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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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  • Optimization of the Interplay of Optical Components for Aberration free Microscopy

    Optical microscopes are used to magnify objects which are otherwise invisible for the human eye. For this purpose high quality optics is necessary to achieve appropriate resolution. However, besides intentional effects, all optical components have also unwanted intrinsic influence on light, resulting in aberrations. This article highlights optical elements and their physical parameters involved in this process. Based on this, it gives a historical overview of philosophies about how to cope with aberration reduction. Seeing the microscope as a whole system turned out to be beneficial, leading to the harmonization of its constituents for optimal microscopic results.
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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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  • Optical Microscopes – Some Basics

    The optical microscope has been a standard tool in life science as well as material science for more than one and a half centuries now. To use this tool economically and effectively, it helps a lot to understand the basics of optics, especially of those essential components which are part of every microscope.
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  • 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...
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  • Digital Cameras

    Manufacturers digital cameras race to outdo each other with ever-increasing numbers of megapixels. The world record for professional medium format digital cameras has now surpassed 60 megapixels per shot using a very large and expensive sensor with a resolution of about 9000 x 6700 pixels. Each time you capture such an image you get about 180 MB of uncompressed data and even more if you switch to 16-bit per colour for full dynamic range.
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  • Beware of "Empty" Magnification

    This article explains how to avoid the phenomen of "empty magnification" in microscopy.
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