Comparative performance of airyscan and structured illumination superresolution microscopy in the study of the surface texture and 3D shape of pollen

The visualization of taxonomically diagnostic features of individual pollen grains can be a challenge for many ecologically and phylogenetically important pollen types. The resolution of traditional optical microscopy is limited by the diffraction of light (250 nm), while high resolution tools such as electron microscopy are limited by laborious preparation and imaging workflows. Airyscan confocal superresolution and structured illumination superresolution (SR-SIM) microscopy are powerful new tools for the study of nanoscale pollen morphology and three-dimensional structure that can overcome these basic limitations. This study demonstrates their utility in capturing morphological details below the diffraction limit of light. Using three distinct pollen morphotypes (Croton hirtus, Dactylis glomerata, and Helianthus sp.) and contrast-enhancing fluorescent staining, we were able to assess the effectiveness of the Airyscan and SR-SIM. We further demonstrate that these new superresolution methods can be easily applied to the study of fossil pollen material

Fluorescence antibunching microscopy

Breaking the diffraction limit in microscopy by utilizing quantum properties of light has been the goal of intense research in the recent years. We propose a quantum superresolution technique based on non-classical emission statistics of fluorescent markers, routinely used as contrast labels for bio-imaging. The technique can be readily implemented using standard fluorescence microscopy equipment

Suppression of quantum noises in coherent atom lithography through squeezing

The Abbe's diffraction limit restricts the resolution of an optical imaging and lithography system. Coherent Rabi oscillation is shown to be able to overcome the diffraction limit in both optical and atom lithography. In previous studies, semiclassical theory is applied where the driving field is treated as a classical light and quantum fluctuation is neglected. Here, we show that the quantum fluctuation may reduce the visibility of the superresolution pattern. However, by squeezing the photon number fluctuation we are able to significantly increase its visibility

Asymptotics of Bayesian Error Probability and 2D Pair Superresolution

This paper employs a recently developed asymptotic Bayesian multi-hypothesis testing (MHT) error analysis to treat the problem of superresolution imaging of a pair of closely spaced, equally bright point sources. The analysis exploits the notion of the minimum probability of error (MPE) in discriminating between two competing equi-probable hypotheses, a single point source of a certain brightness at the origin vs. a pair of point sources, each of half the brightness of the single source and located symmetrically about the origin, as the distance between the source pair is changed. For a Gaussian point-spread function (PSF), the analysis makes predictions on the scaling of the minimum source strength, expressed in units of photon number, required to disambiguate the pair as a function of their separation, in both the signal-dominated and background-dominated regimes. Certain logarithmic corrections to the quartic scaling of the minimum source strength with respect to the degree of superresolution characterize the signal-dominated regime, while the scaling is purely quadratic in the background-dominated regime. For the Gaussian PSF, general results for arbitrary strengths of the signal, background, and sensor noise levels are also presented.Comment: Submitted to Optics Express, March 18, 201

Probing for Binding Regions of the FtsZ Protein Surface through Site-Directed Insertions: Discovery of Fully Functional FtsZ-Fluorescent Proteins

FtsZ, a bacterial tubulin homologue, is a cytoskeletal protein that assembles into protofilaments that are one subunit thick. These protofilaments assemble further to form a “Z ring” at the center of prokaryotic cells. The Z ring generates a constriction force on the inner membrane and also serves as a scaffold to recruit cell wall remodeling proteins for complete cell division in vivo. One model of the Z ring proposes that protofilaments associate via lateral bonds to form ribbons; however, lateral bonds are still only hypothetical. To explore potential lateral bonding sites, we probed the surface of Escherichia coli FtsZ by inserting either small peptides or whole fluorescent proteins (FPs). Among the four lateral surfaces on FtsZ protofilaments, we obtained inserts on the front and back surfaces that were functional for cell division. We concluded that these faces are not sites of essential interactions. Inserts at two sites, G124 and R174, located on the left and right surfaces, completely blocked function, and these sites were identified as possible sites for essential lateral interactions. However, the insert at R174 did not interfere with association of protofilaments into sheets and bundles in vitro. Another goal was to find a location within FtsZ that supported insertion of FP reporter proteins while allowing the FtsZ-FPs to function as the sole source of FtsZ. We discovered one internal site, G55-Q56, where several different FPs could be inserted without impairing function. These FtsZ-FPs may provide advances for imaging Z-ring structure by superresolution techniques. IMPORTANCE One model for the Z-ring structure proposes that protofilaments are assembled into ribbons by lateral bonds between FtsZ subunits. Our study excluded the involvement of the front and back faces of the protofilament in essential interactions in vivo but pointed to two potential lateral bond sites, on the right and left sides. We also identified an FtsZ loop where various fluorescent proteins could be inserted without blocking function; these FtsZ-FPs functioned as the sole source of FtsZ. This advance provides improved tools for all fluorescence imaging of the Z ring and may be especially important for superresolution imaging

Quantum Theory of Superresolution for Two Incoherent Optical Point Sources

Rayleigh's criterion for resolving two incoherent point sources has been the most influential measure of optical imaging resolution for over a century. In the context of statistical image processing, violation of the criterion is especially detrimental to the estimation of the separation between the sources, and modern farfield superresolution techniques rely on suppressing the emission of close sources to enhance the localization precision. Using quantum optics, quantum metrology, and statistical analysis, here we show that, even if two close incoherent sources emit simultaneously, measurements with linear optics and photon counting can estimate their separation from the far field almost as precisely as conventional methods do for isolated sources, rendering Rayleigh's criterion irrelevant to the problem. Our results demonstrate that superresolution can be achieved not only for fluorophores but also for stars.Comment: 18 pages, 11 figures. v1: First draft. v2: Improved the presentation and added a section on the issues of unknown centroid and misalignment. v3: published in Physical Review