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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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  • 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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  • 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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  • Factors to Consider When Selecting a Research Microscope

    An optical microscope is often one of the central devices in a life-science research lab. It can be used for various applications which shed light on many scientific questions. Thereby the configuration and features of the microscope are crucial for its application coverage, ranging from brightfield through fluorescence microscopy to live-cell imaging. This article provides a brief overview of the relevant microscope features and wraps up the key questions one should consider when selecting a research microscope.
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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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  • Infinity Optical Systems

    “Infinity Optics” refers to the concept of a beam path with parallel rays between the objective and the tube lens of a microscope. Flat optical components can be brought into this “Infinity Space” without influencing image formation, which is critical for the utilization of contrast methods such as DIC or fluorescence. Modern microscopy techniques require the addition of multiple optical instruments, such as light sources or laser devices, into the infinite light path. Different approaches to fulfill this need have emerged and are described here.
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  • 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.
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  • What do you Call a Drosophila who Likes to Drink? A Bar Fly!

    The thought of a Drosophoila headed to the local bar for a drink creates a funny joke, but after long hours of sorting flies under a microscope that causes eyestrain or neck pain, you may be the one that wants to head to the local bar for a drink! Unfortunately most users do not know why they experience discomfort when using a stereo microscope.
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  • 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.
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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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  • Video Talk by Joseph Gall: Early History of Microscopy

    Joseph Gall takes us through the history of early microscopes and the discovery of the cell. Compound microscopes were invented alongside the telescope in the 17th century; however these microscopes were not widely used until the late 19th century due to optical aberrations. In the meantime, simple microscopes were used throughout the 1700s and 1800s to make major discoveries in biology, including the first descriptions of the nucleus, cilia, cells, bacteria, and protozoans. Once optics improved in the mid to late 1800s, compound microscopes were used to discover chromosomes, mitosis, and other cellular structures.
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  • Encoding and Decoding Spatio-Temporal Information for Super-Resolution Microscopy

    The challenge of increasing the spatial resolution of an optical microscope beyond the diffraction limit can be reduced to a spectroscopy task by proper manipulation of the molecular states. The nanoscale spatial distribution of the molecules inside the detection volume of a scanning microscope can be encoded within the fluorescence dynamics and decoded by resolving the signal into its dynamics components.
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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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  • 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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  • Light Sheet Fluorescence Microscopy: Beyond the Flatlands

    Light Sheet Fluorescence Microscopy (LISH-M) is a true fluorescence optical sectioning technique, first described by Heinrich Siedentopf in 1902 under the name of Ultramicroscopy. Light sheet microscopy utilises a plane of light to optically section samples. This allows deep imaging within transparent tissues and whole organisms. This book chapter will provide the reader with a comprehensive view on this emerging technology.
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  • How to Clean Microscope Optics

    Clean microscope optics are essential for obtaining good microscope images. If they are dirty, the microscope should be cleaned to avoid a loss of quality. If you decide to do this yourself, you should be extremely careful not to damage the sensitive microscope optics.
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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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  • Selective Plane Illumination Microscopy Techniques in Developmental Biology

    Selective plane illumination microscopy (SPIM) and other fluorescence microscopy techniques in which a focused sheet of light serves to illuminate the sample have become increasingly popular in developmental studies. Fluorescence light-sheet microscopy bridges the gap in image quality between fluorescence stereomicroscopy and high-resolution imaging of fixed tissue sections.
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