Leica Science Lab - Tag : Point Spread Function (PSF) https://www.leica-microsystems.com//science-lab/tag/point-spread-function-psf/ Article tagged with Point Spread Function (PSF) en-US https://www.leica-microsystems.com/48638 THUNDER Imaging Widefield Microscopy Fluorescence Microscopy The Power of Pairing Adaptive Deconvolution with Computational Clearing Deconvolution is a computational method used to restore the image of the object that is corrupted by the point spread function (PSF) along with sources of noise. In this technical brief, learn how the deconvolution algorithm offered by Leica Microsystems allows you to overcome losses in image resolution and contrast in widefield (WF) fluorescence microscopy due to the wave nature of light and the diffraction of light by optical elements. Explore the methods of user-controlled or automated deconvolution to see and resolve more structural detail. https://www.leica-microsystems.com/the-power-of-pairing-adaptive-deconvolution-with-computational-clearing/ Tue, 16 Feb 2021 11:22:00 +0000 PhD Vikram Kohli, PhD James M. Marr, Dr. Oliver Schlicker, Dr. Levi Felts https://www.leica-microsystems.com/28031 Basics in Microscopy Fluorescence Microscopy Widefield Microscopy Getting Sharper 3D Images of Thick Biological Specimens with Widefield Microscopy Widefield fluorescence microscopy is often used to visualize structures in life science specimens and obtain useful information. With the use of fluorescent proteins or dyes, discrete specimen components are marked in a highly specific manner. To fully understand a structure, visualizing it in 3 dimensions can be necessary, but certain challenges are faced when doing so with microscopy. https://www.leica-microsystems.com/science-lab/getting-sharper-3d-images-of-thick-biological-specimens-with-widefield-microscopy/ Tue, 18 Aug 2020 08:00:00 +0000 Dr. Christoph Greb, PhD James DeRose, Dr. Rolf T. Borlinghaus https://www.leica-microsystems.com/24508 Confocal Microscopy Super-Resolution 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/18801 Super-Resolution Fluorescence Microscopy 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/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 https://www.leica-microsystems.com/6417 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/getting-sharper-3d-images-of-thick-biological-specimens-with-widefield-microscopy/ 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