.
Highlights
Structured illumination (SI) is used in fluorescence microscopy to remove out of focus light and create optically sectioned images [1]
Sinusoidal excitation patterns were created using a liquid crystal on silicon spatial light modulator (SLM) configured as a binary phase grating as described in [10]
Removal of out of focus light from 2D-structured illumination microscopy (SIM) images requires a tradeoff between the effective optical section thickness and the signal-to-noise ratio in the reconstructed image
Summary
Structured illumination (SI) is used in fluorescence microscopy to remove out of focus light and create optically sectioned images [1]. By illuminating the specimen with sinusoidal excitation patterns of different phases and exploiting precise knowledge of the frequency and orientation of the excitation pattern, these aliased components can be weighted and shifted to their true location in Fourier space By performing this operation for several (usually 3) orientations of the excitation pattern, the spatial frequency cutoff can be extended, approximately isotropically, up to twice that of an equivalent imaging system employing uniform illumination [3, 4]. These two attributes of SI are often exploited independently, to generate either optically sectioned or super-resolved fluorescence images. The effectiveness of these different reconstruction schemes is compared for SIM images of fluorescent microspheres and a fluorescently labelled cell sample
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