Unit 3 Microscopy
Key unit competence: Interpret observed specimens under microscopes.
Key ideas
- Microscopes produce enlarged images of small objects. Early history: Hooke (1665) described "cells" in cork; van Leeuwenhoek made simple microscopes and saw microorganisms; later compound and electron microscopes were developed.
- Magnification is how many times larger the image is than the real object: magnification = image size ÷ actual size. Resolution (resolving power) is the ability to distinguish two points close together as separate; it limits the detail seen.
- The compound light microscope uses light and glass lenses. Parts: eyepiece (ocular), objective lenses (×4, ×10, ×40, ×100 oil immersion), revolving nosepiece, stage with clips, coarse and fine focus, condenser, iris diaphragm, mirror or lamp, arm and base. Total magnification = eyepiece × objective. Maximum useful magnification about ×1500; resolution about 0.2 µm.
- Units: 1 mm = 1000 µm (micrometres); 1 µm = 1000 nm (nanometres). Always convert to the same unit before calculating magnification.
- Measuring cells: an eyepiece graticule (scale in the eyepiece) is calibrated with a stage micrometer (a slide with a ruled scale, often 1 division = 10 µm). Count how many graticule units match a known length, find the value of one unit, then measure the cell in graticule units.
- Electron microscopes use beams of electrons focused by electromagnets, in a vacuum. Electrons have a much shorter wavelength than light, so resolution is far better (about 0.1–1 nm) and magnification can exceed ×500 000. TEM (transmission): electrons pass through very thin sections, showing internal ultrastructure (2D). SEM (scanning): electrons bounce off the surface, giving 3D surface images.
- Limitations of electron microscopes: specimens must be dead (vacuum), prepared with complex staining with heavy metals, possible artefacts, images in black and white (colour added), very expensive, large and need trained staff.
- Preparing slides: temporary mounts (thin specimen, water or stain, cover slip lowered at an angle to avoid air bubbles); stains: iodine (starch, plant cells), methylene blue (animal cells, nuclei). Permanent slides: fixing, dehydrating with alcohol, clearing, staining and mounting in a resin.
- Care: carry with two hands, start with the lowest power, use only the fine focus at high power, clean lenses with lens tissue, never let objectives touch the slide, store covered.
Common misconceptions
- "Higher magnification always shows more detail." No: detail depends on resolution; beyond the limit, magnification only makes a blurred image bigger ("empty magnification").
- "Electron microscopes can show living cells moving." No: specimens are in a vacuum and must be dead and specially prepared.
- "Magnification = actual size ÷ image size." No: magnification = image size ÷ actual size.
- "1 mm = 100 µm." No: 1 mm = 1000 µm.
Worked examples
Example 1. A drawing of a cell is 60 mm long. The real cell is 30 µm long. Calculate the magnification.
Solution
Convert to the same unit: 60 mm = 60 000 µm. Magnification = image ÷ actual = 60 000 ÷ 30 = ×2000.
Example 2. Ten eyepiece graticule divisions match 4 stage micrometer divisions, each 10 µm. A cell measures 25 graticule divisions. Find its length.
Solution
10 graticule divisions = 4 × 10 = 40 µm, so 1 division = 4 µm. Cell length = 25 × 4 = 100 µm.
Practice questions
Define magnification and resolution.
Show answer
Magnification: how many times larger the image is than the real object (image size ÷ actual size). Resolution: the ability to distinguish two close points as separate; it determines the detail visible.
Common mistake: Treating them as the same thing.
Give four differences between light and electron microscopes.
Show answer
Light: uses light and glass lenses, living specimens possible, colour, low resolution (0.2 µm), magnification up to ×1500, cheap. Electron: electron beam and electromagnets, vacuum so dead specimens, black and white, high resolution (about 1 nm), magnification over ×500 000, expensive.
Common mistake: Saying electron microscopes use glass lenses.
Distinguish between TEM and SEM.
Show answer
TEM: electrons pass through very thin sections, showing internal structures in 2D, higher resolution. SEM: electrons scan and reflect off the surface, giving 3D images of surfaces.
Common mistake: Saying SEM shows inside cells.
Calculate the actual size of a cell whose image is 36 mm at ×600.
Show answer
36 mm = 36 000 µm; actual = 36 000 ÷ 600 = 60 µm.
Common mistake: Forgetting to convert units.
Explain how an eyepiece graticule is calibrated.
Show answer
Place a stage micrometer on the stage; line up the scales; count how many graticule divisions equal a known number of micrometer divisions; calculate the length of one graticule division in µm for that objective.
Common mistake: Using the graticule without calibration.
Describe how to prepare a temporary slide of onion epidermis.
Show answer
Peel a thin layer of epidermis, lay it flat in a drop of water or iodine on a clean slide, lower a cover slip at an angle with a needle, remove excess liquid, view first under low power.
Common mistake: Using a thick piece of onion.
State four ways of caring for a microscope.
Show answer
Carry with two hands (arm and base); clean lenses with lens tissue; start with low power; use fine focus at high power; keep covered and dry; never let the objective touch the slide.
Common mistake: Cleaning with fingers.
State three limitations of electron microscopes.
Show answer
Specimens must be dead; complex preparation may produce artefacts; very expensive; black-and-white images; large and need special rooms and skilled operators.
Common mistake: Saying they have low resolution.
Matching and drag-and-drop
Matching
Match each item in column A with the correct answer in column B. Write the letter next to each number.
Column A Column B 1. A cover slip is lowered at an angle to: a. methylene blue 2. Animal cells are commonly stained with: b. avoid trapping air bubbles 3. Electron beams are focused using: c. electromagnets 4. The scale placed in the eyepiece to measure cells is an: d. eyepiece graticule Show answer
1 → b (avoid trapping air bubbles) · 2 → a (methylene blue) · 3 → c (electromagnets) · 4 → d (eyepiece graticule)
- Bubbles hide the specimen.
- It stains nuclei blue.
- Glass would stop electrons.
- It is calibrated using a stage micrometer.
Drag and drop
Choose the correct word or phrase from the box for each gap. One word in the box is not needed.
actual size ÷ image size · the objective crushing the slide · image size ÷ actual size · ×400- An eyepiece of ×10 and an objective of ×40 give a total magnification of .
- Using only the fine focus at high power prevents .
- Magnification is calculated as .
Show answer
1: ×400 · 2: the objective crushing the slide · 3: image size ÷ actual size
- Multiply the two.
- Clearance is very small at high power.
- Both sizes must be in the same unit.
Matching
Match each item in column A with the correct answer in column B. Write the letter next to each number.
Column A Column B 1. The maximum resolution of a light microscope is about: a. 0.2 µm 2. One millimetre equals: b. 1000 nanometres 3. One micrometre equals: c. vacuum 4. Specimens for electron microscopy must be dead because they are placed in a: d. 1000 micrometres Show answer
1 → a (0.2 µm) · 2 → d (1000 micrometres) · 3 → b (1000 nanometres) · 4 → c (vacuum)
- Limited by the wavelength of light.
- 1 µm = 1000 nm.
- Viruses are measured in nanometres.
- Air would scatter electrons.
Drag and drop
Choose the correct word or phrase from the box for each gap. One word in the box is not needed.
resolution · condenser · transmission electron microscope · hand lens- The electron microscope that shows internal ultrastructure is the .
- The part that focuses light onto the specimen is the .
- The ability to distinguish two close points as separate is .
Show answer
1: transmission electron microscope · 2: condenser · 3: resolution
- Electrons pass through thin sections.
- It is below the stage.
- It limits the detail seen.