Firstly let’s talk through some of the terminology so we understand all of the vocabulary related to depth of field. Some of them sound similar, but have very different meanings, we want to make sure we’re all on the same page.

When you are shooting, depth of field can play a large factor in how your image will be rendered and can change the meaning and intention of the image. You can choose to selectively isolate a subject from its background by having a narrow depth of field or alternatively you can make sure that everything from the foreground to infinity will be in focus, ensuring you have a sharp image throughout your image.

Shallow depth of field photography

When you have a larger aperture (smaller number) you will have a narrower depth of field. The blurry parts that are not in focus are called Bokeh, and many people are quite a fan of the way it will render light.

On the PhotoPills website is a really handy Depth of Field Calculator that will specify hyperfocal distances, as well as all other depth of field calculations.

Figure 3(a) illustrates a hypothetical Airy disk that essentially consists of a diffraction pattern containing a central maximum (typically termed a zeroth order maximum) surrounded by concentric 1st, 2nd, 3rd, etc., order maxima of sequentially decreasing brightness that make up the intensity distribution. Two Airy disks and their intensity distributions at the limit of optical resolution are illustrated in Figure 3(b). In this part of the figure, the separation between the two disks exceeds their radii, and they are resolvable. The limit at which two Airy disks can be resolved into separate entities is often called the Rayleigh criterion. Figure 3(c) shows two Airy disks and their intensity distributions in a situation where the center-to-center distance between the zeroth order maxima is less than the width of these maxima, and the two disks are not individually resolvable by the Rayleigh criterion.

Careful positioning of the substage condenser aperture diaphragm is also critical to the control of numerical aperture and indiscriminate use of this diaphragm can lead to image degradation (as discussed in the section on substage condensers). Other factors, such as contrast and the efficiency of illumination, are also key elements that affect image resolution.

Figure 4 illustrates the effect of numerical aperture on the size of Airy disks imaged with a series of hypothetical objectives of the same focal length, but differing numerical apertures. With small numerical apertures, the Airy disk size is large, as shown in Figure 4(a). As the numerical aperture and light cone angle of an objective increases however, the size of the Airy disk decreases as illustrated in Figure 4(b) and Figure 4(c). The resulting image at the eyepiece diaphragm level is actually a mosaic of Airy disks which we perceive as light and dark. Where two disks are too close together so that their central spots overlap considerably, the two details represented by these overlapping disks are not resolved or separated and thus appear as one, as illustrated above in Figure 3.

The angle m is one-half the angular aperture (A) and is related to the numerical aperture through the following equation:

Where R is resolution (the smallest resolvable distance between two objects), NA equals numerical aperture, equals wavelength, NA(obj) equals the objective numerical aperture, and NA(Cond) is the condenser numerical aperture. Notice that equation (1) and (2) differ by the multiplication factor, which is 0.5 for equation (1) and 0.61 for equation (2). These equations are based upon a number of factors (including a variety of theoretical calculations made by optical physicists) to account for the behavior of objectives and condensers, and should not be considered an absolute value of any one general physical law. In some instances, such as confocal and fluorescence microscopy, the resolution may actually exceed the limits placed by any one of these three equations. Other factors, such as low specimen contrast and improper illumination may serve to lower resolution and, more often than not, the real-world maximum value of R (about 0.25 mm using a mid-spectrum wavelength of 550 nanometers) and a numerical aperture of 1.35 to 1.40 are not realized in practice. Table 2 provides a list resolution (R) and numerical aperture (NA) by objective magnification and correction.

A high-quality zoom lens and some prime lenses will be “faster” and have a maximum aperture of f/2.8. We call them faster lenses because when you can shoot at a larger aperture, you can increase the shutter speed with the same amount of light.

As you can see from the chart, at f/22 the depth of field is from 3m to infinity when the focus is set to 6m. But when the focus is set to 1m, still at f/22, the depth of field is now only from 85cm to 1.2m. When you bring the focus to 0.6m, the depth of field is now only from 55cm to 68cm.

Most objectives in the magnification range between 60x and 100x (and higher) are designed for use with immersion oil. By examining the numerical aperture equation above, we find that the highest theoretical numerical aperture obtainable with immersion oil is 1.51 (when sin (m) = 1). In practice, however, most oil immersion objectives have a maximum numerical aperture of 1.4, with the most common numerical apertures ranging from 1.0 to 1.35.

Look at the example below; the chair has relatively the same amount of blur in each photo, but as we compress the background and make it closer to the image, it gives the impression of being more out of focus, by amplifying the out of focus area in comparison to our subject.

Shallow depth of field

Because the depth of field is a fixed width on an exponential focal plane inherently you will get approximately ⅓ of the distance in front of the focal point in focus and ⅔ of the distance behind the focal point in focus. If you know exactly how much distance you have in front and behind you can focus slightly in front of the focal point, or slightly behind to increase or decrease the depth of field in an image.

Dof explainedphotography

In day-to-day routine observations, most microscopists do not attempt to achieve the highest resolution image possible with their equipment. It is only under specialized circumstances, such as high-magnification brightfield, fluorescence, DIC, and confocal microscopy that we strive to reach the limits of the microscope. In most uses of the microscope, it is not necessary to use objectives of high numerical aperture because the specimen is readily resolved with use of lower numerical aperture objectives. This is particularly important because high numerical aperture and high magnification are accompanied by the disadvantages of very shallow depth of field (this refers to good focus in the area just below or just above the area being examined) and short working distance. Thus, in specimens where resolution is less critical and magnifications can be lower, it is better to use lower magnification objectives of modest numerical aperture in order to yield images with more working distance and more depth of field.

Even though your depth of field in actual terms will be relatively the same at 24mm as 80mm and again as 200mm, the image will appear to be more out of focus when zoomed in. The reason for this is caused by ‘distance compression’; as we walk back and zoom in, keeping our subject the same size within the frame, the background has been brought closer.

The numerical aperture of a microscope objective is a measure of its ability to gather light and resolve fine specimen detail at a fixed object distance. Image-forming light waves pass through the specimen and enter the objective in an inverted cone as illustrated in Figure 1. A longitudinal slice of this cone of light shows the angular aperture, a value that is determined by the focal length of the objective.

About the author: Alexander J.E. Bradley is the founder of Aperture Tours (formally Paris Photography Tours) and heads up the tours in Paris. A professional photographer for over a decade Alexander enjoys shooting the surreal by mixing dreamlike qualities into his conceptual images.

P.S. Why not come on a photography tour with Aperture Tours in London, Paris, or Venice and learn with a professional photographer in a photogenic city.

Note that the D Nikon AF Nikkor 50mm f/1.8D is manual focus only for the following cameras. D5500, D5300, D5200, D5100, D5000, D3300, D3200, D3100, D3000, D60, D40, D40x. For autofocus you will need to buy the $220 Nikon AF-S Nikkor 50mm f/1.8G

You can find more photos and articles like this on the Aperture Tours website, or by following Aperture Tours on Facebook, Twitter, and Instagram. This post was originally published here.

Depth of field definition microscope

Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.

So hopefully you understand how to get great bokeh or to keep everything tack sharp now. Don’t be discouraged if you need to reread this article to set everything in your head. It will take time, and most importantly, practice.

When the microscope is in perfect alignment and has the objectives appropriately matched with the substage condenser, then we can substitute the numerical aperture of the objective into equations (1) and (2), with the added result that equation (3) reduces to equation (2). An important fact to note is that magnification does not appear as a factor in any of these equations, because only numerical aperture and wavelength of the illuminating light determine specimen resolution. As we have mentioned (and can be seen in the equations) the wavelength of light is an important factor in the resolution of a microscope. Shorter wavelengths yield higher resolution (lower values for R) and visa versa. The greatest resolving power in optical microscopy is realized with near-ultraviolet light, the shortest effective imaging wavelength. Near-ultraviolet light is followed by blue, then green, and finally red light in the ability to resolve specimen detail. Under most circumstances, microscopists use white light generated by a tungsten-halogen bulb to illuminate the specimen. The visible light spectrum is centered at about 550 nanometers, the dominant wavelength for green light (our eyes are most sensitive to green light). It is this wavelength that was used to calculate resolution values in Table 2. The numerical aperture value is also important in these equations and higher numerical apertures will also produce higher resolution, as is evident in Table 2. The effect of the wavelength of light on resolution, at a fixed numerical aperture (0.95), is listed in Table 3.

Depth of field photography examples

An important concept to understand in image formation is the nature of diffracted light rays intercepted by the objective. Only in cases where the higher (1st, 2nd, 3rd, etc.) orders of diffracted rays are captured, can interference work to recreate the image in the intermediate image plane of the objective. When only the zeroth order rays are captured, it is virtually impossible to reconstitute a recognizable image of the specimen. When 1st order light rays are added to the zeroth order rays, the image becomes more coherent, but it is still lacking in sufficient detail. It is only when higher order rays are recombined, that the image will represent the true architecture of the specimen. This is the basis for the necessity of large numerical apertures (and subsequent smaller Airy disks) to achieve high-resolution images with an optical microscope.

Visitors are invited to explore changes in numerical aperture with changes in m, using our interactive tutorial that investigates how numerical aperture and magnification are related to the angular aperture of an objective.

Dof explainedcamera

A high-quality prime lens, like a 35mm, 50mm or 85mm will go down as low as f/2 or even f/1.4 giving you a remarkably thin depth of field. For a portrait shoot with a 50mm f/1.4, you can focus on the eyes, and have the tip of the nose and the ears already blurry.

We often talk about the “speed” of a lens or having a “fast” lens. When we are talking about this, we are talking about how large the aperture can open. Many versatile zoom lenses will have a maximum aperture of f4 or f/5.6 which will make it difficult to get a real narrow depth of field.

The smaller the Airy disks projected by an objective in forming the image, the more detail of the specimen that becomes discernible. Objectives of higher correction (fluorites and apochromats) produce smaller Airy disks than do objectives of lower correction. In a similar manner, objectives that have a higher numerical aperture are also capable of producing smaller Airy disks. This is the primary reason that objectives of high numerical aperture and total correction for optical aberration can distinguish finer detail in the specimen.

Depth of field examples

The resolution of a microscope objective is defined as the smallest distance between two points on a specimen that can still be distinguished as two separate entities. Resolution is a somewhat subjective value in microscopy because at high magnification, an image may appear unsharp but still be resolved to the maximum ability of the objective. Numerical aperture determines the resolving power of an objective, but the total resolution of a microscope system is also dependent upon the numerical aperture of the substage condenser. The higher the numerical aperture of the total system, the better the resolution.

But when you have the aperture set to f/2.8 the distance between the two points drastically reduces. When you are shooting a subject 60cm from the camera, the depth of field is only 4cm, as opposed to 1.3m when you were shooting at f/22

The focus barrel in your lens increases exponentially. Each lens is different, but take this example of a 50mm lens below. If we imagine the depth of field as being a fixed width on this exponential scale we can see that the further towards infinity, the larger the depth of field will become, without changing the aperture or focal length of your lens.

This is a little bit of a tricky one. Yes… and no. The depth of field between two identical photos from a full-frame camera and a cropped camera would look the same. Where the blurry line is, is that a full-frame camera has more of the scene in the shot. So to compensate you will need to bring objects closer to achieve the same ratio.

One of the most obvious factors to controlling depth of field is the aperture. When you have a smaller aperture (larger number) you will have a wide depth of field. In this instant, you will have a greater distance between the closest and furthest points in an image.

When you focus your camera, the area around the focal distance will also be in focus. But this can fall off to blur quickly or slowly. The acceptable amount of in-focus area around what’s you are focusing on is called depth of field. Depth of field can be an easy concept to understand, but practicing it isn’t always straightforward. Which aperture you choose, which lens you use, what camera you are shooting with, and even how close something is to your lens all play a large part in controlling depth of field. Throughout this post, the guys from Aperture Photo Tours in Paris, London, and Venice will be exploring all the elements that make up depth of field and how to take control of your camera.

Both Nikon and Canon have very high quality 35mm and 50mm f/1.8 for under $200. The standard focal length means you will have the ability to shoot close and get a narrow depth of field. Most lenses won’t go as fast as f/1.8 and for only a few hundred dollars they should be a standard part of every photographer’s kit.

Dof explainedfor dummies

The wider your lens is, the wider your depth of field will be. On my 8mm f/3.5 lens, for example, I can sit on f/5.6 and my hyperfocal distance is about 50cm, meaning everything from half a meter to infinity will be completely in focus. It can therefore be more difficult to have a shallow depth of field on a wider lens.

If, for example, your subject and background were both in focus in the 24mm image, they would still be in focus in the 200mm image as well. Only when it is out of focus can you amplify the effect.

For example, if you wanted to maximize your depth of field to include infinity, you can focus forward of infinity to include infinity at the end of your depth of field, not in the middle, and bring closer your hyperfocal distance. With our same 50mm example from before, you can set your camera at 30m at f/2.8 and get everything from 15m to infinity in focus. At f/8 if you set the focus to 10m everything from 5.1m until infinity would be in focus. At f/22 if you set your camera’s focus at 6m, everything from 3m to infinity would be in focus.

It is for this reason that if you are shooting anything further than 15m on a 50mm lens, your aperture literally doesn’t affect depth of field. Anything beyond 15m has hit hyperfocal at all aperture stops and everything will be in focus.

This feature of increasing numerical aperture across an increasing optical correction factor in a series of objectives of similar magnification holds true throughout the range of magnifications as shown in Table 1. Most manufacturers strive to ensure that their objectives have the highest correction and numerical aperture that is possible for each class of objective.

When light from the various points of a specimen passes through the objective and is reconstituted as an image, the various points of the specimen appear in the image as small patterns (not points) known as Airy patterns. This phenomenon is caused by diffraction or scattering of the light as it passes through the minute parts and spaces in the specimen and the circular back aperture of the objective. The central maximum of the Airy patterns is often referred to as an Airy disk, which is defined as the region enclosed by the first minimum of the Airy pattern and contains 84 percent of the luminous energy. These Airy disks consist of small concentric light and dark circles as illustrated in Figure 3. This figure shows Airy disks and their intensity distributions as a function of separation distance.

In practice, however, it is difficult to achieve numerical aperture values above 0.95 with dry objectives. Figure 2 illustrates a series of light cones derived from objectives of varying focal length and numerical aperture. As the light cones change, the angle m increases from 7° in Figure 2(a) to 60° in Figure 2(c), with a resulting increase in the numerical aperture from 0.12 to 0.87, nearing the limit when air is the imaging medium.

Correct alignment of the microscope optical system is also of paramount importance to ensure maximum resolution. The substage condenser must be matched to the objective with respect to numerical aperture and adjustment of the aperture iris diaphragm for accurate light cone formation. The wavelength spectrum of light used to image a specimen is also a determining factor in resolution. Shorter wavelengths are capable of resolving details to a greater degree than are the longer wavelengths. There are several equations that have been derived to express the relationship between numerical aperture, wavelength, and resolution:

From what we have learned before, we note that bringing images closer to the camera will give a narrower depth of field, giving the impression that a full frame camera can produce a narrower depth of field.

where n is the refractive index of the imaging medium between the front lens of the objective and the specimen cover glass, a value that ranges from 1.00 for air to 1.51 for specialized immersion oils. Many authors substitute the variable a for m in the numerical aperture equation. From this equation it is obvious that when the imaging medium is air (with a refractive index, n = 1.0), then the numerical aperture is dependent only upon the angle m whose maximum value is 90°. The sin of the angle m, therefore, has a maximum value of 1.0 (sin(90°) = 1), which is the theoretical maximum numerical aperture of a lens operating with air as the imaging medium (using "dry" microscope objectives).

By examining the numerical aperture equation, it is apparent that refractive index is the limiting factor in achieving numerical apertures greater than 1.0. Therefore, in order to obtain higher working numerical apertures, the refractive index of the medium between the front lens of the objective and the specimen must be increased. Microscope objectives are now available that allow imaging in alternative media such as water (refractive index = 1.33), glycerin (refractive index = 1.47), and immersion oil (refractive index = 1.51). Care should be used with these objectives to prevent unwanted artifacts that will arise when an objective is used with a different immersion medium than it was designed for. We suggest that microscopists never use objectives designed for oil immersion with either glycerin or water, although several newer objectives have recently been introduced that will work with multiple media. You should check with the manufacturer if there are any doubts.

The numerical aperture of an objective is also dependent, to a certain degree, upon the amount of correction for optical aberration. Highly corrected objectives tend to have much larger numerical apertures for the respective magnification as illustrated in Table 1 below. If we take a series of typical 10x objectives as an example, we see that for flat-field corrected plan objectives, numerical aperture increases correspond to correction for chromatic and spherical aberration: plan achromat, N.A. = 0.25; plan fluorite, N.A. = 0.30; and plan apochromat, N.A. = 0.45.

Some zoom lenses have a variable maximum aperture. Usually, the maximum aperture is larger at the wider focal length, and smaller at the most telephoto length. So if your lens is a 24-105mm f/3.5-f/5.6, it will mean that at 24mm you can go as low as f/3.5 but when you are zoomed into 105mm, the largest aperture you can have will be f/5.6.

There are some exceptional lenses that are faster than f/1.4, going as fast as f/1 and even f/0.8, but these lenses are very rare and specialist lenses.