A microscope is an instrument that magnifies an object. Because cells and many organisms are too small to see with the naked eye, a microscope is an essential tool in the study of biology. In addition to magnification, microscopes also provide resolution, which is the ability to distinguish two nearby objects as separate. A combination of magnification and resolution is necessary to clearly view specimens under the microscope. The light microscope bends a beam of light at the specimen using a series of lenses to provide a clear image of the specimen to the observer. In this lab, you will learn (or review) how to use a microscope to observe specimens.
Your microscope has 4 objective lenses. On most of the student microscopes, these are 10x, 40x, 63x, and 100x (Oil Immersion). The number indicates the magnification, e.g., the 10x objective will magnify an object by 10 times. Look at each objective lens on the microscope, and write down the magnification of each lens in the table below.
In addition to the objective lenses, the ocular lens (eyepiece) has a magnification. Look at the eyepiece magnification on your microscope and write down the magnification below.
The total magnification is determined by multiplying the magnification of the ocular and objective lenses. For instance, if the ocular lens has a magnification of 20x and the objective lens being used has a magnification of 4x, the total magnification will be 80x.
The number after the magnification on an objective lens indicates the numerical aperture. This is a measure of the light-gathering ability of the objective lens. It is closely related to resolution, which is the shortest distance between two points that be distinguished as separate objects. The higher the numerical aperture, the higher the resolution. A higher numerical aperture also means a shallower depth of field. It also requires a shorter working distance (i.e., you must get the objective lens closer to the specimen).
Resolution is also dependent upon the wavelength of light used to illuminate the specimen. Recall that the visible light spectrum (for humans) ranges from about 390 nm to 700 nm.
Resolution may be calculated as:
r = 0.61λ/NA
r = resolution = minimum distance between points
λ = wavelength
NA = numerical aperture (fixed attribute of a lens) = n sin θ
n = index of refraction (1 for air; 1.33 water; 1.5 for glass; 1.52 for immersion oil)
θ = maximum half angle of the cone of light that enters the lens
Depth of Field – resolution in the z-plane. The range of depth that is in focus.
Field of View – the total circular area you can see through the microscope. Usually expressed as the diameter of the field of view.
Refractive Index – a measure of how much the path of light is bent (or refracted) when entering a material.
Resolution – the ability to distinguish two objects as separate.
Total Magnification – the magnifying power of the objective multiplied by the magnifying power of the objective lens.
Working Distance – the distance between the coverslip and the objective lens when the specimen is in focus.