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  • Introduction to Widefield Microscopy

    One of the most basic microscopy techniques is known as ‘Widefield Microscopy’. It is fundamentally any technique in which the entire specimen of interest is exposed to the light source with the resulting image being viewed either by the observer or a camera (which can also be attached to a computer monitor).
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  • Clarifying Tissue Clearing

    Biological specimens are intrinsically three dimensional; however because of the obscuring effects of light scatter, imaging deep into a tissue volume is problematic. Although efforts to eliminate the scatter by “clearing” the tissue have been ongoing for over a century, there have been a large number of recent innovations. This review introduces the physical basis for light-scatter in tissue, describes the mechanisms underlying various clearing techniques, and discusses several of the major advances in light microscopy for imaging cleared tissue.
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  • Chronic Inflammation Under the Microscope

    In the course of chronic inflammation certain body areas are recurrently inflamed. This goes along with many human diseases. With the help of widefield light microscopy, the underlying processes can be examined from a cellular level to whole organisms. This article presents several widefield microscopy applications such as immunofluorescence, live-cell imaging, histology, and ratiometric analysis to get insight into the development of chronic inflammation, the related diseases, and their treatment.
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  • Methods to Calibrate and Scale Axial Distances in Confocal Microscopy as a Function of Refractive Index

    Application example of HyVolution Super-Resolution - Accurate distance measurement in 3D confocal microscopy is important for quantitative analysis, volume visualization and image restoration. However, axial distances can be distorted by both the point spread function (PSF) and by a refractive-index mismatch between the sample and immersion liquid, which are difficult to separate. Additionally, accurate calibration of the axial distances in confocal microscopy remains cumbersome, although several high-end methods exist. In this paper we present two methods to calibrate axial distances in 3D confocal microscopy that are both accurate and easily implemented.
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  • P53- and Mevalonate Pathway–Driven Malignancies Require Arf6 for Metastasis and Drug Resistance

    Application example of HvYolution Super-Resolution - Drug resistance, metastasis, and a mesenchymal transcriptional program are central features of aggressive breast tumors. The GTPase Arf6, often overexpressed in tumors, is critical to promote epithelial–mesenchymal transition and invasiveness. The metabolic mevalonate pathway (MVP) is associated with tumor invasiveness and known to prenylate proteins, but which prenylated proteins are critical for MVP-driven cancers is unknown. We show here that MVP requires the Arf6-dependent mesenchymal program.
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  • Measuring the 3D STED-PSF with a new Type of Fluorescent Beads

    A new type of fluorescent bead is presented by GATTAquant. These beads, called GATTA-Beads, are characterized by a small diameter (23 nm), high intensity and size uniformity. In combination with state-of the-art STED microscopes such as the Leica TCS SP8 STED 3X and high-end image restoration methods available in the Huygens Software, it is shown that these new beads can be used for accurate STED PSF characterization in 3D. Furthermore, it is shown that the measured 3D STED-PSF can be used to improve image restoration quality in combination with STED deconvolution methods available in the Huygens Software.
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  • Two-Photon Excitation STED Microscopy with Time-Gated Detection

    We report on a novel two-photon excitation stimulated emission depletion (2PE-STED) microscope based on time-gated detection. The time-gated detection allows for the effective silencing of the fluorophores using moderate stimulated emission beam intensity. This opens the possibility of implementing an efficient 2PE-STED microscope with a stimulated emission beam running in a continuous-wave.
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  • HyVolution – Super-Resolution Imaging with a Confocal Microscope

    Since the invention of the microscope, there has been continual discussion about the possibility of showing more detailed features of specimens as compared to just magnifying them. In this article we describe the HyVolution concept and how the combination of confocal multiparameter fluorescence imaging at the confocal super-resolution regime with psf-based real deconvolution allows high-speed multicolor imaging with a resolution down to 140 nm.
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  • HyVolution – the Smart Path to Confocal Super-Resolution

    Super-resolution refers to any device or method that can resolve better than the classical Abbe limit. Apart from infinite super-resolution techniques such as STED (stimulated emission depletion) and SMLM (single-molecule localization methods) that can theoretically resolve to any detail, there are also methods for limited super-resolution. Here we present HyVolution by Leica, which merges optical super-resolution and computational super-resolution. The optical part is provided by confocal microscopy, and the computational part by deconvolution. Lateral resolution of 140 nm is demonstrated. HyVolution offers multiple fluorescence recording in truly simultaneous mode.
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  • Multi-Images Deconvolution Improves Signal-to-Noise Ratio on Gated Stimulated Emission Depletion Microscopy

    Time-gated detection, namely, only collecting the fluorescence photons after a time-delay from the excitation events, reduces complexity, cost, and illumination intensity of a stimulated emission depletion (STED) microscope. In the gated continuous-wave- (CW-) STED implementation, the spatial resolution improves with increased time-delay, but the signal-to-noise ratio (SNR) reduces.
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  • Super-Resolution – On a Heuristic Point of View About the Resolution of a Light Microscope

    Since super-resolution has become one of the most favored methods in biomedical research, the term has become increasingly popular. Still, there is much of confusion about what is super-resolution and what is resolution at all. Here, the classical view of microscopic resolution is discussed and some techniques that resolve better than classical are briefly introduced. The picture on the right shows the intensity distribution of an image of two points whose distance is just the Rayleigh criterion (false color coding).
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  • Huygens STED Deconvolution Quick Guide

    This document is intended to give Leica STED users a brief introduction to deconvolving images on Huygens Professional using images acquired with the Leica TCS SP8 STED 3X microscope. For a more detailed description of Huygens Professional, including additional features and tools, please visit the Manuals section of SVI (www.svi.nl).
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  • Video Tutorial: STED Parameters and STED Deconvolution

    Scientific Volume Imaging has a leading role in deconvolution with the Huygens STED deconvolution option, that takes the specific properties of the STED PSF into account. Huygens is compatible with data from pulsed, CW, and CW-gated STED systems, and reads the microscopic parameters automatically from these Leica LIF files. Most recently, Huygens is also able to handle data from the novel Leica TCS SP8 STED 3X, which can obtain super-resolution in both lateral (x,y) and axial (z) directions.
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  • Huygens STED Deconvolution Increases Signal-to-Noise and Image Resolution towards 22 nm

    STED microscopy has proven to be a valuable super-resolution technique, resolving objects that are smaller than the diffraction-limited resolution. Deconvolution of STED images with Huygens pushes the resolution even further. In a recent publication (see link below), we demonstrate that Huygens offers a two-fold improvements of STED images in X, Y, and Z resolution, and increases signal-to-noise ratios eight times. The presented data also shows that a lateral resolution increase from 50 nm towards 22 nm was obtained by applying Huygens deconvolution on (biological) gated-STED images. Furthermore, we describe that stabilization of 3D STED images is essential for optimal deconvolution, as it corrects for lateral drift which would normally distort the structure of the STED PSF.
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  • Towards Real-time Image Deconvolution: Application to Confocal and STED Microscopy

    Although deconvolution can improve the quality of any type of microscope, the high computational time required has so far limited its massive spreading. Here we demonstrate the ability of the scaled-gradient-projection (SGP) method to provide accelerated versions of the most used algorithms in microscopy. To achieve further increases in efficiency, we also consider implementations on graphic processing units (GPUs).
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  • Abstracts of the 3rd European Super-Resolution User-Club Meeting

    The 3rd meeting of the Leica Super-Resolution User Club was held from June 17th to 19th, 2013 in collaboration with Alberto Diaspro and the Italian Institute of Technology (IIT) in Genoa. Confocal and widefield super-resolution users from ten European countries took three days’ out to deepen their knowledge on super-resolution techniques and applications and make use of an opportunity for full exchange of experiences.
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  • Abstracts of the 2nd European Super-Resolution User-Club Meeting

    The 2nd meeting of the Leica Super-resolution User club was held from September 25 to 27, 2012 in collaboration with the Science for Life Laboratory at the Karolinska Institute, Stockholm, Sweden. With a mixture of engaging talks by key experts in the field of super-resolution microscopy and stimulating discussion sessions, the meeting proved as popular as last year’s event, attracting a wide range of scientists interested in both confocal and widefield super-resolution and sample preparation techniques.
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  • Deconvolution

    Fluorescence microscopy is a modern and steadily evolving tool to bring light to current cell biological questions. With the help of fluorescent proteins or dyes it is possible to make discrete cellular components visible in a highly specific manner. A prerequisite for these kinds of investigations is a powerful fluorescence microscope. One special aim is the three-dimensional illustration of a structure to get an impression of full plasticity. This poses a certain problem for the experimenter using a classical light microscope.
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  • Mapping Billions of Synapses with Microscopy and Mathematics

    A combination of widefield imaging techniques and image segmentation analysis enable researchers to map learning-induced functional changes in individual synapses throughout the hippocampus.
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