Understanding Depth of Field

Observing Sewing Thread Under a Microscope

Have you ever tried to take a picture of someone or something in front of a background, such as a distant mountain, a lake, or some other object or scene? You may have discovered that either the person or object is in focus or the background is in focus, but both are not in focus at the same time.

In both of these pictures the foreground objects (child/leaves) are in sharp focus. The background objects are fuzzy.

A. The lens of a camera is unable to get both background and foreground images into sharp focus at the same time.

Q. What causes this situation?

In this picture, the photographer is focusing on the horse in the foreground. As a result, the horse in the background is not in sharp focus. The portion of a view that appears sharp in the image is called the depth of field.

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The same thing happens with a microscope. You will notice as you gain more experience in the use of the microscope that not everything in your field of view is in focus at the same time.

As you focus on one area of your specimen, another area seems to become fuzzy or unclear. This problem only gets worse when you go to a more powerful magnification.

Depth of Field is the thickness of an object, or a layer in a specimen that is in view and in focus at the same time while viewing it under a microscope or through a camera.

Q. What part of this image of a fly’s foot is in focus, and which part/s are not?

A. Responses will vary.

Q. What causes focusing problems in a tiny area like the field of view of a microscope?

A. Many objects being examined under a microscope are made up of layers or different textures. Only one area can be in the microscope’s depth of field at one time. Everything else will be out of focus - in varying degrees.

These plant cells are three dimensional objects. You can focus up and down to get the best view, but part of the cell will always be out of focus.

Dealing with Depth of Field - A Review

When looking through a lens, focus is possible at only one distance.

In microscopy, as well as photography, the depth of field (DOF) is the portion of a view that appears sharp in the image.

In photography, the decrease in sharpness is gradual on either side of the focused distance and in some cases this decrease in sharpness is not that noticeable. (See horse picture.)

In microscopy, however, even a tiny difference in focus will cause a portion of the image to be unclear or fuzzy.

The goals for today’s lesson are:

1. To use a microscope to observe three crossed threads.

2. To discover the difficulty in focusing specimens which have a number of layers or different textures.

3. To be able to explain the concept of depth of field.

Assessment: Completed lab sheet

Procedure:

• Cut three 1 cm pieces of sewing thread - each in a different color: red, yellow, and blue.

• Arrange the threads in a stack with red on the bottom, yellow in the middle, and blue on top.

• Tape the stacked cross threads onto a blank slide.

• Observe the threads close to the point where all three colored threads intersect.

• Draw detailed descriptions of your observations under three powers of magnification - when the yellow thread is in focus.

• Practice focusing on the different layers of your thread design.

• Think about what happens to the image of the different colored threads when you get just one in perfect focus.

• Discuss your observations with your teammates.

Lab Sheet Format:Using the low power objective and the coarse adjustment knob, locate the point at which all three threads cross. You may have to move your slide to get the best view of all three threads. Observe, draw and label what you see when the yellow thread is in focus.

Object:Magnification:

Using the medium power objective and the coarse adjustment knob, observe, draw and label what you see when the yellow thread is in focus.

Object:Magnification

Using high power objective and the fine adjustment knob, observe, draw and label what you see when the yellow thread is in focus.

Object:Magnification

Questions: Answer the following questions in complete sentences.

1. It is difficult to get a clear, detailed view of all three threads at the same time. Describe your experiences with “depth of field.”2. To determine the positions of all three threads, you must raise and lower your microscope objective lens. This is known as “focusing up and down.” What happens when you “focus up and down”?3. In what instances might “depth of field” be a real problem in microscopy?4. What additional information did you learn about microscopy today?5. What do you still wonder about?

Debrief: Things to Discuss

It is difficult to get a clear, detailed view of all three threads at the same time. Describe your experiences with “depth of field.”

Depth of Field can be understood by focusing on three overlapping colored threads. Notice the textural details visible in the yellow thread. Both the red and blue threads are out of focus. Because the red and blue threads lie either below or above the yellow thread, they are in a different plane of focus. To determine the relative position of all 3 colored threads, you must raise and lower your objective lens slowly (known as "focusing up and down") and observe the order in which the threads go in and out of focus. As your objective lens approaches the slide, the first thread to come into focus is on the top. Understanding the concept of depth of field will help you visualize the structures within a thick section of tissue or a large single-celled organism