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Microscopy Basics

Basic knowledge in optics and contrasting methods is fundamental for microscopic imaging. The precise setup of an optical microscope including correct Koehler illumination improves image quality – the base for further image analysis. Within light microscopy we differentiate between stained and unstained samples influencing the amplitude and the phase of the light waves traversing the sample. For the human eye, differences in the amplitude are visible as brightness differences. Contrasting methods like phase contrast, modulation contrast, differential interference contrast, often used in living samples, convert phase shifts into intensity. Staining and fluorescence techniques, like immunofluorescence or the use of fluorescent proteins, are used to make selected structures or proteins visible. Thus, to optimize the use of a microscope it is reasonable to learn more about its basic characteristics.

  • The Fundamentals and History of Fluorescence and Quantum Dots

    At some point in your research and science career, you will no doubt come across fluorescence microscopy. This ubiquitous technique has transformed the way in which microscopists can image, tag and trace anything from whole organisms to single proteins and beyond. In this article, we will examine what is meant by "fluorescence", the history and basic physics behind its definition, the discovery and application of Green Fluorescent Protein (GFP) and a look at the rapidly expanding field of fluorescent probes including Quantum Dots.
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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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  • Graphene-Based Microbots for Toxic Heavy Metal Removal and Recovery from Water

    Heavy metal contamination in water is a serious risk to the public health and other life forms on earth. Current research in nanotechnology is developing new nanosystems and nanomaterials for the fast and efficient removal of pollutants and heavy metals from water. Here, we report graphene oxide-based microbots (GOx-microbots) as active self-propelled systems for the capture, transfer, and removal of a heavy metal (i.e., lead) and its subsequent recovery for recycling purposes.
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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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  • Introduction to Widefield Microscopy

    One of the most basic microscopy techniques is known as ‘Widefield Microscopy’. It is fundamentally any technique in which the entire specimen of interest is exposed to the light source with the resulting image being viewed either by the observer or a camera (which can also be attached to a computer monitor).
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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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  • Milestones in Incident Light Fluorescence Microscopy

    Since the middle of the last century, fluorescence microscopy developed into a bio scientific tool with one of the biggest impacts on our understanding of life. Watching cells and proteins with the help of fluorescence molecules is a standard method in nearly every life science discipline today. This broad application range goes back to the technical work of some researchers who wanted to improve and simplify fluorescence microscopic labor. One person who was involved in that development was the Dutch medic Johann Sebastiaan Ploem.
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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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  • Definitions of Basic Technical Terms for Digital Microscope Cameras and Image Analysis

    Most microscopes today are operated with a camera. The characteristics of the camera often decide whether the acquired image will reveal what a researcher wants to see. But when diving into camera terminology, the technical terms can be overwhelming. We have compiled the most important terms with a concise explanation to provide orientation.
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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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  • Introduction to Digital Camera Technology

    A significant majority of modern optical microscopy techniques require the use of a digital camera. By working with digital devices researchers can observe specimens on a screen in real time or acquire and store images and quantifiable data. Here we introduce the basic principles behind digital camera technologies commonly encountered in scientific imaging.
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  • Color Infidelity: Why Using a Light Source Incorrectly is Cheating on your Data

    There are many influences on color in the imaging process including lighting, optics, sensor, and monitor, and ultimately print. The first, and generally most important, is lighting. There are plenty of options for light sources, Halogen, LED, and arc lamps are among the most popular for microscopes. Each light source has its own advantages and disadvantages and it is up to the user to learn which is best for the sample and application.
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  • Fluorescent Proteins Illuminate Cell Biology

    Green fluorescent protein (GFP) isolated from the jellyfish Aequorea victoria and GFP-like fluorescent proteins from other animals have had an important role in the technical innovations that have driven these advances. This poster provides a comprehensive user's guide to fluorescent proteins and sensors , their key properties and the cell biological questions to which they can be applied.
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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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  • 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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  • Microscopes Put to the Test with Severe Conditions: ISO Standard for Resistance of Optical Instruments to Fungus and Mold Growth

    Microscopes and other optical instruments can be affected during use by environmental factors. The environment depends on the geographic location and conditions of the place where the instrument is put to use. Usually, microscopes are manufactured in a way to ensure instrument robustness to a variety of conditions.
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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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  • Video Talk by Bo Huang: What is Light?

    This lecture discusses the many aspects of light. Light can be described as rays, waves, or particles. Various aspects of all three of these descriptions are presented, such as ray tracing, wavelength, frequency, refraction, dispersion, diffraction, interference, the photoelectric effect and photons as the quantized energy of light.
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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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  • Micropolarimetric Analysis of Guard Cell Walls in M. Koenigii versus De Novo Localized Starch Granules in Freshly-Isolated Potato Tuber

    Polarized light microscopy (PLM) has often been utilized in the fields of geology and the material sciences with significant implications in determining the mineral compositions and structures of composite materials (e.g. fibers) through the use of typical quartz wedge compensators, mica quarter-waveplates and de Sénarmont compensation.
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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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  • Video Talk by Daniel Fletcher: Lenses and Image Formation

    Light microscopes use lenses. The basic properties of light, how light interacts with matter, the principles behind refractive lenses and how lenses form (magnified) images will be introduced in this talk.
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  • Hunting Down the Hay Bacillus – Educational Microscopes in Biology Teaching

    Learning begins with perception. Sensory impressions are branded in our mind and become the building blocks of knowledge. The more intensively young people are involved in the lesson and the more experiences they can make themselves, the easier they find it to learn. Hands-on microscopy is therefore a key component of modern science teaching, for example at the Philippinum Grammar School in Weilburg, where students love working with Leica educational microscopes.
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  • Bird Park Gives Fascinating Insights into the Variety of Nature

    The Vogel- und Naturschutz-Tierpark Herborn to the north of Frankfurt may be small, but it’s always a great experience for school classes – and not only because it’s home to more than 300 animals of 80 different species, from South African blue cranes and white storks to meerkats and muntjac deer. Another special highlight for schoolchildren is the opportunity to look through a stereomicroscope and gain fascinating insights into nature.
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  • Step by Step Guide to Fluorescence Microscopy

    Fluorescence Microscopy is a special form of light microscopy. It uses fluorescence to highlight structures in fixed and living biological specimens instead of using absorption, phase or interference effects. The fluorescence is delivered either by inorganic dyes, proteins, synthetic beads or by autofluorescent structures within a sample. In this tutorial the principles of fluorescence microscopy will be explained.
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Interactive Tutorials

Useful Links

Communities and Web Sources network for scientists tools, video lectures on biology and microscopy

bitesizebio.comOnline magazine and community for molecular and cell biology researchers

www.somersault1824.comResource for high-end scientific illustrations, images and animations

Search Engines and Data Bases

www.cellimagelibrary.orgPublic resource database of images, videos, and animations of cells meta search engine for genes and proteins

www.gopubmed.comSearch interface for pubmed of academic databases and search engines of Google's search engine for scientific article abstracts

Journals of open access journals

www.lifescied.orgCBE-Life Sciences Education – an ASCB online journal publication of exceptional research articles of Cell Science Journal of Experimental Biology Disease Models & Mechanisms Journal of Life Science Methods of Journals and Proceedings in Optics and Photonics - peer-reviewed journals on applied research in optics and photonics of Biophotonics, peer-reviewed, open-access, online publication B - the Royal Society's biological research journal Journal for microscopists and features on biomedical microscopy

Organizations / Institutes Society of America Microscopy Society Microscopical Society on microscopy for materials science; University of Cambridge, Department of Materials Science and Metallurgy American Society of Cell Biology company of biologists

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