Lab 2 The compound light microscope

The compound light microscope

is an essential tool in the study of human anatomy. The human eye is not able to perceive objects smaller than 0.1 mm in diameter, so a microscope is needed to study small structures such as a typical cell. As mention in Lab 1, we call the study of such small structures microscopic anatomy.

The microscope that you will be using in this lab is called a compound light microscope for two reasons:

1. The term compound is applied because it uses two different types of lenses. It has ocular lenses found within the eyepieces and objective lenses of various powers of magnification found within the objectives.

2. The term light is applied because it requires a light source in order to view an object. A compound light microscope shines light through the material you wish to view; therefore, the material must be very thin so the light can pass through it to see the details within the material. In comparison, a dissecting light microscope is used to examine larger material or opaque material and therefore shines the light on the surface of the object instead of through it.

The microscope is a very important tool used in the study of anatomy to examine cells and tissues that are too small to be seen with the naked eye. Microscopes are also invaluable diagnostic tools in the clinical setting. Describe two specific ways in which microscopes are used in the diagnosis and treatment of disease.

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They can be used to find cancerous cells (biopsy)

They can be used to determine which types of bacteria are causing an illness

parts of microscope: base, on/off, light intensity knob, stage, slide positioning knobs, stage clip

The base rests on the bench and contains the light source.

There is an on/off switch for the light and a light intensity knob to adjust its brightness. The arm rises from the base and supports the stage and the body tube.

The stage is the flat surface where the specimen slide is placed.

The stage has slide-positioning knobs, which allow precise positioning of the slide.

It also has a stage clip that will keep your slide in place.

parts of microscope, coarse/fine adjustment knobs, condensor, iris diaphragm, body tube

Other adjustments (coarse and fine adjustment knobs) change the vertical position of the stage itself.

The condenser is found beneath the stage. It focuses light on the slide and its position is adjusted by turning the knob next to it.

Built into the base of the condenser is an iris diaphragm. The small arm located on the condenser may be used to open or close the diaphragm; this regulates the amount of light entering the condenser and lens system.

The body tube is supported by the arm and has one or two eyepieces containing ocular lenses at one end and a revolving nosepiece with attached objective lenses at the other end.

Magnification: lenses

All of our microscopes have at least three objective lenses but some also have a fourth lens.

The shortest lens is the low power lens with a magnification of 4X or 5X, depending on the particular microscope.

The medium power lens has a 10X magnification.  The high power lens has a magnification of 40X.

If present, the fourth objective lens will have a black line near its tip. This lens has a 100X magnification and is designed for a technique called oil immersion that involves suspending a drop of oil between the lens and the microscope slide to improve resolution. You will not be using this lens in this lab.

Focusing adjustment knobs

The coarse adjustment knob is used to bring objects into rough focus when using the 4X and 10X objectives.

The fine adjustment knob is the only focusing knob to be used with the 40X and 100X objectives. Please note that these focusing knobs change the vertical position of the stage and if not used properly will result in you smashing through a slide with the higher power objectives.

Total magnification

of objects under the microscope is found by multiplying the magnification of the ocular lens (10X) by the magnification of the objective lens you are using. For example, the total magnification obtained when using the low power objective lens would be 10X (ocular lens) multiplied by 4X (objective lens) = 40X.

Field of view diameter

Field of view is the area of the slide or specimen that is visible at any given time through the microscope. It is important to know the size of the field of view for each objective lens because it will allow you to estimate the size of specimen you are examining.

Low power (4X) 4,5mm

Medium power (10X) 1.8mm

High Power (40X) 0.45mm

Oil Immersion (100X) 0.18mm

What is the relationship between the Field of View Diameter and the Total Magnification?

They are inversely related, the higher the magnification the lower the field of view diameter

Should you increase or decrease the amount of light when moving up to a higher magnification?

The procedure to focusing your slide stated that once you have the cells in focus, use the slide positioning knobs to centre the cells in the field of view before moving to the next higher objective lens. Why is this important?

1. You should increase the amount of light when moving to a higher power magnification. The f.o.v.d. gets smaller and cuts out light

2. As you move to a higher objective the field of view diameter gets smaller so if cells are centered before moving up, it ensures they stay in the field of view when you move to the next objective.

DEPTH OF FOCUS

The vertical distance in focus at any given time is called depth of focus. All specimens that you will be viewing are 3-dimensional; that is, they have thickness or depth to them. You cannot appreciate the thickness of a specimen unless you scan up and down through it using the fine focus. Beginner microscopists, often make the mistake of getting their specimen in focus and then putting their hands behind their backs so they don’t mess things up. Instead, adjust the fine focus – that way you get to appreciate the 3-dimensional aspect of your specimen.

How to fix if you are seeing two circles. How to fix if you are seeing a partially black circle.

1. adjust the ocular lenses

2. adjust the objective lens