Abstract
Three-dimensional microscopy suffers from sample-induced aberrations that reduce the resolution and lead to misinterpretations of the object distribution. In this paper, the resolution of a three-dimensional fluorescent microscope is significantly improved by introducing an amplitude diversity in the form of a binary amplitude mask positioned in several different orientations within the pupil, followed by computer processing of the diversity images. The method has proved to be fast, easy to implement, and cost-effective in high-resolution imaging of casper fli:GFP zebrafish.
Highlights
Imaging fluorophores inside large three-dimensional samples with sub-cellular resolution is of a great interest in life sciences, the inherent optical inhomogeneity of biological tissues distorts the image
Whilst light-sheet fluorescence microscopy (LSFM) [3, 4] is a technique that has been developed for imaging large three-dimensional samples, the aberrations, scattering and absorption only generally allow for the use of lower numerical apertures (NA) that dampen the effect of aberrations at the cost of resolution
The benefits of this new approach may be summarised at this point: firstly, it is not necessary to determine what are the control settings that optimise the image, this means that the amount of expertise required to use adaptive optics well is reduced dramatically; secondly, the method requires less frames incurring less photo-bleaching and toxicity than would be required by model-free or model-based optimisation of the images with most devices used for microscopy
Summary
H. Stelzer, “Multi-view image fusion improves resolution in three-dimensional microscopy,” Opt. Express 15, 8029–8042 (2007). H. Stelzer, “High-resolution three-dimensional imaging of large specimens with light sheet–based microscopy,” Nat. methods, 311 4 (2007). D. Love, “3d adaptive optics in a light sheet microscope,” Opt. Express 20, 13252–13261 (2012). “Adaptive illumination based on direct wavefront sensing in a light-sheet fluorescence microscope,” Opt. Express 24, 24896–24906 (2016). “Adaptive optics confocal microscopy using direct wavefront sensing,” Opt. letters 36, 1062–1064 (2011). “Model-based wavefront sensorless adaptive optics system for large aberrations and extended objects,” Opt. Express 23, 24587–24601 (2015). “Experiments on speckle imaging using projection methods,” in “SPIE Optical Engineering+ Applications,” J. “Blind multi-frame deconvolution by tangential iterative projections (TIP),” Opt. Express 25, 32305 (2017). “Model-based sensor-less wavefront aberration correction in optical coherence tomography,” Opt. Lett.
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