The compound light microscope: parts, handling and preparing specimens – Microscopy (Senior 4 Biology) | Ganzaa

Microscopy

Lessons

Senior 4 · Biology

Microscopy

4 lessons

Lesson 2 of 4

The compound light microscope: parts, handling and preparing specimens

25 min

Seven pieces of glass

Before light from the lamp of a school microscope reaches your eye, it passes through a condenser lens, a glass slide, the specimen, a coverslip, several lenses inside the objective and more inside the eyepiece. A fingerprint on any one of these surfaces, a crack in a slide, or a condenser set at the wrong height can turn a sharp image of cells into a grey blur. A light microscope also costs a school a great deal, and an objective lens driven down onto a slide can be scratched in a second.

In S1 you named the main parts and looked at onion and cheek cells. At A-level you need to know what each part does to the light, how to get the best possible image at each magnification, and how to prepare a specimen so that the structures you want are visible.

Definition

Objective lens: the lens nearest the specimen, which forms the first magnified image; most microscopes carry ×4, ×10, ×40 and sometimes ×100 (oil-immersion) objectives on a revolving nosepiece.

Eyepiece (ocular) lens: the lens you look through, which magnifies the image formed by the objective, usually ×10.

Condenser: a lens under the stage that focuses light onto the specimen. The iris diaphragm beneath it controls the width of the cone of light, and so the brightness and contrast.

Field of view: the circular area of the specimen that you can see at one time. Depth of field: the thickness of the specimen that is in focus at once.

Temporary mount: a specimen placed in a drop of liquid on a slide under a coverslip, for viewing on the same day.

The parts and the path of light

Part	Function
Light source (lamp or mirror)	Supplies light that passes up through the specimen
Condenser and iris diaphragm	Concentrate light on the specimen; closing the diaphragm increases contrast and depth of field but reduces brightness and resolution
Stage and clips	Hold the slide over the hole through which light passes
Objective lenses on a revolving nosepiece	Form the first magnified image; turning the nosepiece changes magnification
Body tube	Holds the eyepiece at the correct distance from the objectives
Eyepiece	Magnifies the image again and brings it to the eye
Coarse focus knob	Moves the stage (or tube) a long way quickly; used only with low-power objectives
Fine focus knob	Moves it a tiny distance for sharp focus at high power
Arm and base	Support the microscope; carry it by these

The total magnification is the product of the two lenses:

\text{total magnification} = \text{eyepiece magnification} \times \text{objective magnification}

As magnification increases, three things fall: the field of view (its diameter falls in proportion to the magnification), the brightness, and the depth of field. At ×400 you see a smaller, dimmer, thinner slice of the specimen, so you must re-centre, open the diaphragm a little, and use only the fine focus.

Worked example 1: using the field of view to estimate size

With a ×10 eyepiece and a ×10 objective, the field of view of a school microscope is 1.6 mm across. A student sees a filament of the green alga Spirogyra lying across the whole field, with 10 cells end to end. (a) Estimate the length of one cell in micrometres. (b) Estimate the diameter of the field of view with the ×40 objective.

(a) Each cell is $\frac{1.6\,\mathrm{mm}}{10} = 0.16\,\mathrm{mm}$ . Since $1\,\mathrm{mm} = 1000\,\mu\mathrm{m}$ , one cell is $0.16 \times 1000 = 160\,\mu\mathrm{m}$ long.
(b) Total magnification rises from $10 \times 10 = 100$ to $10 \times 40 = 400$ , four times larger, so the diameter of the field falls to $\frac{1.6}{4} = 0.40\,\mathrm{mm}$ , or $400\,\mu\mathrm{m}$ .
This method gives only an estimate. For accurate measurement you calibrate an eyepiece graticule (Lesson 3).

Handling and focusing, step by step

Carry the microscope upright with two hands, one on the arm and one under the base. Place it at least 10 cm from the edge of the bench.
Turn the nosepiece until the lowest-power objective clicks into place. Switch on the lamp and open the diaphragm.
Clip the slide on the stage with the specimen over the hole.
Looking from the side, turn the coarse knob to bring the objective close to the slide, without touching it.
Now look through the eyepiece and turn the coarse knob to move the objective and slide apart until the image appears. Sharpen it with the fine knob. (Focusing upward means the lens can never hit the slide.)
Move the part you want into the centre of the field, then turn to the next objective. Use only the fine knob at ×40 and above, and adjust the diaphragm for the best contrast.
For ×100 oil immersion: place one drop of immersion oil on the coverslip, swing the ×100 objective into the oil, and focus with the fine knob only. Clean the oil from the lens with lens tissue afterwards.
When you finish: lowest-power objective in place, stage lowered, slide removed, lamp off, cover on. Clean lenses only with lens tissue, never with a cloth or paper.

Preparing a temporary mount

Specimens must be thin, so that light passes through, and most need a stain, because cell contents are nearly colourless.

Place a drop of water or stain in the centre of a clean slide.
Put a thin piece of specimen in it (a single layer of cells, a thin section cut with a sharp blade, or a smear).
Touch one edge of a coverslip to the drop at about 45° and lower it slowly with a mounted needle, so that the liquid spreads out and pushes the air ahead of it: no air bubbles.
Draw off extra liquid with filter paper at the edge of the coverslip. To add stain afterwards, put a drop at one edge and draw it under the coverslip with filter paper at the opposite edge (irrigation).

Stain	What it shows	Colour
Iodine solution	Starch grains; also makes nuclei and cell walls visible	Starch blue-black; other parts yellow-brown
Methylene blue	Nuclei of animal cells; bacteria	Blue
Eosin	Cytoplasm	Pink
Safranin	Lignified walls of xylem; nuclei; Gram-negative bacteria (Unit 2)	Red
Acetocarmine	Chromosomes in dividing cells	Red

Worked example 2: improving a poor image

A student viewing a stained section of a leaf at ×400 reports: "The image is very dark, and when the top layer of cells is sharp, the cells below are blurred." Explain both observations and say what to do.

Darkness: at high power the objective collects light from a much smaller area of the specimen, so less light reaches the eye. Open the iris diaphragm a little, or turn up the lamp.
Blurring: the depth of field at ×400 is only a few micrometres, thinner than the section, so only one layer can be in focus at a time.
Remedy: turn the fine focus knob slowly up and down to focus through the layers one at a time ("optical sectioning"), or cut a thinner section.
Do not switch to the coarse knob at ×400: its large movement could drive the lens into the slide.

Tami Guy: "How To Use a Compound Light Microscope: Biology Lab Tutorial". Watch the order of the focusing steps, and how the instructor uses the diaphragm and stores the microscope at the end.

Warning

Safety and care

Never use the coarse focus with the ×40 or ×100 objective, and never lower an objective while looking through the eyepiece.
Glass slides and coverslips break into sharp pieces: report breakages and clear them up with a brush and pan, not your fingers.
Iodine and some stains irritate skin and eyes and stain clothes. Wear a lab coat and wash off splashes with plenty of water.
Never use a mirror to collect direct sunlight: it can damage your eye. Keep water away from the lamp and its cable.
If you take your own cells (cheek cells), use a new sterile cotton bud and put used buds and slides in disinfectant.

Tip

Memory hook. "Low, side, up, fine": start on low power; lower the lens watching from the side; focus by moving up (apart); finish with the fine knob. More magnification means a smaller, dimmer, shallower view.

✦

Practice: using the light microscope

State the function of (a) the condenser; (b) the iris diaphragm; (c) the fine focus knob.
Calculate the total magnification with a ×15 eyepiece and a ×40 objective.
At ×40 the field of view is 4.0 mm across. What is its diameter at ×400?
Explain why you should focus by moving the objective and slide apart, not together, while looking through the eyepiece.
Give two reasons for staining a specimen, and name a stain that shows starch.
Air bubbles appear as dark-edged circles under a coverslip. Explain how to avoid them.
A pupil says: "I can't see the cells clearly at ×400, so I will use the ×100 oil-immersion objective to make them clearer." Evaluate this decision.

Show answersHide answers

(a) Focuses light onto the specimen; (b) controls the amount and angle of light, and so brightness and contrast; (c) makes small focusing movements for a sharp image at high power.
$15 \times 40 = 600$ , so ×600.
Magnification increases $\frac{400}{40} = 10$ times, so the diameter falls to $\frac{4.0}{10} = 0.40\,\mathrm{mm}$ .
If you move them together while looking through the eyepiece you cannot see how close the lens is, and it may crash into the slide, breaking it and scratching the lens. Moving them apart can never cause a collision.
Cell contents are nearly transparent, so stains add contrast; different stains bind to different structures (nucleus, starch, cell wall), so you can identify them. Iodine solution stains starch blue-black.
Lower the coverslip at an angle of about 45° from one edge of the drop, slowly, with a mounted needle, so the liquid spreads and pushes air out; use enough liquid.
The decision might help only if the image was poor because of magnification. If the problem is a thick, unstained or poorly lit specimen, the ×100 objective will make it worse: dimmer, shallower depth of field, and it needs oil. First improve the specimen (thinner, stained), the lighting (diaphragm) and focus at ×400; oil immersion improves resolution only when used correctly.

Quick check

Match each part to its function.

Tap one on the left, then its partner on the right.

Key points

Light passes from the lamp through condenser, specimen, objective and eyepiece; total magnification = eyepiece × objective.
Higher power gives a smaller, dimmer field with less depth of field: centre first, use fine focus, adjust the diaphragm.
Start on low power, lower the lens watching from the side, focus by moving apart, use oil only with the ×100 objective.
Specimens must be thin and usually stained; lower the coverslip at 45° to avoid air bubbles.

Sources and credits

2.1.2: Studying Cells, from Biology 2e by OpenStax (Mary Ann Clark, Matthew Douglas, Jung Choi), via LibreTexts Biology, licensed under CC BY 4.0. Used for the light microscope, magnification and staining. Adapted by Ganzaa.
Figure 1, the procedures, worked examples (the Spirogyra count is illustrative), practice and quick check: written by Ganzaa.
Photos: Wiedehopf20, CC BY-SA 4.0; Nico10310, CC BY-SA 3.0; Jeremyida002, CC BY-SA 4.0; all via Wikimedia Commons.
Video: "How To Use a Compound Light Microscope: Biology Lab Tutorial", Tami Guy, embedded from YouTube.

1Seven pieces of glass

2The parts and the path of light

3Handling and focusing, step by step

4Preparing a temporary mount

Seven pieces of glass

The parts and the path of light

Handling and focusing, step by step

Preparing a temporary mount