Diffraction Limit
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Video Talk by Kurt Thorn: The Abbe Diffraction Experiment
This lecture describes the famous experiments of Ernst Abbe which showed how diffraction of light by a specimen (and interference with the illuminating light) gives rise to an image and how collection of diffracted light defines the resolution of the microscope. These concepts are demonstrated by using a diffraction grating as a specimen and visualizing and comparing the diffraction pattern in the back focal plane as well as the image in the image plane.Read article -
TIRF Publication List
This monthly updated references list presents current papers using Leica AM TIRF in the major application fields for TIRF microscopy.Read article -
Microscope Resolution: Concepts, Factors and Calculation
In microscopy, the term ‘resolution’ is used to describe the ability of a microscope to distinguish detail. In other words, this is the minimum distance at which two distinct points of a specimen can still be seen - either by the observer or the microscope camera - as separate entities. The resolution of a microscope is intrinsically linked to the numerical aperture (NA) of the optical components as well as the wavelength of light which is used to examine a specimen. In addition, we have to consider the limit of diffraction which was first described in 1873 by Ernst Abbe. This article covers some of the history behind these concepts as well as explaining each using relatively simple terminology.Read article -
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.Read article -
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.Read article -
Video: Fluorescence is a State of Mind
How to break a fundamental law of physics and win a Nobel Prize to boot. Stefan Hell explains super-resolved fluorescence microscopy for which he shared the 2014 Nobel Prize in chemistry.Read article -
A Brief History of Light Microscopy – From the Medieval Reading Stone to Super-Resolution
The history of microscopy begins in the Middle Ages. As far back as the 11th century, plano-convex lenses made of polished beryl were used in the Arab world as reading stones to magnify manuscripts. However, the further development of these lenses into the first microscopes cannot be attributed to any one person. It took the ideas and designs of many scientists and scholars to produce instruments capable of strong magnification.Read article -
Gated STED Microscopy with Time-gated Single-photon Avalanche Diode
The maximization of the useful (within the time gate) photon flux is then an important aspect to obtain super-resolved STED images. Here we show that by using a fast-gated single-photon avalanche diode (SPAD), i.e. a detector able to rapidly (hundreds picoseconds) switch-on and -off can improve significantly the signal-to-noise ratio (SNR) of the gated STED image. In addition to an enhancement of the image SNR, the use of the fast-gated SPAD reduces also the system complexity. We demonstrate these abilities both on calibration and biological sample.Read article -
STED Nanoscopy: A Glimpse into the Future
The well-known saying of "Seeing is believing" became even more apt in biology when stimulated emission depletion (STED) nanoscopy was introduced in 1994 by the Nobel laureate S. Hell and coworkers. This article gives an overview of the various cutting-edge implementations of STED nanoscopy and tries to shine a light into the future: imaging everything faster with unprecedented sensitivity and label-free.Read article -
Pathways to Optical STED Microscopy
STED nanoscopy has evolved to a highly versatile tool for the observation of the living cell, more and more finding its way into state-of-the-art optical imaging facilities in biomedical research institutes.Read article -
Video Talk by Daniel Axelrod: Total Internal Reflection Fluorescence (TIRF) Microscopy
Total Internal Reflection Fluorescence (TIRF) Microscopy is a technique that only illuminates dye molecules near a surface. In this video, the pioneer of TIRF Microscopy describes what this technique is used for, explains the principles of the evanescent wave, gives many examples of different microscope configurations used in TIRF, and shows how polarized light TIRF can be used to image membrane orientation.Read article -
GSDIM Publication List
GSDIM microscopy is a widefield super-resolution technique based on the localization of fluorophores with nanometer precision. With its help a lateral resolution of down to 20 nm can be achieved, whereas the new 3D feature even shrinks axial resolution to 50 nm. Here we provide a collection of publications around that super-resolution microscopy method also called dSTORM.Read article -
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).Read article -
Finding, Defining and Breaking the Diffraction Barrier in Microscopy – A Historical Perspective
Diffraction plays a crucial role in microscopy as it prevents the recording of arbitrarily sharp images with conventional light microscopes. Many names are connected with the notion of diffraction and the definition of resolution. An overview over the contributions of the different scientists to the recognition and definition of the diffraction barrier in the past centuries is given and the recent developments that led to breaking this barrier are portrayed.Read article -
Which Super-Resolution Method is Right for You?
Super-resolution microscopy has dramatically improved our understanding of intracellular dynamics, redefining what is possible in biological research. This infographic gives a compact overview on the different super-resolution techniques such as localization, structured illumination and stimulated emission depletion and will help you to choose the technology that best fulfills your individual research needs.Read article -
Video Interview with Rainer Pepperkok
Rainer Pepperkok is Head of the Advanced Light Microscopy Core Facility and Senior Scientist at the EMBL in Heidelberg (Germany). In the course of his studies he is interested in membrane traffic of the early secretory pathway in mammalian cells which he is trying to analyze with the help of most modern light microcopy techniques.Read article -
Super-Resolution Microscopy – Get Your Free e-Book for Download
Until recently, the diffraction of light had placed a fundamental limit on how far biologists could peer into cells with optical microscopes, preventing them from resolving features less than 250 nm in size, missing critical structures within cells. Over the past 20 years scientists have developed several ingenious techniques allowing them to resolve features as small as 20 nm.Read article -
3D STED (Stimulated Emission Depletion) Microscopy
The resolution needed to image subcellular architecture and dynamics in light microscopy is hindered by the diffraction limits as described by Ernst Abbe. Simply stated, structures smaller than 200 nanometers are lost in a blur. However, the field of super-resolution microscopy has produced methods to obtain resolution beyond this limit. Leica Microsystems has pioneered this field and offers the Leica TCS SP8 STED 3X for 3D Stimulated Emission Depletion microscopy. STED instantly produces super-resolution images, compatible with the dynamics of living cells, without the need for post-processing.Read article -
Nobel Prize in Chemistry for Achievements in Super-Resolution Microscopy
On October 8th 2014, The Royal Swedish Academy of Sciences has decided to award the Nobel Prize in Chemistry for 2014 to Eric Betzig, Stefan W. Hell and William E. Moerner "for the development of super-resolved fluorescence microscopy". For a long time optical microscopy was held back by a presumed limitation: that it would never obtain a better resolution than half the wavelength of light. Helped by fluorescent molecules the Nobel Laureates in Chemistry 2014 ingeniously circumvented this limitation. Their ground-breaking work has brought optical microscopy into the nanodimension.Read article -
Video Interview with Stefan Hell, the Inventor of Super-Resolution
Professor Stefan Hell is director at the Max Planck Institute for Biophysical Chemistry and head of the department of NanoBiophotonics in Goettingen and widely considered as the father of super-resolution. His inventions of 4Pi and STED microscopy were turned into the first commercial super-resolution microscopes available by Leica Microsystems in 2004 and 2007.Read article -
Video Talk by Jeff Lichtman: Resolution in Microscopy – Wave Optics and the Diffraction Limit
Light has properties of particles and waves. Understanding the wave nature of light is essential to understanding the workings of a microscope. This lecture describes Huygens Wavelets, constructive/destructive interference, and diffraction.Read article -
STED Microscopy of Living Cells – New Frontiers in Membrane and Neurobiology
Recent developments in fluorescence far-field microscopy such as STED microscopy have accomplished observation of the living cell with a spatial resolution far below the diffraction limit. Here, we briefly review the current approaches to super-resolution optical microscopy and present the implementation of STED microscopy for novel insights into live cell mechanisms, with a focus on neurobiology and plasma membrane dynamics.Read article -
STED Publication List
It is already 20 years ago that STED microscopy was first described by Stephan Hell. In the meantime it has become an important tool to study subcellular structures beyond the diffraction limit. This reference list gives an overview about the technological development of STED nanoscopy and also shows selected publications performed on a Leica STED instrument.Read article -
Video Talk on Super-Resolution: Overview and Stimulated Emission Depletion (STED) Microscopy
Historically, light microscopy has been limited in its ability to resolve closely spaced objects, with the best microscopes only able to resolve objects separated by 200 nm or more. This limit is known as the diffraction limit. In the last twenty years, a number of techniques have been developed that allow resolution beyond the diffraction limit.Read article -
Interview with Stefan Hell: Breaking Nanoscale Boundaries
Stefan Hell is credited with having conceived and applied the first viable concept for breaking Abbe’s diffraction limit in a light-focusing microscope. He is a scientific member of the Max Planck Society and a director at the Max Planck Institute for Biophysical Chemistry in Göttingen, Germany, where he currently leads the department of nanobiophotonics.Read article -
Video: STED Microscopy at Turku BioImaging
Today, Turku BioImaging embraces state-of-the-art imaging technologies - and of course this includes STED microscopy. In this video the background, principles and inventors of STED super-resolution microscopy as well as the applications STED microscopy is used for in Turku are explained.Read article -
STED and GSDIM: Diffraction Unlimited Resolution for all Types of Fluorescence Imaging
This article gives an overview of two different types of superresolution techniques. Stimulated emission depletion (STED) microscopy is a versatile and fast method that is based on point scanning microscopy – usually an extension of a confocal microscope. Ground state depletion imaging (GSDIM) is a parallel recording widefield approach that explores inherent switching of fluorochromes and typically comes with a TIRF microscope. The two methods use very different approaches to reach the same goal: to see more details in light microscopes than possible when diffraction limited.Read article -
Widefield Super-Resolution with GSDIM
Great advancements in biology have been possible by using fluorescence microscopy. So far, the resolution of the images was limited due to physical constraints. In the past couple of years, new methods evolved circumventing these limitations and bringing fluorescence microscopy to a new level of resolution, boosting the possibilities in science with fluorescence microscopes.Read article -
Depletion and Emission Beam Paths in STED Microscopy
STED is one technology, and it requires already additional beam-routings as compared to pure fluorescence imaging. The Leica TCS SP5 offers configurations with STED, confocal and multiphoton imaging in the very same instrument. This unique concept unites technologies of super-resolution fluorescence, multichannel confocal fluorescence, multiphoton-excited fluorescence and second-harmonic or higher order nonlinear image generation. The various beam-paths are shown and explained in this tutorial.Read article -
Stimulated Emission Depletion (STED)
Among the new super-resolution technologies, stimulated emission depletion (STED) is the most versatile concept. Whether the sample is tissue, e.g. muscle striation details, classical cytoskeletons, nuclear proteins, yeast or bacterial details: STED serves for understanding structure and function by showing the very finest details.Read article
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