Question

1. Give an example of an observation made with the microscope which illustrates the concepts/terms:

a. Depth of field

b. Contrast

c. Field of view

Answer 1

1(a)Depth of field:- It is the resolution in the longitudinal plane ( parallel to optical axis). It is measured as the distance between the nearest object plane in focus to the farthest object plane, which is simultaneously in focus and is measured in microns.

Example – When two hairs present crisscrossing each other on a microscopic slide at lower magnification, it is possible to get both the hairs in focus at the same time, but at higher magnification, when one hair is in focus other appears blurry and vice versa. Another example is in case of tissues of plants or animals which are two or more layers deep, cells in one layer will be in focus at higher magnification, but cells at other layers will appear blurry.

(b)Contrast – Contrast is the difference in light intensity between the image and the adjacent background relative to the overall background. Usually a minimum contrast value of 0.2 or (2%) is required by human eye to distinguish between the image and its background.

Contrast is the ability of a specimen by absorbing light, brightness etc . so that it can be seen against its background. Contrast is the darkness of the background relative to the specimen. Lighter specimens are easy to see under dark background, but in case of transparent or colorlesss specimens a special microscope is required, that is a phase contrast microscope.

Bright light microscope cannot change the contrast beyond a certain point, if it goes beyond than the object appears distorted. To adjust contrast in bright light microscope, the condenser is moved closer to the stage as possible, aperture is closed throughout and the specimen is seen through the eyepiece to check contrast. Slowly the aperture is opened, while seeing through the eyepiece and stopped when image appears bright and clear, if image looks distorted than the aperture is opened more.

In Phase contrast microscopy contrast is used to help illuminate details of the living cells and other microscopic organisms. Here to adjust either we have to slide the condenser or rotate depending on the built of the microscope. It is slowly done by looking through the eyepiece and should be stopped when image is sharp and bright.

(C) Field of view:- When we look through a microscope a circular area is seen, the diameter of it is known as field of view. It is how much a specimen is visible at a given time, in the lateral plane (perpendicular to the optical axis). It is considered that the higher the magnification less is the field of view.

To calculate field of view it is required to know the field number and the magnification of the objective lens. Field number is the diameter of the image area when seen under an eyepiece. This is usually written on the side of the eyepiece.

So field of view = Field number (FN)/ objective magnification

Suppose field number is 20mm and objective magnification is 10X than field of view will be 20/10 = 2.0mm, this is the field view in this example. The more the magnification the smaller is the field of view.

Example:- If an insect is seen under a microscope than in lower magnification whole insect can be seen, but if magnification increased than if the focus is on leg we can see that part only, but not the whole specimen, so when magnification increases the field of view decreases and vice versa.