Fourier ring correlation simplifies image restoration in fluorescence microscopy

Fourier ring correlation (FRC) has recently gained popularity among fluorescence microscopists as a straightforward and objective method to measure the effective image resolution. While the knowledge of the numeric resolution value is helpful in e.g., interpreting imaging results, much more practical use can be made of FRC analysis\u2014in this article we propose blind image restoration methods enabled by it. We apply FRC to perform image de-noising by frequency domain filtering. We propose novel blind linear and non-linear image deconvolution methods that use FRC to estimate the effective point-spread-function, directly from the images. We show how FRC can be used as a powerful metric to observe the progress of iterative deconvolution. We also address two important limitations in FRC that may be of more general interest: how to make FRC work with single images (within certain practical limits) and with three-dimensional images with highly anisotropic resolution

A new filtering technique for removing anti-Stokes emission background in gated CW-STED microscopy

Stimulated emission depletion (STED) microscopy is a prominent approach of super-resolution optical microscopy, which allows cellular imaging with so far unprecedented unlimited spatial resolution. The introduction of time-gated detection in STED microscopy significantly reduces the (instantaneous) intensity required to obtain sub-diffraction spatial resolution. If the time-gating is combined with a STED beam operating in continuous wave (CW), a cheap and low labour demand implementation is obtained, the so called gated CW-STED microscope. However, time-gating also reduces the fluorescence signal which forms the image. Thereby, background sources such as fluorescence emission excited by the STED laser (anti-Stokes fluorescence) can reduce the effective resolution of the system. We propose a straightforward method for subtraction of anti-Stokes background. The method hinges on the uncorrelated nature of the anti-Stokes emission background with respect to the wanted fluorescence signal. The specific importance of the method towards the combination of two-photon-excitation with gated CW-STED microscopy is demonstrated. © 2014 The Authors. J. Biophotonics

On the Advent of Super-Resolution Microscopy in the Realm of Polycomb Proteins

Simple Summary The genomes of metazoans are organized at multiple spatial scales, ranging from the double helix of DNA to whole chromosomes. The intermediate genomic scale of kilobases to megabases, which corresponds to the 50-300 nm spatial scale, is particularly interesting because the tridimensional arrangement of chromatin is implicated in multiple regulatory mechanisms. Indeed, a crucial hallmark of cellular life is the widespread ordering of many biological processes in nano-/mesoscopic domains (10-200 nm), which now may be revealed by an imaging toolbox referred to as super-resolution microscopy. In this context, polycomb proteins stand as major epigenetic modulators of chromatin function, acting prevalently as repressors of gene transcription. This work reviews the current state-of-the-art super-resolution microscopy applied to polycomb proteins. Of note, super-resolution data have complemented cutting-edge molecular biology methods in providing a rational framework for understanding how polycomb proteins may shape 3D chromatin topologies and functions. The genomes of metazoans are organized at multiple spatial scales, ranging from the double helix of DNA to whole chromosomes. The intermediate genomic scale of kilobases to megabases, which corresponds to the 50-300 nm spatial scale, is particularly interesting, as the 3D arrangement of chromatin is implicated in multiple regulatory mechanisms. In this context, polycomb group (PcG) proteins stand as major epigenetic modulators of chromatin function, acting prevalently as repressors of gene transcription by combining chemical modifications of target histones with physical crosslinking of distal genomic regions and phase separation. The recent development of super-resolution microscopy (SRM) has strongly contributed to improving our comprehension of several aspects of nano-/mesoscale (10-200 nm) chromatin domains. Here, we review the current state-of-the-art SRM applied to PcG proteins, showing that the application of SRM to PcG activity and organization is still quite limited and mainly focused on the 3D assembly of PcG-controlled genomic loci. In this context, SRM approaches have mostly been applied to multilabel fluorescence in situ hybridization (FISH). However, SRM data have complemented the maps obtained from chromosome capture experiments and have opened a new window to observe how 3D chromatin topology is modulated by PcGs

Synthesis of highly luminescent wurtzite CdSe/CdS giant-shell nanocrystals using a fast continuous injection route

We synthesized CdSe/CdS giant-shell nanocrystals, with a CdSe core diameter between 2.8 nm and 5.5 nm, and a CdS shell thickness of up to 7–8 nm (equivalent to about 20 monolayers of CdS). Both the core and shell have a wurtzite crystal structure, yielding epitaxial growth of the shell and nearly defect-free crystals. As a result, the photoluminescence (PL) quantum efficiency (QE) is as high as 90%. Quantitative PL measurements at various excitation wavelengths allow us to separate the nonradiative decay into contributions from interface and surface trapping, giving us pathways for future optimization of the structure. In addition, the NCs do not blink, and the giant shell and concurring strong electron delocalization efficiently suppress Auger recombination, yielding a biexciton lifetime of about 15 ns. The corresponding biexciton PL QE equals 11% in 5.5/18.1 nm CdSe/CdS. Variable-temperature time-resolved PL and PL under magnetic fields further reveal that the emission at cryogenic temperature originates from a negative trion-state, in agreement with other CdSe/CdS giant-shell systems reported in the literature

Muscular cystic hydatidosis: case report

BACKGROUND: Hydatidosis is a zoonosis caused by Echinococcus granulosus, and ingesting eggs released through the faeces from infected dogs infects humans. The location of the hydatid cysts is mostly hepatic and/or pulmonary, whereas musculoskeletal hydatidosis is very rare. CASE PRESENTATION: We report an unusual case of primary muscular hydatidosis in proximity of the big adductor in a young Sicilian man. The patient, 34 years old, was admitted to the Department of Infectious and Tropical Diseases for ultrasonographic detection, with successive confirmation by magnetic resonance imaging, of an ovular mass (13 × 8 cm) in the big adductor of the left thigh, cyst-like, and containing several small cystic formations. Serological tests for hydatidosis gave negative results. A second drawing of blood was done 10 days after the first one and showed an increase in the antibody titer for hydatidosis. The patient was submitted to surgical excision of the lesion with perioperatory prophylaxis with albendazole. The histopathological examination of the bioptic material was not diriment in the diagnosis, therefore further tests were performed: additional serological tests for hydatidosis for the evaluation of IgE and IgG serotype (Western Blot and REAST), and molecular analysis of the excised material. These more specific serological tests gave positive results for hydatidosis, and the sequencing of the polymerase chain reaction products from the cyst evidenced E. granulosus DNA, genotype G1. Any post-surgery complications was observed during 6 following months. CONCLUSION: Cystic hydatidosis should always be considered in the differential diagnosis of any cystic mass, regardless of its location, also in epidemiological contests less suggestive of the disease. The diagnosis should be achieved by taking into consideration the clinical aspects, the epidemiology of the disease, the imaging and immunological tests but, as demonstrated in this case, without neglecting the numerous possibilities offered by new serological devices and modern day molecular biology techniques

High quality RNA isolation from Aedes aegypti midguts using laser microdissection microscopy

Background: Laser microdissection microscopy (LMM) has potential as a research tool because it allows precise excision of target tissues or cells from a complex biological specimen, and facilitates tissue-specific sample preparation. However, this method has not been used in mosquito vectors to date. To this end, we have developed an LMM method to isolate midgut RNA using Aedes aegypti

Intensity Weighted Subtraction Microscopy Approach for Image Contrast and Resolution Enhancement

We propose and demonstrate a novel subtraction microscopy algorithm, exploiting fluorescence emission difference or switching laser mode and their derivatives for image enhancement. The key novelty of the proposed approach lies in the weighted subtraction coefficient, adjusted pixel-by-pixel with respect to the intensity distributions of initial images. This method produces significant resolution enhancement and minimizes image distortions. Our theoretical and experimental studies demonstrate that this approach can be applied to any optical microscopy techniques, including label free and non-linear methods, where common super-resolution techniques cannot be used