Three-dimensional super-resolution imaging for fluorescence emission difference microscopy

Abstract

We propose a method theoretically to break the diffraction limit and to improve the resolution in all three dimensions for fluorescence emission difference microscopy. We produce two kinds of hollow focal spot by phase modulation. By incoherent superposition, these two kinds of focal spot yield a 3D hollow focal spot. The optimal proportion of these two kinds of spot is given in the paper. By employing 3D hollow focal spot, super-resolution image can be yielded by means of fluorescence emission difference microscopy, with resolution enhanced both laterally and axially. According to computation result, size of point spread function of three-dimensional super-resolution imaging is reduced by about 40% in all three spatial directions with respect to confocal imaging.

Resolution Enhancement and Background Suppression in Optical Super‐Resolution Imaging for Biological Applications

“…Improper subtraction caused by sample drifting can be avoided utilizing the one‐scan technique (Figure 3e). [ 178,179 ] This technique holds potential in the axial and depth visualization of organisms, like zebrafish. [ 180 ] Factors, like hardware, probes, algorithm, interact with each other and play an important role in enhancing the axial the resolution and reducing the background noise.…”

Enhancement of fluorescence emission difference microscopy using conjugated vortex phase modulation

“…In the last few decades, several optical microscopy methods have been proposed to improve the resolution to the nanoscale (Hell & Wichmann, 1994;Gustafsson, 2000;Betzig et al, 2006;Rust et al, 2006). As the resolution of optical microscopy is being enhanced to higher levels, some other properties of optical microscopy are also being improved in order to provide better-performance instruments to researchers, including imaging speed (Shao et al, 2010;Chmyrov et al, 2013;Bergermann et al, 2015), photobleaching protection (Mondal, 2008;Staudt et al, 2011;Balzarotti et al, 2017;Heine et al, 2017;Oracz et al, 2017), three-dimensional superresolution (Harke et al, 2008;Gould et al, 2012;Sonnen et al, 2012;You et al, 2015;Zhao et al, 2016b;Zhu et al, 2017), fewer image artefacts and even label-free imaging.…”

Preface to Special Topic: Emerging Techniques in Fluorescence Microscopy and Imaging

“…Three dimensional super-resolution imaging (all, along x,y and z) has been reported by You et al 23 by means of fluorescence emission difference microscopy. Another important milestone is the study of rapidly occurring biological events that can only be studied by enabling fast and real-time imaging techniques.…”

“…Specifically the papers published in this special issue include, fast fluorescence imaging techniques (Jabbar et al, 21 Cha et al 22 ), Super-resolution (You et al 23 ), portable Microscopy (Jagannadh et al 24 ), Multiphoton Microscopy (Ehmke et al 25 ), Image Reconstruction Techniques (Venkatesh et al 26 ) and application in the field of Medicine/Biology (Mandal et al, 27 Rai et. Al.…”