Resolution
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What Does 30,000:1 Magnification Really Mean?
One important criterion concerning the performance of an optical microscope is magnification. This report will offer digital microscopy users helpful guidelines to determine the useful range of magnification values.Read article -
Eyepieces, Objectives and Optical Aberrations
For most microscope applications, there are generally only two sets of optics which are adjusted by the user, namely, the objectives and the eyepieces. Of course, this is assuming that the microscope is already corrected for Koehler Illumination during which the condenser and diaphragms are…Read article -
Koehler Illumination: A Brief History and a Practical Set Up in Five Easy Steps
The technique of Koehler Illumination is one of the most important and fundamental techniques in achieving optimum imaging in any given light microscope set-up. Although it should be routinely used as part of setting up a microscope, many microscopists are put off by thinking that the correct set-up…Read article -
Immersion Objectives: Using Oil, Glycerol, or Water to Overcome some of the Limits of Resolution
To examine specimens at high magnifications using the microscope, there are a number of factors which need to be taken into consideration. These include resolution, numerical aperture (NA), the working distance of objectives and the refractive index of the medium through which the image is collected…Read article -
Collecting Light: The Importance of Numerical Aperture in Microscopy
Numerical aperture (abbreviated as ‘NA’) is an important consideration when trying to distinguish detail in a specimen viewed down the microscope. NA is a number without units and is related to the angles of light which are collected by a lens. In calculating NA (see below), the refractive index of…Read article -
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…Read article -
Super-Resolution Optical Microscopy of Lipid Plasma Membrane Dynamics
Plasma membrane dynamics are an important ruler of cellular activity, particularly through the interaction and diffusion dynamics of membrane-embedded proteins and lipids. FCS (fluorescence correlation spectroscopy) on an optical (confocal) microscope is a popular tool for investigating such…Read article -
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…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…Read article -
How Digital Microscopy can Contribute to Efficient Workflows for Microelectronics and Electronics
This report explains how users can benefit from the digital microscope portfolio of Leica Microsystems to attain cost-effectiveness over entire workflows in research and development (R&D), product innovation, process engineering, production, quality control and assurance (QC/QA), and failure…Read article -
Visualization of Membrane Dynamics with Millisecond Temporal Resolution
Application Note for Leica EM ICE, Leica EM AFS2 - Electrical stimulation of neurons combined with high-pressure freezing allows physiological activation of synaptic activity and precise control over the time frame of the induced synaptic activity.Read article -
Practical Guide for Excellent GSDIM Super-Resolution Images
Do you know that most protists and bacteria lack in one feature that each of our body cell has? Our cells are touch and communicate with one another. They send and receive a variety of signals that coordinate their behavior to act together as a functional multicellular organism. Exploring the way of…Read article -
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…Read article -
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…Read article -
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.Read article -
Super-Resolution Mapping of Neuronal Circuitry With an Index-Optimized Clearing Agent
Super-resolution imaging deep inside tissues has been challenging, as it is extremely sensitive to light scattering and spherical aberrations. Here, we report an optimized optical clearing agent for high-resolution fluorescence imaging (SeeDB2). SeeDB2 matches the refractive indices of fixed tissues…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…Read article -
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…Read article -
Image Gallery: Increasing Confocal Resolution Down to 140 nm – HyVolution Confocal Super-Resolution Reveals More Details in Crisp Images
Super-sensitive detection with low noise plus Huygens deconvolution is the key to resolving structures down to 140 nm with a confocal microscope. The result is crisp images full of contrast revealing details, which cannot be seen with confocal microscopy.Read article -
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…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 -
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…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…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…Read article -
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…Read article -
FusionOptics in Neurosurgery and Ophthalmology – for a Larger 3D Area in Focus
Neurosurgeons and ophthalmologists deal with delicate structures, deep or narow cavities and tiny structures with vitally important functions. A clear, three-dimensional view on the surgical field is thus indispensable for the outcome of the operation and the patient’s safety. Until now, an…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 -
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…Read article -
Factors to Consider When Selecting a Stereo Microscope
Stereo microscopes are often nicknamed the workhorse of the lab or the production department. Users spend many hours behind the ocular inspecting, observing, documenting or dissecting samples. Which factors need to be considered when selecting...Read article -
Unlimited Resolution - STED
STED uses a differential method of two different diffraction patterns, where one pattern excites and the second pattern de-excites fluorochromes. The residual excited area is controllable by intensity down to (theoretically) zero – unlimited resolution.Read article
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