Abstract
Applying structured instead of plane illumination in widefield optical fluorescence microscopy can improve the spatial resolution beyond what is known as the Abbe limit. In general it is not only the resolution of an imaging system that is of interest but also its field of view (FOV). These two parameters are expressed in the space-bandwidth product (SBP). Here we introduce a modified structured illumination microscopy (SIM) approach that offers a larger SBP than any other available implementation. This is achieved through a transillumination geometry instead of the typical epifluorescent configuration. Compared to conventional SIM, the illumination path is decoupled from the objective lens by using a multi-mirror setup to generate the sinusoidal interference pattern for structured illumination in transmission mode. The spatial frequency of the illumination pattern can be controlled by changing the angle of the mirrors, achieving comparably fine patterns over a large FOV. In this work simulation results demonstrate the potential resolution improvement to be expected by the suggested implementation. Preliminary experimental results demonstrate phase-shifting ability and the stability of fringe frequencies over a large FOV of (16 mm2) at different numerical apertures, fulfilling the prerequisites for SIM acquisition.
Highlights
In general the resolution of a light microscope is limited due to the wave-like nature of light and the objective’s finite aperture
We introduce a modified structured illumination microscopy (SIM) approach that offers a larger space-bandwidth product (SBP) than any other available implementation
In the first two sections we demonstrate the capability of the mirror mounts to generate uniform high frequency illumination patterns over a large field of view (FOV)
Summary
In general the resolution of a light microscope is limited due to the wave-like nature of light and the objective’s finite aperture. Expressed in the Fourier domain it means that high-frequency sample information that lies outside the support region of the system’s optical transfer function (OTF) is shifted into that region by structured illumination [9] It is this down conversion of frequency components and computational unmixing and relocation of shifted components that can be used to reconstruct the image with improved resolution. Generates illumination patterns with higher spatial frequencies than can be achieved using a low magnification/NA imaging objective lens. While a low magnification/NA objective lens enables imaging a large FOV, an independent multi-mirror mount enables to push the resolution limit beyond what is supported by the NA of the objective. The proposed transillumination SIM setup is shown to be capable of generating illumination patterns with different fringe periods (different spatial frequency) and supporting isotropic resolution enhancement by filling the Fourier space. The capability to introduce well defined phase steps is demonstrated
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