You can see in the photos below how when at the same aperture the photo with the wider focal length has less depth of field.

In fact, if you are shooting with your kit lens this is a great trick to give you a more narrow depth of field when you feel restricted by how wide your aperture can go.

For a more in-depth overview of considerations relevant to the design of a fluorescence multiplexing system, click here.

To be able to explain depth of field we have to start with defining certain terms. Then I will get into describing some things that affect depth of field.

Shallowdepth offield photography

Aperture, focal length, and distance from your subject. Using a combination of those 3 things you can create exactly what you need.

Notice in these next few photos how the subject is in focus but the majority of the rest of the photo is not sharp. This is showing narrow or a shallow depth of field.

Fluorescence guided surgery (FGS) uses optical filters to assist surgeons in visualizing organs or structures during surgery. In this example, the patient drank a fluid containing the fluorophore, and the surgeon used a special visualizing system that sent light into the body to excite the fluorophore. In the photo, the green area highlights an anatomically complex location, and the surgeon knows precisely where to operate. Without FGS, this patient’s surgery would be difficult and dangerous.

Shallowdepth offield

The main difference between an excitation filter vs emission filter? An excitation filter lets in light to excite a molecule, while an emission filter transmits the light the molecule emits.

Focal length can affect the depth of field as well since the longer you focal length the more narrow your depth of field is.

An optimal optical filter set of the above three individual filters blocks unwanted excitation light (including in the UV and IR) as well as unwanted emitted light (including autofluorescence) to ensure high signal and low background.

Fluorescence microscopy, and most instruments that use fluorescence, including fluorescence microscopes, rely on optical filters. A typical microscope has three basic filters: an excitation filter (or exciter), a dichroic beamsplitter (or dichroic mirror), and an emission filter (or emitter). These three filters form what is referred to as a “filter set” and are often housed in a special assembly called a “filter cube” that can be quickly mounted in a microscope.

DOF is a range of distances on either side of the focal plane that are “acceptably sharp.” (“Acceptably sharp” is where the confusion begins.) Another way to state this would be, the area behind and in front of your focus point that is in focus as well.

One day it just clicked. I’m not sure where or when, but I have a much better grasp on it now. I love using that knowledge to positively impact my own personal art as well as the art I create for my clients.

Semrock employs all these advantages in the industry-leading Avant™ filter sets that increase emission signal from short Stokes’ shift (low “Gap”) fluorophores by collecting the emission signal over the peak emission ranges and blocking excitation crosstalk. Learn more.

Depth offield photography

I hope after reading this you have a better understanding of depth of field. Now go get your camera out and experiment with DOF.

The excitation filter is usually a bandpass filter designed to pass only those light source wavelengths that are to be absorbed by the fluorophore, thereby minimizing excitation of other sources of fluorescence, and especially blocking excitation light in the fluorescence emission band.

Fluorescence occurs when a molecule absorbs light at wavelengths within its absorption band, and then emits light at longer wavelengths within its emission band. For example, brightly fluorescent molecules (called fluorophores) can be attached to biologically significant molecules in e.g. cell membranes, in the brain, or even on subunits of DNA, whose structures become visible in a fluorescence microscope that allows us to track the way cells function in health and disease. Fluorescence is widely used in biology, biotechnology, and medicine, due to its extraordinary sensitivity, high specificity, and simplicity of usage.

Read more: Creating Depth in Your ImagesHow Focus Stacking Will Help You Create Shaper Photos11 Composition Rules to Know to Improve your PhotographyWhy a Wide Angle Lens Will Be Your New Favorite Lens

Depth offield photography aperture

In each of the photos below you can see that the focus is on the bunny but how blurry the background is (DOF) changes drastically depending on the aperture.

The team at IDEX Health & Science has specific experience and expertise in design of optical filter sets and optimizes each set to make sure the filters work well together. Semrock fluorescence optical filters and fluorescence filter sets therefore offer a number of critical advantages to users and developers of fluorescence-based optical platforms/p>

The filter set described above can only be used for one fluorophore, since it has only one passband each for excitation and for emission. If for example two fluorophores are used, the set will need to have two passbands each for exciter, dichroic, and emitter. These are called multiband sets; for more information, review our White Paper.

Our flagship BrightLine® filter sets follow fluorophore usage trends, light source innovation, and detector enhancements. These sets balance filter set needs and cost and offer set performance over a wide range of fluorescent probes and microscopy applications, based on our years of experience designing optical filters.

Depth offield photography examples

Understanding DOF will allow you to add interest in your photos. If you want your subject to really pop you need to know how to create a shallow DOF. If you want your whole image in focus, like in landscape photography, you will need to create a deeper DOF.

The dichroic is an edge filter (usually passing longer wavelength light and reflecting shorter wavelength light) used at an oblique angle of incidence (typically 45°) to efficiently redirect light in the excitation band towards the sample and transmit light in the emission band towards the detector.

The distance that is sharp around your focus point. Shallow DOF means not much of your image other than your subject is sharp. Deep DOF means a lot of your image other than your subject is sharp.

As you can see from the example above the 200mm lens really compressed DOF. Even though these images were both shot at the exact same place this lens makes the subject and background seem more pushed together.

Depth offield examples

If you are struggling to fit everything in the frame is it because you have crop sensor? See here: Everything you Need to Know About Crop Sensor vs Full Frame

I’m not really sure what James (the above commenter) is talking about as he really doesn’t elaborate what he takes issues with in regards to this article.

Depth offield photography settings

When you are shooting more wide open (smaller aperture number) less of the photos will be in focus. Then you will have a narrow depth of field where the background is blurred.

The only thing that confuses me is the area where you talk about sensor sizes where it seems that you may be contradicting your own argument by stating:

Sensor Size: “I believe this has the least impact on depth of field” and then you state “Changes in DOF depend on whether you use a crop or full frame;”

If you are anything like me, understanding depth of field (DOF) was one of the biggest hurdles you had to overcome when learning the technical aspects of photography and shooting in manual mode. I would read explanations online, see links to calculators, hear it described as “shallow” or “deep” and couldn’t make sense of it all.

Depth offield photography formula

In the photos below you can see how the focal plane affects how much of the photo is in focus. The first photo all the toys are in focus because they are on the same focal plane.

Hi! wonderful article today. Is there any chance you could post the same pics of your “studly” husband with the 50 mm and 85 mm with the same camera settings, to see the difference? Can you tell I’m an an amateur? LOL

Hey Adam! Thanks for the comment. To clarify sensor size does affect depth of field, but since most people use one camera consistently (and thus one sensor size) I didn’t want to go into too much detail about how changing sensor size affects DOF. That subject could be a whole article on its own but since most readers of this particular blog probably don’t switch between full frame and crop, I felt it was the least important factor to discuss. Hope that makes more sense! Thanks for asking!

To summarize, filter sets strike a balance between high brightness and high contrast, typically by using narrower or wider passbands in combination with center wavelength placement.

The emitter is usually a bandpass filter that passes only the wavelengths emitted by the fluorophore and blocks all undesired light outside this band – especially the excitation light.

The below photos were taken with the same settings and same focal length. The only difference is I moved the stool closer to the plant in the second photo.

I know a lot of people who initially struggle with getting a grasp on aperture and depth of field especially when starting and and you have laid out it nicely in this article.

The next photo only the wolf is in focus. The other toys are on different focal planes. They have been moved to a different focal plane therefor they are not blurry. You can see this more clearly with the turtle, half of the turtle in in focus while the other half is on a different plane.

Even though every photograph is two-dimensional, it portrays a three-dimensional world. The distance between the camera and the subject and the distance behind the subject to the “end” of the photo is the “depth” of the photo. The amount of depth that is in focus is the depth of field.