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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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  • 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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  • How to Correct Aberration in Stereo Microscopy by Using the Right Objective Lenses

    For samples/specimens immersed in a liquid or embedded in a polymer, high quality microscopic observation can be hindered as a result of spherical aberration. An objective which can correct for refractive index mismatch allows images with greatly reduced spherical aberration and sharper focus to be obtained.
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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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