Abstract
The deconvolution of widefield fluorescence images provides only guesses of spatial frequency information along the optical axis due to the so called missing cone in the optical transfer function. Retaining the single-shot imaging speed of deconvolution microscopy while gaining access to missing cone information is thus highly desirable for microscopy of volumetric samples. Here, we present a concept that superimposes two orthogonally polarized excitation lattices with a phase-shift of p between them. In conjunction with a non-iterative image reconstruction algorithm this permits the restoration of missing cone information. We show how fluorescence anisotropy could be used as a method to encode and decode the patterns simultaneously and develop a rigorous theoretical framework for the method. Through in-silico experiments and imaging of fixed biological cells on a structured illumination microscope that emulates the proposed setup we validate the feasibility of the method.
Highlights
Optical sectioning (OS) is a key requirement to provide contrast in volumetric imaging and is commonly realized via confocal detection or two-photon excitation
To produce a suitable data set for the proposed single shot optical sectioning (ssOS) algorithm, the first phase of the first orientation in each slices was used as image i′ and the two images with phase-stepped patterns were averaged to yield the required raw image i′′ phase-shifted by
As deconvolution of widefield images can only guess axial spatial frequency information due to the missing cone, a method of optical sectioning microscopy was sought that can be implemented as a single-shot imaging technique akin to deconvolution microscopy
Summary
Optical sectioning (OS) is a key requirement to provide contrast in volumetric imaging and is commonly realized via confocal detection or two-photon excitation. These scanning microscopies are significantly slower than widefield alternatives, such as spinning disk, light-sheet, or structured illumination microscopy, SIM1. Despite the linear processing procedures of SR SIM, the comparatively many raw frames (9–15) render these approaches less useful when minimal excitation doses or maximal frame-rates are desired To address this problem, Wicker and Heintzmann pioneered the idea of single shot optical sectioning (ssOS), which builds on the homodyne OS SIM concept[3]. A conceptual optical set-up is outlined, which focuses on the generation of orthogonally polarized excitation lattices and volumetric image capture
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