The imaging medium between the objective front lens and the specimen cover slip is another important element in respect to correction for spherical aberration and coma in the design of lens elements for objectives. Lower power objectives are designed to be used with only air as the imaging medium between the objective front lens and the coverslip. The maximum theoretical numerical aperture obtainable with air is 1.0, however in practice it is virtually impossible to produce a dry objective with a numerical aperture above 0.95. The effect of coverslip thickness variation is negligible for dry objectives having numerical apertures less than 0.4, but such deviation becomes significant at numerical apertures exceeding 0.65, where fluctuations as small as 0.01 millimeter can introduce spherical aberration.

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Older objectives typically have lower numerical apertures, and are subject to chromatic difference of magnification, an aberration that requires correction by the use of specially designed compensating oculars or eyepieces. This type of correction was prevalent during the popularity of fixed tube length microscopes, but is not necessary with modern infinity-corrected objectives and microscopes. Recently, correction for chromatic difference of magnification is either built into the modern microscope objectives themselves (Olympus and Nikon), or corrected in the tube lens (Leica and Zeiss). The intermediate image in an infinity-corrected system appears behind the tube lens in the optical pathway at the reference focal length. The tube lens focal length varies between 160 and 250 millimeters, depending upon design constraints imposed by the manufacturer. By dividing the reference focal length by the focal length of the objective lens, the magnification of an infinity-corrected objective can be calculated.

It is possible to correct for variations in coverslip thickness. Several high-performance apochromat dry objectives are fitted with correction collars that allow adjustment by a rotating collar, which causes two of the lens element groups in the objective to move closer together or farther apart (see Figure 4). Various specialized phase contrast objectives that are designed for tissue culture observation with an inverted microscope have an even broader compensation range of between 0 to 2 millimeters. In this way, specimens can be viewed through the bottom of most culture vessels, which in this size range, often have dramatic thickness fluctuations.

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When rays of light are focused precisely on the camera sensor, they form a point (top). However, when the rays come from an area of the shot that is not precisely in focus, such as an object in the foreground or the background, they may converge to a point in front of the sensor or behind it (middle and bottom). The result is that a circle of light (shown in red), instead of a point, is formed on the sensor. If this circle is small enough, it is still perceived as a point. The largest circle that is still perceived as a point is known as the circle of confusion.

More extensive depth of field – comparatively speaking, much more of the image looks sharp, although still only a narrow area is perfectly in focus. Taken on a Canon EOS 80D with a Canon EF 24-70mm f/4L IS USM lens at 67mm, 0.5 sec, f/11 and ISO6400.

While using a small aperture delivers extensive depth of field, it's important to bear in mind that this also makes the impact of diffraction (the bending of light as it passes over the edge aperture blades) more evident. You can see this for yourself if you scrutinise a series of images shot from exactly the same position with the aperture being adjusted from its widest to its narrowest setting. Although closing down from the widest aperture may initially result in sharper images, when you examine the images shot at the smallest apertures, you'll see that they are not quite as sharp – even at the focus point. That's because the bent light can't be focused to a small point. Canon's Diffraction Correction feature can mitigate the worst effects of diffraction to produce sharper images at small aperture settings. It is available in-camera in some cameras when you're shooting JPEGs or HEIFs and can be applied using Canon's Digital Photo Professional (DPP) software post shoot when you're shooting RAW. Diffraction Correction is also part of Canon's Digital Lens Optimizer (DLO) technology in cameras that have this and in DPP.

There are three vital design characteristics of the objective that set the ultimate resolution limit of the microscope: The wavelength of light used to illuminate the specimen, the angular aperture of the light cone captured by the objective, and the refractive index in the object space between the objective front lens and the specimen. Resolution for a diffraction-limited optical microscope can be described as the minimum visible distance between two closely spaced specimen points:

Shallowdepth of field

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Your aperture setting has a profound effect on depth of field. At f/1.8, only our subject is sharp, with the background pleasingly blurred. Taken on a Canon EOS 1300D with Canon EF 50mm f/1.8 STM lens at 50mm, 1/60 sec, f/1.8 and ISO250.

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As you can see, defining depth of field is a rather arbitrary affair. So how can you hope to control the results produced by your camera? Here is a range of options. The rough guide If you want an extensive depth of field, set a small lens aperture (higher f-number), such as f/16 or f/22. Using a small aperture may require a slow shutter speed for correct exposure, so use a tripod to reduce the effects of camera shake. Also, use a wide-angle lens for maximum effect. If you want shallow depth of field, set a wide aperture (lower f-number), such as f/2.8 or f/4, and use a telephoto lens for maximum effect. If depth of field is not a critical factor in your composition, use an aperture of around f/5.6, f/8 or f/11. Your lens will usually give optimum performance at these settings. Basic modes You might think that using one of the Basic mode settings available on EOS cameras would save you time and trouble. You might assume that the Landscape mode will give wide depth of field, while the Portrait mode will give an out-of-focus background. Unfortunately not. The Basic shooting modes are designed to give foolproof settings for beginners, avoiding the extremes of apertures or shutter speeds which give true creative control. The best advice for controlling depth of field while keeping things relatively simple is to shoot in Aperture priority (Av) mode. Depth of Field preview and Focus Peaking On a DSLR, the image you see in the viewfinder is normally the view at the largest aperture available on the lens you're using, meaning you can't visually assess the depth of field before taking a shot. However, if your camera has a Depth of Field Preview button then pressing this will stop down to the lens's current aperture setting, so you can see how much of the scene is in focus through the viewfinder and even more clearly on the Live View image on the LCD screen. If your camera doesn't have a dedicated Depth of Field Preview button, you can assign this function to the camera's SET button with a custom function while using P, Tv, Av or M mode. On the EOS 90D in Live View and on mirrorless cameras including the EOS R5, EOS R6, EOS R, EOS RP, EOS M6 Mark II and EOS M50 Mark II, you can also enable manual focus peaking (MF peaking), a visual aid to show which parts of the image are in sharpest focus. In theory, areas in focus will coincide with the greatest contrast, so the image is evaluated for contrast and these areas are highlighted on the display in a bright colour of your choice. You can see the highlighted areas of the scene change as you change the focus. Hyperfocal distance focusing Depth of field extends in front of the point of focus and behind it. In fact, apart from when the subject is very close, it extends roughly twice as far behind the focus point as it does in front. This means that if you focus at infinity or on the horizon you'll actually "waste" some depth of field and not get the widest sharp zone possible in your image. Hyperfocal distance focusing is a technique that enables you to capture the maximum depth of field possible in a photograph. The aim is to focus so that the far limit of depth of field just reaches infinity (or the furthest point in the scene). The point on which you need to focus to achieve this is known as the hyperfocal distance. The hyperfocal distance is the near limit of depth of field when you are focused on infinity. And when you focus on the hyperfocal distance, the depth of field extends from roughly half the hyperfocal distance to infinity. There are depth of field tables widely available on the internet that tell you where the hyperfocal distance is for any given lens and camera combination, but hyperfocal distance is not a fixed value for a lens – it changes with the aperture and the focal length – so the easiest way to work it out is to use the depth of field and hyperfocal distance calculator in Canon's free Photo Companion app. You'll find this under Skills - Calculators. Then set your camera lens to manual focusing (there is an AF/MF switch on the side of most Canon lenses) and turn the focusing ring to this distance. If you don't have time for calculations, a rough rule of thumb is to focus approximately one third of the way into a scene.

In situations where the specimen is designed to be imaged without a coverslip, the working distance is measured at the actual surface of the specimen. Working distance typically decreases in a series of matched objectives as the magnification and numerical aperture increase. Objectives intended to view specimens with air as the imaging medium should have comparatively long working distances providing that numerical aperture requirements are satisfied. Alternatively, immersion objectives should have shallower working distances in order to keep the immersion liquid between the front lens and the specimen in place. Many objectives designed with similar working distances have a spring-loaded retraction stopper that allows the front lens assembly to be withdrawn by pushing it into the objective body and twisting to secure its place. Twisting the retraction stopper in the opposite direction releases the lens assembly for use. In some applications (see below), a long free working distance is indispensable, and special objectives are designed for such use despite how difficult it is to achieve large numerical apertures and the necessary degree of optical correction.

F number depth of fieldphotography

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All three types of objectives suffer from pronounced field curvature, thus they project curved images rather than flat ones. Such artifact increases in severity with higher magnification. To overcome this inherent condition, optical designers have produced flat-field corrected objectives, which yield images that are in common focus throughout the viewfield. Objectives that have flat-field correction and low distortion are called plan achromats, plan fluorites, or plan apochromats, depending upon their degree of residual aberration. This correction, although expensive, is extremely valuable in digital imaging and conventional photomicrography.

In many biological and petrographic applications, when mounting the specimen, a glass coverslip is used to both protect the integrity of the specimen and to provide a clear window for observation. The coverslip acts to converge the light cones originating from each point in the specimen. But it also introduces chromatic and spherical aberration that must be corrected by the objective. The refractive index, dispersion, and thickness of the coverslip determine the degree to which light rays are converged. An additional concern is the aqueous solvent or excess mounting medium that lies between the specimen and coverslip in wet or thickly mounted preparations, which add to the variations in refractive index and thickness of the cover slip.

Depth offocus

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When the objective is assembled, spherical aberration is corrected by selecting the best set of spacers to fit between the hemispherical and meniscus lens (the lower lens mounts). The objective is parfocalized by translating the entire lens cluster upward or downward within the sleeve with locking nuts so that focus will not be lost while objectives housed on a multiple nosepiece are interchanged. Adjustment for coma is accomplished with three centering screws that optimize the position of internal lens groups with respect to the optical axis of the objective.

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The common design of a practical oil immersion objective includes a hemispherical front lens element, followed by a positive meniscus lens and a doublet lens group. Aplanatic refractions occur at the first two lens elements in a typical apochromatic oil immersion objective. Oil immersion objective lenses can also correct for chromatic defects that are introduced by the first two lens elements, while initiating a minimum amount of spherical aberration. Employing an oil immersion objective without oil between the cover slip and first lens element will result in defective images due to refraction that cannot be corrected by subsequent lens components within the objective.

The most common objectives used on laboratory microscopes are the achromatic objectives. Such objectives are corrected for axial chromatic aberration in blue and red wavelengths, which are about 486 and 656 nanometers, respectively. Both are brought into a single common focal point. Achromatic objectives are also corrected for spherical aberration in the color green (546 nanometers; see Table 1). Achromatic objectives' limited correction can result in images with a magenta halo if focus is chosen in the green region of the spectrum. The lack of correction for flatness of field (or field curvature) presents a further problem. Plan achromats provide flat-field corrections for achromat objectives (Figure 2). An even higher level of correction and cost is found in objectives called fluorites or semi-apochromats (illustrated by center objective in Figure 2), named for the mineral fluorite, which was originally used in their construction.

An example of shallow depth of field – only a narrow area is in sharp focus, with the rest of the image rapidly blurring away. Taken on a Canon EOS 80D with a Canon EF 24-70mm f/4L IS USM lens at 67mm, 1/12 sec, f/4 and ISO6400.

The third type of objective, the apochromatic objective, possesses the highest level of correction (Figure 2). Lower power apochromat objectives (5x, 10x, and 20x) have a longer working distance than higher power (40x and 100x) apochromat objectives. Apochromats almost eliminate chromatic aberration, are usually corrected chromatically for three colors (red, green, and blue), and are corrected spherically for either two or three wavelengths (see Table 1). Apochromatic objectives are the best choice for color photomicrography in white light. Because of their high level of correction, apochromat objectives usually have, for a given magnification, higher numerical apertures than do achromats or fluorites. Many of the newer high-performance fluorite and apochromat objectives are corrected for four (dark blue, blue, green, and red) or more colors chromatically and four colors spherically.

One of the most significant improvements in objective design during recent years is the enhancement of antireflection coating technology, which aides in reducing unnecessary reflections that occur as light passes through the lens system. Each uncoated air-glass interface is capable of reflecting between four and five percent of an incident light beam normal to the surface, resulting in a transmission value of 95-96 percent at normal incidence. If a quarter-wavelength thick antireflection coating with the appropriate refractive index is applied, it can increase this value by three to four percent. Multilayer coatings, which produce transmission values exceeding 99.9 percent in the visible spectral range, have replaced the single-layer lens coatings once used to reduce glare and improve transmission.

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For several years, most manufacturers conformed to an international standard of parfocal distance when designing objective lenses for biological applications. As a result, a majority of objectives had a parfocal distance of 45.0 millimeters and were considered interchangeable. As it became commonplace to produce infinity-corrected tube lengths, a new set of design criteria was created to correct for aberrations in the objective and tube lenses. Alongside a demand for greater flexibility to accommodate the requirement of expanding working distances with higher numerical apertures and field sizes, interchangeability between objective lenses from different manufacturers is now more limited.

Microscope manufacturers produce objectives with restricted tolerances to refractive index and dispersion. This means they require matching values in the liquid placed between the coverslip and objective front lens. It is advisable to employ only the oil intended by the objective manufacturer, and to not mix immersion oils between manufacturers. Additionally, objectives that use water and/or glycerin as an imaging medium are also available for applications with living cells in culture or sections of tissue immersed in physiological saline solution.

where Resolution is the minimum separation distance between two point objects that are clearly resolved, λ is the illumination wavelength, n is the imaging medium refractive index, and θ is equal to one-half of the objective angular aperture. With this in mind, it is apparent that resolution is directly proportional to the illumination wavelength. The human eye responds to the wavelength region between 400 and 700 nanometers, which represents the visible light spectrum that is utilized for a majority of microscope observations. Resolution is also dependent upon the refractive index of the imaging medium and the objective angular aperture. Objectives are intended to image specimens either through air or a medium of higher refractive index between the front lens and the specimen. The field of view is often highly restricted, and the front lens element of the objective is placed close to the specimen with which it must lie in optical contact. A gain in resolution by a factor of about 1.5 is attained when immersion oil is substituted for air as the imaging medium.

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Getting very close to your subject results in a very shallow depth of field (left). To get more of a small subject in focus (right), macro photographers might shoot from further away or sometimes use techniques such as focus stacking to combine multiple images with different parts of the subject in focus. These component images might be captured using focus bracketing on cameras that offer this feature, including EOS R5, EOS R6, EOS RP, EOS 90D, EOS M6 Mark II, PowerShot G5 X Mark II and PowerShot G7 X Mark III. With this feature, the camera takes a sequence of shots, automatically changing the focus point by very small increments each time so that different areas are in focus. Whether you use this feature or take a set of shots manually, you can then use the Depth Compositing function in Canon's Digital Photo Professional (DPP) software to combine the component images into a single image in which more of the scene is sharp.

Depth of fieldphotography examples

By the time we reach an aperture setting of f/16, the background is almost as distinct as the subject. The depth of field extends from the foreground all the way through to the background. Taken on a Canon EOS 1300D with Canon EF 50mm f/1.8 STM lens at 50mm, 1/12 sec, f/16 and ISO3200.

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A camera lens can focus precisely on only one plane (shown here in red). This is the only area of the scene that is really sharp. However, a wider area of the scene – some nearer the lens and some further from it – may appear to be sharp. The extent of this area of apparent sharpness, shown here in blue, is called the depth of field.

A wide lens aperture produces a large circle of confusion (shown in red) from an out-of-focus area of the subject (top). A smaller lens aperture produces a smaller circle of confusion from the same area (below).

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At an aperture setting of f/4, our subject still stands out from the background but more background detail is becoming discernible. Taken on a Canon EOS 1300D with Canon EF 50mm f/1.8 STM lens at 50mm, 1/85 sec, f/4 and ISO1250.

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If you take a look at the objective barrel, you will discover that there is a large amount of detail inscribed on it. Each objective is inscribed with the magnification; the tube length for which the objective was designed to give its finest images; and the thickness of coverslip protecting the specimen, which the designer assumed to have a constant value, correcting for spherical aberration. The objective will be engraved OIL or OEL or HI if the objective is designed to function with immersion oil. If not, the objective is meant to be used dry. Objectives are also always engraved with their numerical aperture value. If the objective does not indicate a higher correction, it is most likely an achromatic objective (more highly corrected objectives have inscriptions such as apochromat or apo, plan, FL, fluor, etc).

Depth of fieldcalculator

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The rear aperture or exit pupil of the objective restricts the light rays as they pass through an objective. The diameter of this aperture varies between 12 millimeters for low magnification objectives down to around 5 millimeters for the highest power apochromatic objectives. Close consideration of aperture size is absolutely imperative for epi-illumination applications that rely on the objective to act as both an imaging system and condenser, where the exit pupil also becomes an entrance pupil. The image of the light source must entirely fill the objective rear aperture to produce even illumination across the viewfield. If the light source image is smaller than the aperture, the viewfield will experience vignetting from uneven illumination. Conversely, if the light source image is larger than the rear aperture, all of the light will not enter the objective and the intensity of illumination is reduced.

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Fluorite objectives are fashioned from advanced glass formulations that contain materials such as fluorspar or newer synthetic substitutes that allow for greatly improved correction of optical aberration. Similar to the achromats, the fluorite objectives are also corrected chromatically for red and blue light, however, the fluorites are also spherically corrected for two or three colors instead of a single color, as are achromats. Compared to achromats, fluorite objectives are made with a higher numerical aperture, which results in brighter images. Fluorite objectives also have better resolving power than achromats and provide a higher degree of contrast, making them better suited for color photomicrography in white light.

The most important imaging component in the optical microscope is the objective, a complex multi-lens assembly that focuses light waves originating from the specimen and forms an intermediate image that is subsequently magnified by the eyepieces. 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. Furthermore, the magnification of a particular specimen and the resolution under which fine specimen detail also heavily depends on microscope objectives. The most difficult component of an optical microscope to design and assemble, the objective is the first element that light encounters as it passes from the specimen to the image plane. Objectives received name from the fact that they are, by proximity, the closest component to the object, or specimen, being imaged.

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F number depth of fieldnikon

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There are a few factors that govern depth of field or our perception of it: Aperture The lens aperture is the easiest way to control depth of field. The rule is simple: the smaller the aperture (that is, the bigger the f-number), the greater the depth of field. For example, f/16 will give you a more extensive depth of field than f/4. That's because a smaller aperture enables a narrower beam of light from any given point on the subject to reach the sensor. This means that, other things being equal, the circle of light from an area beyond the point of focus will be smaller, making that part of the image look sharper than at a wide aperture. As a very general rule, use apertures between about f/2.8 and f/8 for portraits where you want the background to be out of focus. Use an aperture between about f/11 and f/22 for landscapes where you want everything from the foreground to the far distance to appear sharp. Subject distance The greater the distance between the lens and the subject, the greater the depth of field is. This is because the further you are from a subject, the more perpendicular to the sensor (or less divergent) the light is as it enters the lens. This means that out-of-focus areas form a smaller circle on the sensor than when the lens is focused on a closer subject. A closer subject reflects more divergent light into the lens, which, after passing through the lens elements, forms a relatively large circle on the sensor. Anyone who has tried close-up photography will have seen how getting very close to a subject results in very shallow depth of field. At life-size magnification, little more than the subject in the plane of focus will appear sharp, and the point you focus on is critical to the success of the photograph.

For many years, field curvature went uncorrected as the most severe optical aberration that occurred in fluorite (semi-apochromat) and apochromat objectives, tolerated as an unavoidable artifact. The introduction of flat-field (plan) correction to objectives perfected their use for photomicrography and video microscopy, and today these corrections are standard in both general use and high-performance objectives. Figure 3 illustrates how correction for field curvature (for a simple achromat) adds a considerable number of lens elements to the objective. The significant increase in lens elements for plan correction also occurs with fluorite and apochromat objectives, frequently resulting in an extremely tight fit of lens elements (see Figure 1) within the internal objective sleeve.

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A majority of the microscope objectives being produced today offer extraordinarily low degrees of aberration and other imperfections, assuming the appropriate objective is selected and utilized properly. Even still, the microscopist must be conscious of the fact that objectives are not perfectly crafted from every standpoint, but are designed to meet a certain set of qualifications depending on intended use, constraints on physical dimensions, and price ranges. Consequently, objectives are made with degrees of correction that differ for chromatic and spherical aberration, field size and flatness, transmission wavelengths, freedom from fluorescence, birefringence, and additional factors contributing to background noise. Additionally, they are intended to be used under certain limited conditions, such as with particular tube lengths and tube lenses, type and thickness of immersion media and coverslips, wavelength ranges, field sizes, ocular types, and special condensers.

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A number 1½ coverslip is standard, with a thickness of 0.17 millimeters. Unfortunately, not all 1½ coverslips are manufactured to this standard (they range from 0.16 to 0.19 millimeters), and many specimens have media between them and the coverslip. By adjusting the mechanical tube length of the microscope, or by the utilization of specialized correction collars, compensation for coverslip thickness can be provided. Objective numerical aperture can be radically increased if the objective is used with an immersion medium such as oil, glycerin, or water. Typical immersion oils have a refractive index of 1.51 and a dispersion profile similar to that of glass cover slips. An immersion medium with a refractive index similar to that of the glass cover slip will practically eliminate image degradation due to thickness variations of the coverslip whereby rays of wide obliquity no longer undergo refraction and are more readily grasped by the objective. Light rays passing through the specimen encounter a homogeneous medium between the cover slip and immersion oil and are not refracted as they enter the lens, but only as they leave its upper surface. Therefore, if the specimen is placed at the aplanatic point of the first objective lens, imaging this portion of the lens system is totally free of spherical aberration.

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F number depth of fieldcanon

is known as the numerical aperture (NA), and provides an important indicator of the resolution for any particular objective. Other than magnification, numerical aperture is generally the most important design criteria when considering which microscope objective to choose. Values range from 0.025 for very low magnification objectives (1x to 4x) to as much as 1.6 for high-performance objectives that employ specialized immersion oils. As numerical aperture values increase for a series of objectives of the same magnification, a greater light-gathering ability and increase in resolution occurs. Under the best circumstances, detail that is just resolved should be enlarged sufficiently to be viewed with comfort, but not to the point that empty magnification obstructs observation of fine specimen detail. The microscopist should carefully choose the numerical aperture of an objective to match the magnification produced in the final image. Magnifications higher than this value will yield no additional useful information (or finer resolution of image detail), and will lead to image degradation. Exceeding the limit of useful magnification causes the image to suffer from empty magnification, where increasing magnification will simply cause the image to become more magnified with no corresponding increase in resolution.

Finally, the last but perhaps most important factor in determining the resolution of an objective is the angular aperture, which has a practical upper limit of about 72 degrees (with a sine value of 0.95). When combined with refractive index, the product:

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Depth of field exists because our eyes can't resolve the difference between a point and a very small circle of light. When a lens focuses, each point of the subject in the plane of focus is projected as a point onto the camera's sensor. All these points create a sharp image of the subject. If the subject were flat, like a cardboard cut-out of a person perfectly perpendicular to the lens, then all of it would genuinely be in focus. However, parts of the scene that are not in the plane of focus do not form image points on the sensor. The rays of light from these points focus to a point in front of the sensor or behind it, which means that they form a circle when they hit the sensor. It's just like focusing the sun's rays on a piece of paper using a magnifying glass – at the right distance, the cone of light focuses to a point, but otherwise you get a larger or smaller circle of light, as if you had sliced across the cone. If the circle on the sensor is so small that it still appears as a point to our eyes, then that part of the subject will still appear sharp in the image. If our eyes see it as a circle, then that part of the subject will appear unsharp. The largest circle that is still perceived to be a point is called the circle of confusion, and it's a key factor in defining depth of field.

For many landscape images, the ideal is sharpness from foreground to horizon. For maximum depth of field, photographers might use a relatively wide-angle setting plus a relatively small aperture (high f-number), but other factors come into play – including the optical characteristics of the lens – and this shot taken at f/10 looks sharp from the foreground trees to the distant shoreline in the background. Taken on a Canon EOS RP with a Canon RF 24-240mm F4-6.3 IS USM lens at 83mm, 1/500 sec, f/10 and ISO400.

Focal length Most photographers usually choose the focal length of the lens to suit the subject or the shooting conditions rather than for the depth of field. However, the accepted rule is that you get greater depth of field with wide-angle lenses than with telephoto lenses. In fact, this rule is misleading. What actually happens is that a wide-angle lens magnifies the subject less than a telephoto lens, which means that more of the image appears sharper. A simple test is to take two photographs of the same subject from the same position, one with a wide-angle and one with a telephoto focal length lens. Then enlarge the centre of the wide-angle image to match the view of the telephoto image. You'll find that the depth of field will be identical. However, depth of field is all about acceptable sharpness, and a wide-angle shot will give the appearance of greater sharpness across a scene. Alternatively, try creating the same composition and framing using a wide-angle and a telephoto lens. With the wide-angle lens, you have to move much closer to the subject to get the same framing as with the telephoto lens, and as a consequence, the depth of field is very similar at the same aperture. As a very general rule, wide-angle lenses are good for landscapes where you want sharpness from front to back. A medium telephoto lens (around 100mm or 135mm) is good for portraits if you want an out-of-focus background.

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Just as the brightness of illumination in a microscope is directed by the square of the working numerical aperture of the condenser, the brightness of an image produced by the objective is determined by the square of its numerical aperture. Additionally, objective magnification also plays a role in determining image brightness, which is inversely proportional to the square of the lateral magnification. The square of the numerical aperture/magnification ratio expresses the light-gathering power of the objective when used with transmitted illumination. High numerical aperture objectives collect more light and produce a brighter, more corrected image that is highly resolved because they also are often better corrected for aberration. In cases where the light level is a limiting factor (image brightness decreases rapidly as the magnification increases), choose an objective with the highest numerical aperture with the lowest magnification factor capable of producing sufficient resolution.

The distance from the lens center to a point where parallel rays are focused on the optical axis is defined as the focal length of a lens system. An imaginary plane perpendicular to the principal focal point is called the focal plane of the lens system. There are two principal focal points, one in front and one at the rear, for light entering each side of every lens. Conventionally, the objective focal plane found nearer to the front lens element is known as the front focal plane and the focal plane located behind the objective is known as the rear focal plane. The specific position of the rear focal plane varies with construction of the objective, but is usually situated somewhere inside the objective barrel for high magnification objectives. Lower magnification objectives often have a rear focal plane that is located on the exterior, in the thread area or within the microscope nosepiece.

So what is the diameter of this circle? Well, that's where some of the confusion begins, because there are several factors to take into consideration. For example, how good is your eyesight? And what distance are you viewing from? With perfect vision, under ideal lighting and at a normal reading distance, a circle of confusion might be as small as 0.06mm. But these conditions are far too strict for the real world, and a figure of around 0.17mm is often used in photography as the largest circle that most viewers would still perceive as a point. However, there is another factor to consider. You may have noticed that when you look at a thumbnail of a digital image, or look at it on the screen on the back of the camera, it appears sharp, but when you open it on your computer monitor it doesn't look as sharp as you thought. The issue here is one of viewing size. The actual image is the size of the sensor – 36x24mm in the case of a full-frame sensor, the same size as a 35mm film negative – but this is rarely viewed at its original size. Traditionally it would be enlarged to make a 5x7-inch print. This is a 5x enlargement of the original image, so the 0.17mm circle of confusion is enlarged to around 0.85mm – easily visible as a circle to most people. So if we want a circle that still looks like a point at this conventional viewing size, what we need on the sensor is a circle that gives a size of 0.17mm after being enlarged five times. A quick tap on a calculator shows this size to be about 0.034mm. A circle of confusion is based on perception – it's not something that can be calculated precisely. This is why different depth-of-field charts and tables often give different results – they are based on different circle of confusion values. Canon uses a value of 0.035mm in depth-of-field calculations for its full-frame cameras. On EOS cameras with the smaller APS-C format sensor, the image must be enlarged more to produce a 7x5 inch print, which means a smaller circle of confusion is needed on the sensor. Canon uses 0.019mm in its calculations.

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In the past 100 years, construction techniques and materials used to manufacture objectives have greatly improved. Composed up of numerous internal glass lens elements, modern objectives have reached a high state of quality and performance considering the extent of correction for aberrations and flatness of field. Objectives are currently designed with the assistance of Computer-Aided-Design (CAD) systems, which use advanced rare-element glass formulations of uniform composition and quality characterized by highly specific refractive indices. These advanced techniques have allowed manufacturers to produce objectives that are very low in dispersion and corrected for most of the common optical artifacts such as coma, astigmatism, geometrical distortion, field curvature, spherical and chromatic aberration. Not only are microscope objectives now corrected for more aberrations over wider fields, but image flare has been dramatically reduced thanks to modern coating technologies, with a substantial increase in light transmission, yielding images that are remarkably bright, sharp, and crisp.

A dramatic improvement in contrast and transmission of visible wavelengths is the result of most microscope manufacturers currently producing their own proprietary formulations, along with a simultaneous destructive interference in harmonically-related frequencies lying outside the transmission band. The microscopist should be aware of the fact that these specialized coatings can be easily damaged by mis-handling. A good rule to employ in order to distinguish between coatings is that multilayer antireflection coatings have a slightly greenish tint, as opposed to the purplish tint of single-layer coatings. Also, the surface layer of antireflection coatings used on internal lenses is often much softer than corresponding coatings. Special care should be taken when cleaning optical surfaces that have been coated with thin films, especially if the microscope has been disassembled and the internal lens elements are subject to inspection.

Confocal microscopy consists of a reciprocal focusing process of excitation and emission light, which in principle allows for greater resolution than a single ...

At f/8, much more detail is visible in the background and our subject does not stand out to anything near the same degree. Taken on a Canon EOS 1300D with Canon EF 50mm f/1.8 STM lens at 50mm, 1/50 sec, f/8 and ISO3200.

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Major microscope manufacturers offer a wide range of objective designs that feature excellent optical characteristics under a wide spectrum of illumination conditions and provide various degrees of correction for the primary optical aberrations. The objective illustrated in Figure 1 is a 20x multi-immersion media plan-apochromat, which contains 9 optical elements that are cemented together into two groups of lens doublets, a movable lens triplet group, and two individual internal single-element lenses. The objective also has a hemispherical front lens and a meniscus second lens, which work synchronously to assist in capturing light rays at high numerical aperture with a minimum of spherical aberration. Many high magnification objectives are equipped with a spring-loaded retractable nosecone assembly that protects the front lens elements and the specimen from collision damage. Internal lens elements are carefully oriented and tightly packed into a tubular brass housing that is encapsulated by the decorative objective barrel. Specific objective parameters such as numerical aperture, magnification, optical tube length, degree of aberration correction, and other important characteristics are imprinted or engraved on the external portion of the barrel. The objective featured in Figure 1 is designed to operate utilizing water, glycerin, or a specialized hydrocarbon-based oil as the imaging medium.

2024125 — Photographer Kyle van Bavel packs two Sony Alpha cameras and two Sony lenses in his kit to capture the littlest details in our big world.

When any lens is focused on a point, there's an area in front of that point (closer to the camera) and behind it (further from the camera) that looks sharp. The extent of this area of apparent sharpness is known as the depth of field (DOF), and it can be made shallower or deeper to creative effect. In fact, depth of field is one of the most important creative tools for photographers, because it enables you to control where in the image is sharp and where is blurred. In a portrait, for example, you may want to restrict the depth of field so that just the subject's face is sharp while the cluttered, distracting background beyond is blurred. Conversely, landscape photographers often want extensive depth of field so that everything from the foreground to the background looks sharp.