Leica Science Lab - Tag : Point Spread Function (PSF) https://www.leica-microsystems.com//science-lab/tag/tags/point-spread-function-psf/show/Tag/ Article tagged with Point Spread Function (PSF) en-US https://www.leica-microsystems.com/24508 Super-Resolution Confocal Microscopy Live-Cell Imaging Simultaneously Measuring Image Features and Resolution in Live-Cell STED Images Reliable interpretation and quantification of cellular features in fluorescence microscopy requires an accurate estimate of microscope resolution. This is typically obtained by measuring the image of a nonbiological proxy for a point-like object, such as a fluorescent bead. Although appropriate for confocal microscopy, bead-based measurements are problematic for stimulated emission depletion microscopy and similar techniques where the resolution depends critically on the choice of fluorophore and acquisition parameters. In this article, we demonstrate that for a known geometry (e.g., tubules), the resolution can be measured in situ by fitting a model that accounts for both the point spread function (PSF) and the fluorophore distribution. https://www.leica-microsystems.com//science-lab/simultaneously-measuring-image-features-and-resolution-in-live-cell-sted-images/ Mon, 12 Nov 2018 23:00:00 +0000 https://www.leica-microsystems.com/18925 Super-Resolution Confocal Microscopy Image Restoration and Deconvolution 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. https://www.leica-microsystems.com//science-lab/methods-to-calibrate-and-scale-axial-distances-in-confocal-microscopy-as-a-function-of-refractive-index/ Fri, 09 Dec 2016 08:29:00 +0000 https://www.leica-microsystems.com/18801 Super-Resolution Fluorescence Microscopy Image Restoration and Deconvolution 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. https://www.leica-microsystems.com//science-lab/measuring-the-3d-sted-psf-with-a-new-type-of-fluorescent-beads/ Wed, 21 Sep 2016 06:54:00 +0000 PhD Jürgen J. Schmied, MSc Remko Dijkstra, Ph.D. Max B. Scheible, Ph.D. Giulia M. R. De Luca, PhD Jochen J. Sieber https://www.leica-microsystems.com/19026 Super-Resolution Mirror-Enhanced Super-Resolution Microscopy Axial excitation confinement beyond the diffraction limit is crucial to the development of next-generation, super-resolution microscopy. STimulated Emission Depletion (STED) nanoscopy offers lateral super-resolution using a donut-beam depletion, but its axial resolution is still over 500 nm. Total internal reflection fluorescence microscopy is widely used for single-molecule localization, but its ability to detect molecules is limited to within the evanescent field of ~100 nm from the cell attachment surface. We find here that the axial thickness of the point spread function (PSF) during confocal excitation can be easily improved to 110 nm by replacing the microscopy slide with a mirror. The interference of the local electromagnetic field confined the confocal PSF to a 110-nm spot axially, which enables axial super-resolution with all laser-scanning microscopes. https://www.leica-microsystems.com//science-lab/mirror-enhanced-super-resolution-microscopy/ Thu, 18 Aug 2016 09:29:00 +0000 https://www.leica-microsystems.com/18088 Super-Resolution Translation Microscopy (TRAM) for Super-Resolution Imaging Super-resolution microscopy is transforming our understanding of biology but accessibility is limited by its technical complexity, high costs and the requirement for bespoke sample preparation. We present a novel, simple and multi-color super-resolution microscopy technique, called translation microscopy (TRAM), in which a super-resolution image is restored from multiple diffraction-limited resolution observations using a conventional microscope whilst translating the sample in the image plane. https://www.leica-microsystems.com//science-lab/translation-microscopy-tram-for-super-resolution-imaging/ Fri, 22 Jul 2016 17:51:00 +0000 https://www.leica-microsystems.com/16064 Super-Resolution Quantifying the Resolution of a Leica SR GSD 3D Localization Microscopy System with 2D and 3D Nanorulers DNA origami based nanorulers produced by GATTAquant are common standards to test the achievable spatial resolution of super-resolution microscopes. Recently the nanorulers were used to test the performance of the Leica SR GSD 3D microscope. https://www.leica-microsystems.com//science-lab/quantifying-the-resolution-of-a-leica-sr-gsd-3d-localization-microscopy-system-with-2d-and-3d-nanorulers/ Wed, 11 May 2016 07:32:00 +0000 PhD Carsten Forthmann, PhD Jürgen J. Schmied, PhD Tamara Straube https://www.leica-microsystems.com/15458 Confocal Microscopy Fluorescence Microscopy Improving Axial Resolution in Confocal Microscopy with New High Refractive Index Mounting Media Resolution, high signal intensity and elevated signal to noise ratio (SNR) are key issues for biologists who aim at studying the localisation of biological structures at the cellular and subcellular levels using confocal microscopy. The resolution required to separate sub-cellular biological structures is often near to the resolving power of the microscope. https://www.leica-microsystems.com//science-lab/improving-axial-resolution-in-confocal-microscopy-with-new-high-refractive-index-mounting-media/ Thu, 11 Jun 2015 13:04:00 +0000 https://www.leica-microsystems.com/15079 Super-Resolution Confocal Microscopy Image Restoration and Deconvolution 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). https://www.leica-microsystems.com//science-lab/super-resolution-on-a-heuristic-point-of-view-about-the-resolution-of-a-light-microscope/ Wed, 21 Jan 2015 16:00:00 +0000 Dr. Rolf T. Borlinghaus https://www.leica-microsystems.com/13927 Basics in Microscopy Video Talk by Jeff Lichtman: Point Spread Function An infinitesimally small point appears in the microscope as a spot with a certain size, blurred in the z-direction and with concentric rings around it. This "point spread function" reveals many of the optical properties of your microscope. This lecture explains why and how the microscope images a point as a point spread function. https://www.leica-microsystems.com//science-lab/video-talk-by-jeff-lichtman-point-spread-function/ Thu, 08 Jan 2015 13:23:00 +0000 Dr. Jeff Lichtman https://www.leica-microsystems.com/9680 Confocal Microscopy Pinhole Geometry: Four Corners are Perfect Square and hexagonal pinholes provide identical image signal levels, if the geometries are compared in a sensible manner. The amount of light passing the pinhole depends on the area of that aperture, consequently the area is the parameter that must be compared when discussing brightness of focus images. The use of incommensurable edge lengths is meant to confuse the reader and thus dishonest and reprehensible. In this article, the signal level as a function of geometry and size in confocal microscopes is described. https://www.leica-microsystems.com//science-lab/pinhole-geometry-four-corners-are-perfect/ Thu, 23 May 2013 12:59:00 +0000 Dr. Rolf T. Borlinghaus https://www.leica-microsystems.com/6417 Widefield Microscopy Fluorescence Microscopy Live-Cell Imaging Image Restoration and Deconvolution Image Processing for Widefield Microscopy 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. https://www.leica-microsystems.com//science-lab/deconvolution/ Wed, 04 Jul 2012 22:00:00 +0000 Dr. Christoph Greb https://www.leica-microsystems.com/4411 Super-Resolution A Guide to Super-Resolution Fluorescence Microscopy For centuries, cell biology has been based on light microscopy and at the same time been limited by its optical resolution. However, several new technologies have been developed recently that bypass this limit. https://www.leica-microsystems.com//science-lab/a-guide-to-super-resolution-fluorescence-microscopy/ Sun, 18 Jul 2010 22:00:00 +0000