Firstly let’s discuss optical or lens distortion which is created due to the optics of the glass inside the lens. If you’ve not seen the glass inside they are not flat but instead have curves. The curve varies on each according to the lens focal width. Commonly wide angle lenses create barrel distortion and telephoto lens pincushion distortion. A small selection of mainly older, but some modern lenses create moustache distortion.

The amount of distortion all three create is actually normally quite minimal except at the very widest focal lengths. Optical distortion is often blamed incorrectly for the effects of perspective distortion which is far more noticeable.

Numerical Aperture and Light Cone Geometry - The light-gathering ability of a microscope objective is expressed in terms of the numerical aperture, which is a measure of the number of highly diffracted image-forming light rays captured by the objective. This interactive tutorial explores the effect of numerical aperture on light cone geometry.

Microscope Alignment for Köhler Illumination - Illumination of the specimen is the most important variable in achieving high-quality images in microscopy and critical photomicrography or digital imaging. This interactive tutorial explores how to establish Köhler illumination on a transmitted light microscope.

Field Iris Diaphragm Function - When the microscope is properly configured for Köhler illumination, the field diaphragm is imaged in the same conjugate plane as the specimen, and in fact, all of the image-forming conjugate planes are simultaneously imaged into each other and can collectively be observed while examining a specimen in the eyepieces.

Feb 20, 2022 — The direction of polarization is defined to be the direction parallel to the electric field of the EM wave. Unpolarized light is composed of ...

Infinity Corrected Microscope Conjugate Field Planes - A majority of modern research microscopes are equipped with infinity-corrected objectives that no longer project the intermediate image directly into the intermediate image plane. Light emerging from these objectives is instead focused to infinity, and a second lens, known as a tube lens, forms the image at its focal plane.

The Point Spread Function - The ideal point spread function (PSF) is the three-dimensional diffraction pattern of light emitted from an infinitely small point source in the specimen and transmitted to the image plane of a microscope (or other diffraction-limited optical instrument) through a high numerical aperture (NA) objective or lens system.

Fluorescence Microscopy - The application of fluorescence illumination and detection in optical microscopy has ushered in a wide range of advanced applications for live-cell imaging and in vivo observations. The articles tabulated in this section discuss the basic aspects of fluorescence, microscope configuration, fluorescent probes, software, light sources, detectors, objectives, filter sets, and a variety of other pertinent topics.

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Specimen Contrast - Control of image contrast in a microscope optical system is dependent upon several factors, primarily the setting of aperture diaphragms, degree of aberration in the optical system, the optical contrast system employed, the type of specimen, and the optical detector.

If you’re still not sure about what lens you should be using or how close to stand do the following. If you’re shooting a portrait use a lens somewhere 70-200mm and then stand somewhere where you can fit all of the horse/person in the frame. Humans for a close up portrait look closest to reality at around 70mm, for horses aim a little longer at 100mm. No matter your subject up to 200mm you can’t go far wrong and overall you may get overall a more aesthetically pleasing image.

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Condenser Numerical Aperture - The size and numerical aperture of the light cone emitted by a substage condenser is determined by adjustment of the aperture diaphragm. This interactive tutorial examines how changing the aperture iris diaphragm opening size alters the size and angle of the light cone.

Modern compound microscopes operate using a dual stage magnifying design that incorporates a primary imaging lens, the objective, coupled to a secondary visualizing lens system known as the eyepiece or ocular mounted at the opposite ends of a body tube. The objective is responsible for primary image formation at varying magnifications, while the eyepiece is used to observe the image created by the objective. Advanced microscopes feature infinity optical systems that project a parallel bundle of wavefronts from the objective rear aperture to a tube or telan lens, which in turn focuses the image at the intermediate image plane in the eyepieces. The microscopist is able to observe a greatly enlarged virtual image of the specimen by peering through the eyepieces. Magnification is determined by multiplying the individual values of the objective and eyepiece. Resolution and contrast in optical microscopy are derived through a number of optical strategies and is strongly coupled to the types of reagents used to prepare the specimen. This section discusses the basic concepts necessary for a complete understanding of microscopy, including objectives, eyepieces, condensers, magnification, numerical aperture, resolution, contrast, and optical aberrations, along with a wide spectrum of additional considerations.

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You may be wondering what focal length do we see in? The answer is the eye sees at 22mm. BUT given to lots of clever science in reality it is closest to the effects of a 50mm lens. I would though rarely shoot a portrait at 50mm as it is not the most flattering! For humans for a head only portrait I would shoot at at least 70mm. For horses 100mm, but I often shoot at 200mm as I like the subtle effects of compression. At 200mm you will get more background blur/bokeh than you will at 70mm. You must weigh up which elements are most important to your artistic vision. The right/below image was shot at 200mm but with the angle etc does not show notable compression distortion.

A low beam divergence is useful for applications like pointing or free-space optical communications. Beam Diameter: The width of the laser beam @ aperture ...

People get confused and think the lens is creating the distortion which is inaccurate. If you choose a position and then take several photos at different focal lengths in the same position, you will see the same level of distortion in each photo. The photo to the right/below shows an image taken at 24mm with a crop window applied. That same image cropped is then below next to a 42mm image slightly cropped and a 70mm uncropped image. As you can see they are essentially identical. There is no change in distortion, because my distance between the horse did not change between them.

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Microscopy in Everyday Use - Although conventional microscope design has not necessarily been a problem for short-term use, long-term sessions have in the past created problems for scientists and technicians who used the instruments. Ergonomics is concerned with finding a better fit between people microscopes.

Condenser Aperture Diaphragm Function - The size and numerical aperture of the light cone produced by the condenser is determined by adjustment of the aperture diaphragm. Appropriate use of the adjustable aperture iris diaphragm (incorporated into the condenser or just below it) is of significant importance in securing correct illumination, contrast, and depth of field.

The often misunderstood distortion is perspective. It is not caused by the camera or lens, but your position in relation to the subject. Often people call it lens or focal length distortion which is incorrect.

Focal length and distance to subject have an effect, but they directly cancel each other out when we keep the object being photographed the same size. Think of ...

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If you look at the above photo of the robin, the fence closest to the camera is big, furthest away it is small. All of it is exactly the same height but it looks smaller because it is further away. The closer you are to the object the more dramatic the distortion fall off will be from front to back.

Fixed Tube Length Microscope Conjugate Field Planes - The geometrical relationship between image planes in the traditional fixed tube length (usually 160 millimeters) optical microscope is explored in this tutorial. In most of the imaging steps in the microscope optical train, the image is real and inverted, but a virtual image is also produced in one of the image planes.

The suggested ideal focal length where optical distortion is minimal is somewhere around 200mm but honestly is minimal from 50mm up.

Inverted Microscope Lightpaths - Microscopes featuring an inverted-style frame are designed primarily for live-cell imaging applications and are capable of producing fluorescence illumination through an episcopic and optical pathway. This interactive tutorial explores illumination pathways in the Zeiss Axio Observer research-level inverted tissue culture microscope.

Take this image right (below) if you look carefully the horse is actually jumping the middle element of a combination, not the last. You can see poles from all three parts which are between 7-11m between one another. As this was shot at 400mm the gaps between them look smaller than they actually are. 99% of the time compression is not going to be an issue for you. It can make a horse/object look ‘stouter’ than it actually is. I would leave your 300mm+ lenses for sport images when you really need them. Aka when you can’t stand close enough to your subject to use a 70-200mm!

Phase Contrast Microscopy - Phase contrast was introduced in the 1930's for testing of telescope mirrors, and was adapted by the Carl Zeiss laboratories into a commercial microscope for the first time several years later. The technique provides an excellent method of improving contrast in unstained biological specimens.

Image below left is a straight out of camera 24mm image showing barrel distortion and associated vignette. Below right (or second image if viewing on a mobile) shows the corrected image using only Lightroom corrections. Even at 24mm, as there are no straight lines to highlight it the barrel distortion is minimal.

With all three types of optical distortion, if you were to imagine a grid of squares overlaid on the image you would see a different distortions pattern. In all though the centre vertical and horizontal lines will be straight.

The Concept of Magnification - A simple microscope or magnifying glass (lens) produces an image of the specimen upon which the microscope or magnifying glass is focused. This interactive tutorial explores how a simple magnifying lens operates to create a virtual image of the specimen on the retina of the human eye.

Have you noticed when taking a photo of your horse with your phone that their head is huge, and I mean HUGE? Yes you’re not going mad it will look disproportionally hug. This is due to something called perspective distortion. There are two main types of distortion that occur when taking a photo and they have both similar and different linked effects. The first is perspective distortion and the second optical. Optical is broken into the sub categories of barrel, pin and moustache distortion.

Infinity Optical System Basics - Infinity-corrected microscope optical systems are designed to enable the insertion of auxiliary optical devices into the optical pathway between the objective and eyepieces without introducing spherical aberration, requiring focus corrections, or creating other image problems.

Depth of field refers to the area of the image that is clearly in focus. This varies depending on various conditions such as the focal length of the lens, ...

Reflected Light Microscope Optical Pathways - Reflected light microscopy is often referred to as incident light, epi-illumination, or metallurgical microscopy, and is the method of choice for fluorescence and for imaging specimens that remain opaque even when ground to a thickness of 30 micrometers.

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If you don't have other micro-b devices around, you'll have to keep track of those cables with the enclosures. Type-c you'll likely have many ...

Spatial Frequency and Image Resolution - When a line grating is imaged in the microscope, a series of conoscopic images representing the condenser iris opening can be seen at the objective rear focal plane. This tutorial explores the relationship between the distance separating these iris opening images and the periodic spacing (spatial frequency) of lines in the grating.

Coverslip Thickness Correction - High magnification dry objectives are often subject to aberration artifacts due to variations in cover glass thickness and dispersion. This tutorial demonstrates how internal lens elements in such an objective may be adjusted to correct for these fluctuations.

If you don’t have a lens circa 70-200mm you will need to still stand in the same position then crop the resulting image out of camera. Do not stand closer to the object to compensate.

Numerical Aperture and Resolution - The numerical aperture of a microscope objective is the measure of its ability to gather light and to resolve fine specimen detail while working at a fixed object (or specimen) distance. Resolution is determined by the number of diffracted wavefront orders captured by the objective.

Differential Interference Contrast (DIC) Microscopy - Differential interference contrast converts gradients in specimen optical path length into amplitude differences that can be visualized as improved contrast in the resulting image. Images produced in DIC microscopy have a distinctive shadow-cast appearance

Katie Mortimore is a creative wedding & equestrian photographer based on the Wiltshire Hampshire border who travels across Britain for projects.

The modulation transfer function (MTF) curve is an information-dense metric that reflects how a lens reproduces contrast as a function of spatial frequency ( ...

Basic Principles in Optical Microscopy - A list of the best textbooks that provide a general knowledge of the principles and practice of optical microscopy, as well as an introduction to contrast-enhancing techniques. The volumes listed here are useful in both the classroom and the research laboratory.

Microscope Optical Systems - The microscope optical train typically consists of an illuminator (including the light source and collector lens), a condenser, specimen, objective, eyepiece, and detector, which is either some form of camera or the observer's eye. These components are often supplemented with contrast-enhancing optical elements.

The bigger the object the further back you will need to stand. For buildings you are likely to meet issues with vertical perspective distortion wherever you stand, unless miles away! On the most part unless a specialist architectural photographer it’s not a huge worry. If it is, you can use a tilt-shift lens which is designed to specifically counteract the effects of perspective distortion. Be warned though they are not cheap!

Fundamental Aspects of Airy Disk Patterns - This tutorial explores how Airy disk pattern size changes with objective numerical aperture and the wavelength of illumination. It also simulates the close approach of two Airy patterns as they approach the Rayleigh criterion for determining the ability to resolve two closely spaced objects in the microscope.

Enhancing Contrast in Optical Microscopy - The contrast-enhancing techniques for transmitted light microscopy described in this section represent a variety of methods in sample preparation as well as optical tricks that generate intensity changes which are useful for observation and imaging.

Microscope Ergonomics - Although conventional microscope design has not necessarily been a problem for short-term use, long-term sessions have in the past created problems for scientists and technicians who used the instruments. Microscope operators often must assume an unusual and challenging position.

Oil Immersion and Refractive Index - One way of increasing the optical resolving power of the microscope is to use immersion liquids between the front lens of the objective and the cover slip. This tutorial explores how changes in the refractive index of the imaging medium can affect how light rays are captured by the objective, which has an arbitrarily fixed angular aperture of 65 degrees.

Focus Depth and Spherical Aberration - Explore the three-dimensional aspects of spherical aberration that is generated when imaging deep into specimens using the meridional section of a point spread function with this interactive tutorial. Spherical aberration is a significant problem when imaging specimens in aqueous media.

Microscope Optical Systems - Microscope objectives are perhaps the most important components of an optical microscope because they are responsible for primary image formation and play a central role in determining the quality of images that the microscope is capable of producing. Other components include the illumination collector, condenser, and eyepieces.

Polarized Light Microscopy - The polarized light microscope is designed to observe specimens that are visible due to their birefringent character. Polarizing microscopes must be equipped with a polarizer, positioned in the light path before the specimen, and an analyzer placed in the optical pathway between the objective rear aperture and the observation tubes or camera port.

Practical Use of the Microscope - If certain simple guidelines are followed, it will be a short matter of time before a beginner is able to obtain an image of high quality. In fact, you may be surprised at how easy it is to set up the microscope correctly so that it will produce beautiful, sharp images.

Conoscopic Images of Periodic Structures - This tutorial explores the reciprocal relationship between line spacings in a periodic grid (simulating a specimen) and the separation of the conoscopic image at the objective aperture plane. When the line grating has broad periodic spacings, several images of the condenser iris aperture appear in the objective rear focal plane.

Microscope Conjugate Planes - The conjugate planes critical for establishing proper illumination in the microscope are examined in this interactive tutorial. Four conjugate planes can be brought simultaneously into focus: the field diaphragm, the specimen plane, the intermediate image plane (where the reticule is positioned), and the human eye.

If we step away from cameras entirely and think about what we see with our own eyes instead it can be easier to understand. If you stood next to a very long wall, say the great wall of china, the bit of the wall you are next to will look utterly enormous, but a bit of wall a mile away will look tiny. We all know the wall is still enormous a mile away but because of our perspective to it, it looks small.

Objective Specifications - Microscope objectives are precision optical systems that feature a wide range of magnifications, numerical aperture, immersion media, specialized contrast applications, and other properties. This interactive tutorial examines the specifications found on typical objectives.

But fear not optical distortion is normally very easy to correct. Most mainstream lenses will have a lens correction profile. They are available with popular editing programs to apply to an image with the click of a button. The metadata from your camera will tell the program the lens used so you only need to click to confirm application.

Reflected Light Microscopy - Reflected light microscopy is often referred to as incident light, epi-illumination, or metallurgical microscopy, and is the method of choice for fluorescence and for imaging specimens that remain opaque even when ground to a thickness of 30 micrometers using conventional contrast-enhancing techniques.

Once you understand how perspective distortion affects how you see an object, it allows you to select the appropriate lens for your subject. The simplest way is walk back until your subject looks in proportion and normal. For a horse I would want to be standing at least 10 metres back possibly a bit more. It will depend on if I’m shooting the whole horse or just it’s head. Now I am in position I think about what I want my image to look like in the frame. Do I want the horse to fill the frame? Do I want it with lots of surroundings? If I want it to fill the frame I am going to want to use something 70mm+. If I want to include scenery I’m going to rock a 24mm!

Optical Pathways in the Transmitted Light Microscope - The design of an optical microscope must ensure that the light rays are organized and precisely guided through the instrument. This interactive tutorial explores the function of the field and condenser aperture diaphragms of a transmitted light microscope.

Introduction to Microscopy - Microscopes are specialized optical instruments designed to produce magnified images of specimens that are too small to be seen with the naked eye. In addition to complex designs featuring objectives and condensers, microscopes also consists of very simple single-lens instruments that are often hand-held, such as a common magnifying glass.

Microscope Objectives - The most important component of an optical microscope is the microscope objective. Objectives are responsible for primary image formation and play a central role in establishing the quality of images that the microscope is capable of producing.

If you have to stand further away than the ‘ideal’ viewing point you will start seeing compression. This won’t be that noticeable unless using a much longer focal length. The compression effect basically makes two points seem closer together than they actually are. Again this is caused by your position in relation to the objects, not by the lens. Look at a mountain range from many many miles away and the mountain peaks will look like they are on top of each other. In reality they are potentially miles apart. All you do with a long zoom lens (300mm+) is make the effect more noticeable. The further away you are from something the closer two objects in front of one another will look, but it’s most obvious when you zoom in.

Contrast Modes in Reflected Light Microscopy - In its standard configuration, a typical reflected light microscope is readily equipped to examine amplitude (absorption) specimens using brightfield incident light. Through the addition of auxiliary components, a variety of contrast-enhancing mechanisms can be introduced.

Illumination and the Microscope Optical Train - The design of an optical microscope must ensure that the light rays are organized and precisely guided through the instrument. Illumination of the specimen is the most important controllable variable in achieving high-quality images in microscopy and digital imaging.

Barrel distortion is as it sounds, shaped like a barrel. Imagine the middle of each side being pulled out away from the centre. Pincushion is the opposite where the centre of each side is pushed in. Moustache is a combination of the two and the hardest to deal with as the distortion can be very irregular.

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But actually sometimes distortion is good and desirable so you don’t always have to correct it. The prime example of this is when using a fisheye lens. They are designed to push barrel distortion to the limits as a desired effect. You wouldn’t purposefully use a fisheye lens, then try and correct the distortion, it’s an artistic choice.

Filters are applied to your image from the Filters menu (destructive) or Layer menu (non-destructive) and mostly include customizable settings alongside general ...

Köhler Illumination - Illumination of the specimen is the most important variable in achieving high-quality images in microscopy and critical photomicrography. Köhler illumination was first introduced in 1893 by August Köhler of the Carl Zeiss corporation as a method of providing the optimum specimen illumination.

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Numerical Aperture and Image Resolution - The image formed by an objective at the intermediate image plane of a microscope is a diffraction pattern produced by spherical waves exiting the rear aperture and converging on the focal point. This tutorial explores the effects of objective numerical aperture on the size of Airy disk patterns.

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Fluorescence Microscopy - Because of the highly sensitive emission profiles, spatial resolution, and high specificity with regards to signal-to-noise and contrast, fluorescence microscopy is rapidly becoming an important tool in genetics and cell biology, and remains at the forefront of biomedical research with the continuous introduction of new techniques.

Understanding the Digital Image - This discussion is intended to aid in understanding the basics of light detection, the fundamental properties of digital images, and the criteria relevant to selecting a suitable detector for specific applications.

Condenser Light Cones - It is critical that the condenser light cone be properly adjusted to optimize the intensity and angle of light entering the objective front lens. Each time the objective is changed, a corresponding adjustment must be performed on the condenser to provide the proper light cone to match the numerical aperture of the new objective.

I have a range of articles covering both the basics and more in depth areas of photography (see below!). Plus I also offer 1-2-1 mentoring and training.

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Microscopy: Historical Perspective - For many centuries, the construction of microscopes and the underpinning optical systems was entirely an issue of exterior cosmetic craftsmanship, with the design of optical components lagging seriously behind advances in the fabrication of microscope bodies and frames.

Airy Disk Formation - When an image is formed in the focused image plane of an optical microscope, every point in the specimen is represented by an Airy diffraction pattern having a finite spread. This interactive tutorial explores the origin of Airy diffraction patterns formed by the rear aperture of the microscope objective and observed at the intermediate image plane.

How the Microscope Forms Images - Optical microscopes belong to a class of instruments that are said to be diffraction limited, meaning that resolution is determined in part by the number of diffraction orders created by the specimen that can be successfully captured by the objective and imaged by the optical system.

Care and Maintenance of the Microscope - Microscopes often represent a significant investment of funds and are sophisticated optical instruments that require periodic maintenance and cleaning to guarantee successful microscopy and perfect images.