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

3D HDO-CLEM: cellular compartment analysis by correlative light-electron microscopy on cryosections.

Fundamental to obtaining a depth-understanding of the function and structure of cells is the ability to study and correlate their molecular topography with the ultrastructural morphology, for example, to visualize the position of a given protein relative to a given cell compartment and its morphology. Standard fluorescence light microscopy (FLM) relies on simple sample preparations, and localizes proteins in living or fixed cells with a resolution in the range of few hundred nanometers, allowing large field of view. However, FLM is unable to visualize the unlabeled cellular context. On the other hand, electron microscopy (EM) techniques reveal protein topology with the resolution in a range of a few tens of nanometer, retains the cellular context, but can only be applied on a limited field of view. Therefore, both approaches present shortcomings, in terms of field of view, statistical output, resolution, sample preparation, and context analysis, that can likely complement each other. To bridge the gap between FLM imaging and EM, several laboratories have developed methods for correlative light-electron microscopy (CLEM). In a nutshell, CLEM enables one to investigate the same exact region of interest utilizing the two microscope platforms, and thereby virtually combine their capabilities. We describe a protocol based on immunolabeling of Tokuyasu cryosections that allows correlation of LM and EM images with excellent preservation of cellular ultrastructure. We will refer to this method as high-data-output CLEM (HDO-CLEM). The major benefits of HDO-CLEM are the possibility to (1) correlate several hundreds of events at the same time, (2) perform three-dimensional (3D) correlation, (3) immunolabel both endogenous and recombinantly tagged proteins at the same time, and (4) combine the high data analysis capability of FLM with the high precision of transmission EM in a CLEM hybrid morphometric analysis. We have identified and optimized critical steps in sample preparation, defined routines for sample analysis and retracing of regions of interest, developed software for semi/fully automatic 3D FLM reconstruction and set the basis for a hybrid light/EM morphometry approach

Diagnostic and prognostic role of liquid biopsy in non-small cell lung cancer: evaluation of circulating biomarkers

Lung cancer is still one of the main causes of cancer-related death, together with prostate and colorectal cancers in males and breast and colorectal cancers in females. The prognosis for non-small cell lung cancer (NSCLC) is strictly dependent on feasibility of a complete surgical resection of the tumor at diagnosis. Since surgery is indicated only in early stages tumors, it is necessary to anticipate the timing of diagnosis in clinical practice. In the diagnostic and therapeutic pathway for NSCLC, sampling of neoplastic tissue is usually obtained using invasive methods that are not free from disadvantages and complications. A valid alternative to the standard biopsy is the liquid biopsy (LB), that is, the analysis of samples from peripheral blood, urine, and other biological fluids, with a simple and non-invasive collection. In particular, it is possible to detect in the blood different tumor derivatives, such as cell-free DNA (cfDNA) with its subtype circulating tumor DNA (ctDNA), cell-free RNA (cfRNA), and circulating tumor cells (CTCs). Plasma-based testing seems to have several advantages over tumor tissue biopsy; firstly, it reduces medical costs, risk of complications related to invasive procedures, and turnaround times; moreover, the analysis of genes alteration, such as EGFR, ALK, ROS1, and BRAF is faster and safer with this method, compared to tissue biopsy. Despite all these advantages, the evidences in literatures indicate that assays performed on liquid biopsies have a low sensitivity, making them unsuitable for screening in lung cancer at the current state. This is caused by lack of standardization in sampling and preparation of specimen and by the low concentration of biomarkers in the bloodstream. Instead, routinely use of LB should be preferred in revaluation of patients with advanced NSCLC resistant to chemotherapy, due to onset of new mutations

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

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

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