Bio 220 Lab week 1

Why is immersion oil used with the 100X objective?

Because the oil has the same angle of refraction as the glass in the slide and in the objective lens and too much light would be lost if air was between the slide and lens instead of oil.

The ocular lens on your microscope is 10x. If a 5x ocular were used with the same three objectives lenses (i.e., 10x, 40x, and 100x), what magnification would be achieved?

Remember that the microscope has two sets of lenses - the ocular lens, which is unchangeable, and the objective lenses, which are changeable (you can rotate the 4x, 10x, 40x, or 100x into place - note that we don't use the 4x in microbiology because we are looking at very small things, so we have to use the other more powerful lenses). To calculate the total magnification, you have to multiply the two lenses being using together.

How would a decrease in the wavelength of light effect the Resolving Power? (i.e., Would a shorter wavelength increase or decrease resolution?) Remember: RP = (l)/(2 x n.a.), where "l" represents wavelength of light. Wavelength of light also is often represented by the symbol "λ." "n.a." represents the numerical aperture of the lens used.

Remember, in lecture that we talk about the fact that shorter wavelengths (blue range of visible light or even shorter UV light or electrons, which have an even shorter wavelength than light) and larger numerical aperture provide the greatest resolution. So, even without using the formula, you can deduce that a decrease in wavelength would increase resolution. However, you can also see this mathematically by playing with the formula: For example: RP = 400 nm = 160 nm 400 nm is the wavelength of light in the violet range and 1.25 is the 2 x 1.25 numerical aperture of the 100x objective lens. RP = 200 nm = 80 nm 200 nm is the wavelength of light in the UV range and 1.25 is the 2 x 1.25 numerical aperture of the 100x objective lens. Also, remember that nanometers (nm) are very small units of measurement, below micrometers (µm), millimeters (mm), meters (m), etc. We usually measure microbes in micrometers (µm).

Resolving Power

The ability to distinguish between two adjacent (side-by-side) objects or the ability to see one object. Resolving power is calculated by a formula that considers the wavelength of light (shorter blue wavelengths have greater resolving power than longer red wavelengths), and the size of the lens. The resolving power value will tell you how far apart two things have to be in order to know that they are 2 separate things and not view them as one (because they appear to overlap). This value also tells you how big something must be in order to be able to distinguish it from the background (an example of this would be if you have had trouble resolving a dim and distant star in the night sky).

Ocular Lens

eyepiece (10x)

Condenser

Condenses or focuses the diffuse cone of light from the light source into a narrow beam through a hole in the stage.

Iris Diaphragm

Controls the amount of light passing through the specimen.

Objective Lens

One of 3 or 4 lenses that can be rotated into place for viewing specimens at different magnifications (4X, 10X, 40X, 100X).

What is the function of the iris diaphragm?

(Same as definition above.) It controls the amount of light passing through the specimen.

What is the advantage of the parfocal lenses?

Once you get your specimen in focus on one objective, then you should be able to switch to any other objective and the specimen should still be in focus (with possible minor adjustment of the fine focus knob only - our microscope repairman has the microscopes parfocal to within a quarter-turn of the fine focus knob each year in August).

Oil immersion

The 100X objective (total magnification when used is 1000X) that is used with immersion oil. The oil immersion (100X) objective is immersed in optical grade oil because this oil refracts light at the same angle as the glass in the slide and the glass in the microscope lenses, so you don't lose light that is bent at other angles through air. Optical oil is only useful at higher magnifications such as this (1000X total mag.). You will not detect the loss of light through the air at the lower magnifications (using the less powerful objectives, such as 40X and 10X). *Note that only the oil immersion objective is specially sealed to be immersed in oil - the 40X and 10X objectives are NOT sealed and will actually be ruined by immersion in oil. *Also note that the oil must be removed from the microscope lens after each lab period's use because it will thicken and become yellow if left on the microscope and you will not be able to see very well or remove it as easily next time.

Working Distance

The distance between the specimen on the stage and the objective lens (how much room you have to move the stage up and down = how much room you have to focus on the image of the specimen).

Total Magnification

The total amount that an image is magnified, calculated by multiplying the magnification of individual lenses together, ex. 10X ocular x 40X objective = 400X total magnification.