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  • 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 adjusted.
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  • 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 is complex and time consuming and it is therefore still not widely practised. By getting to know the two main components of the microscope which are adjusted in this technique (the diaphragms and sub-stage condenser) in reality, correct set-up should only take a matter of minutes. A correctly aligned microscope can result in greatly improved images of uniform contrast and illumination as well as higher resolution and more detail. In this article, we will look at the history of the technique in addition to how to adjust the components in five easy steps.
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  • 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 by the front lens of an objective. In this article, we will briefly look at how using an immersion medium between the coverslip and the objective front lens helps to increase the NA and resolution.
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  • 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 a medium is also taken into account and by matching the refractive index of a slide or cell culture container with an immersion medium, then more of the detail of a specimen will be resolved. The way in which light behaves when travelling from one medium to another is also related to NA (and termed ‘refraction’). This article also covers a brief history of refraction and how this concept is a limiting factor in achieving high NA.
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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.
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  • 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 dynamics. Unfortunately, its full applicability is constrained by the limited spatial resolution of a conventional optical microscope. The present chapter depicts the combination of optical super-resolution STED (stimulated emission depletion) microscopy with FCS , and why it is an important tool for investigating molecular membrane dynamics in living cells. Compared with conventional FCS , the STED- FCS approach demonstrates an improved possibility to distinguish free from anomalous molecular diffusion, and thus to give new insights into lipid–protein interactions and the traditional lipid ‘raft’ theory.
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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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  • 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.
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  • 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 analysis (FA).
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  • Webinar: STED Nanoscopy in Combination with Optical Clearing Reveals Localization of Slit Diaphragm Proteins in the Kidney

    In this webinar, we show that optical clearing drastically increases the signal-to-noise ratio and staining quality, thus enabling STED nanoscopy of the subtlest elements of the kidney. In this way we show that optical clearing is not only a sample preparation technique to consider when imaging large mm-scale samples, but could also be fruitful when imaging at the nanoscale. Furthermore, the increased transparency of the optically cleared sample enables volumetric 3D STED imaging at sub-diffraction-limited resolution.
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  • 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.
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  • 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 cellular communication and the ways how the cell surface interacts to organize tissues and body structures is of great interest. Kees Jalink and his team of scientists at the Netherlands Cancer Institute (NKI) in Amsterdam obtained new scientific insights into the molecular architecture of hemidesmosomes, cytoskeletal components, cell surface receptors and vesicular proteins with the help of Ground-State-Depletion (GSD)/ dSTORM microscopy. In this interview, Kees Jalink comments on their developments in imaging chambers, buffer conditions and image analysis to get the perfect super resolution image.
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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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  • 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.
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  • Webinar on Demand: High Pressure Freezing With Leica EM ICE - The Tool For Understanding Dynamic Processes

    In this webinar, we will introduce the features and applications of the Leica EM ICE high pressure freezer with light stimulation, emphasizing the physics and technical solutions behind the user interface. We will discuss parameters such as dwell time, pressure and temperature correlation, how they are measured and what the displayed data means. We will also explain how light stimulation is synchronized with the freezing process to give you millisecond precision in your experiments.
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  • 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.
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  • 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 to that of immersion oil (1.518), thus minimizing both light scattering and spherical aberrations.
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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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  • 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.
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  • 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.
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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.
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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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  • 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.
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  • 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.
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  • 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 increased depth of field for a larger three-dimensional area in focus was only achievable by reducing the resolution. A new technology is able to overcome this challenge.
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  • 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.
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  • 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.
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