Microscopy - Optical and Electron — AQA A-level Biology

SPEC 3.2.1.3 - METHODS OF STUDYING CELLS

Microscopy, Magnification & Cell Fractionation

Optical and electron microscopes, resolution vs magnification, calculation skills, artefacts, and the full cell fractionation procedure — with mark-scheme precision

Magnification vs Resolution

Definition

Magnification: The degree to which an image is enlarged compared to the actual object.

Formula: Magnification = Image size ÷ Actual size

Definition

Resolution: The ability to distinguish two points as separate objects. The minimum distance at which two points can be seen as distinct. Also called resolving power.

Key Distinction

Magnification makes things bigger. Resolution makes things clearer. A high-magnification image that is blurry has poor resolution. Increasing magnification beyond the resolving power of the microscope just produces a larger blurry image — “empty magnification.”

Why Resolution Matters

The resolution of a microscope is limited by the wavelength of radiation used. Shorter wavelength = better resolution. Visible light (~400–700 nm) gives a resolution of ~200 nm. Electrons have a wavelength of ~0.004 nm, giving resolution ~0.1 nm — 2000× better than light.

Three Microscope Types

FeatureOptical (Light)TEMSEM

Radiation sourceVisible lightElectrons (transmitted)Electrons (reflected)

Max magnification×1,500×500,000×100,000

Resolution~200 nm~0.1 nm~1–20 nm

Image type2D; colour possible2D; black and white3D surface; black and white

SpecimenLiving or dead; stained or unstainedDead; very thin sections; heavy metal stainedDead; coated in thin layer of metal (e.g. gold)

Internal structure?Limited (large organelles only)Yes — detailed internal ultrastructureNo — surface only

Key limitationLow resolution; cannot see most organellesArtefacts possible; cannot view living specimens; expensive; time-consuming preparationSurface view only; cannot see internal structure; cannot use living material

Why Electron Microscopes Resolve Better

Resolution is limited by the wavelength of radiation used. Shorter wavelength = better resolution because shorter wavelengths diffract less around objects, allowing finer detail to be distinguished.

Examiner Alert — Resolution vs Magnification

Do NOT say “electron microscopes have higher magnification so they show more detail.” The correct reason is: electrons have a much shorter wavelength than visible light, giving much greater resolution. Resolution is the limiting factor, not magnification alone.

Magnification Calculations

Magnification = Image size ÷ Actual size | Actual size = Image size ÷ Magnification

Unit Conversions — Must Know

1 mm = 1,000 μm | 1 μm = 1,000 nm | 1 mm = 1,000,000 nm

Always convert both measurements to the same unit before calculating. The most common error is mixing mm and μm in the same calculation.

Worked Example 1

Image size = 40 mm

Actual size = 20 μm

Convert: 40 mm = 40,000 μm

Magnification = 40,000 ÷ 20

= ×2,000

Worked Example 2

Magnification = ×500,000

Image size = 50 mm

Convert: 50 mm = 50,000,000 nm

Actual size = 50,000,000 ÷ 500,000

= 100 nm

Exam Tip

Always show your unit conversion as a separate step. Even if the final answer is wrong, you can gain a method mark. Never write “mm” and “μm” in the same division — convert first.

Using the Optical Microscope — Practical Skills

AQA spec explicitly states: “Measuring the size of an object viewed with an optical microscope.” This is a required practical skill tested in Paper 1 and Paper 3.

StepActionWhy

1Place slide on stage; use low-power objective firstWider field of view; safer; reduces risk of cracking slide

2Use coarse focus, then fine focusBrings specimen into focus without damaging slide or lens

3Switch to higher power objectiveIncreases magnification to see organelles or cell detail

4Use stage micrometer to calibrate eyepiece graticuleAllows accurate measurement of object size

5Measure object in graticule units; convert using calibrationGives actual size in μm; allows magnification to be calculated

Eyepiece Graticule

A glass disc with a scale inserted into the eyepiece. The stage micrometer (a slide with a scale of known length, e.g. 1 mm = 100 divisions) is used to calibrate the graticule at each objective magnification.

Calculating Actual Size

Once calibrated: if 1 graticule unit = 10 μm and an object spans 8 units, the object is 80 μm. Then: magnification = image size (in μm) ÷ 80. Must state units.

Artefacts — A Historical Perspective

Definition

Artefact: A structure visible under a microscope that is NOT present in the living cell — it has been introduced during specimen preparation (e.g. fixation, staining, sectioning, dehydration, or metal coating).

The AQA specification explicitly states: “Students should be able to appreciate that there was a considerable period of time during which the scientific community distinguished between artefacts and cell organelles.”

How Artefacts Arise

Chemical fixation can distort membranes

Staining with heavy metals (osmium, uranyl acetate) adds electron-dense material

Dehydration and embedding in resin can shrink or distort tissue

Thin sectioning can slice through structures, producing circular profiles

Scientific Importance

Early electron microscopists could not always distinguish between genuine organelles and artefacts of preparation. Structures like the endoplasmic reticulum and ribosomes were initially debated. Establishing whether a structure is genuine requires multiple preparation methods and correlating with biochemical evidence.

Exam Point

AQA may ask you to suggest how scientists confirmed a structure was real and not an artefact. Answer: using different fixation/staining methods; isolating the structure by cell fractionation and confirming its biochemical activity; comparing with live fluorescence microscopy.

Cell Fractionation — Purpose & Principles

Definition

Cell fractionation: The process of breaking up cells and separating their organelles by differential centrifugation, allowing individual organelles to be studied in isolation.

Definition

Ultracentrifugation: Repeated centrifugation at progressively increasing speeds to separate cell organelles by size and density. Larger, denser organelles sediment (pellet) at lower speeds; smaller organelles require higher speeds.

Principle

When a suspension of broken cells (homogenate) is centrifuged, organelles are pelleted in order of decreasing size and density. The pellet contains the sedimented organelles; the remaining liquid (supernatant) is transferred to a new tube and centrifuged at a higher speed to pellet the next fraction. This is differential centrifugation.

Cell Fractionation — Buffer Conditions

Before centrifugation, cells are homogenised in a carefully prepared solution. Three specific conditions are required, each with a specific reason. AQA mark schemes require all three with their correct justifications.

ConditionWhy it is requiredCommon Wrong Answer

Ice-coldPrevents enzyme activity — specifically, prevents proteases from digesting organelle proteins and lipases from degrading membranes. Enzymes are released when cells are broken; cold temperatures reduce their activity.“To prevent damage to cells” — vague, scores zero. Must name enzyme activity or specific enzymes.

IsotonicPrevents osmotic damage to organelles. If the solution were hypotonic, water would enter organelles by osmosis causing them to swell and burst (lysis). If hypertonic, organelles would shrink (crenation).“To prevent damage to organelles” — must state osmosis/water potential and lysis specifically.

Buffered (correct pH)Maintains a constant, appropriate pH . Prevents changes in pH that would alter the charge on organelle membrane proteins, potentially denaturing them or disrupting membrane integrity.“To prevent enzymes denaturing” or “to prevent damage to organelles” — too vague. Must state maintenance of pH/protein structure.

2024 Examiner Report

The AQA 2024 Examiner Report explicitly flagged that students write “buffering prevents damage to organelles” or “buffering prevents organelles denaturing” — these are insufficient. Required: “to maintain a constant/appropriate pH to prevent changes in protein structure.”

Differential Centrifugation — Steps

After homogenisation and filtering to remove cell debris, the homogenate is centrifuged at increasing speeds. At each speed, one fraction pellets while the others remain in the supernatant.

Order of Pelleting

Nuclei (largest, densest) → Mitochondria + chloroplasts → Ribosomes + membrane fragments. Each pellet is washed and resuspended for study. The final supernatant contains soluble cytosol proteins.

Cell Fractionation — Exam Precision

The most commonly examined aspects are: (1) justifying buffer conditions, (2) ordering the fractions correctly, (3) explaining what happens if conditions are wrong.

Error 1 — Vague buffer justifications

Wrong: “isotonic to prevent damage to organelles”

Right: “isotonic to prevent osmotic lysis of organelles — if the solution were hypotonic, water would enter organelles by osmosis, causing them to burst”

Error 2 — Wrong order of fractions

Students frequently place mitochondria in the first (low-speed) pellet or ribosomes in the second pellet. Always: Pellet 1 = nuclei (low speed); Pellet 2 = mitochondria/chloroplasts (medium speed); Pellet 3 = ribosomes/ER fragments (high speed).

Error 3 — Describing “increasing speeds” to get nuclei

The 2024 AQA Examiner Report noted students wrote “increase spin speed to get nuclei” — this implies nuclei pellet last, which is wrong. Nuclei pellet at the lowest speed. Dense structures sediment first at low speed; smaller structures require progressively higher speeds.

Memory Aid

Order by size: Nuclei (largest) → Mitochondria → Ribosomes (smallest). Or: “Never Miss Ribosomes”. Speed order: Low → Medium → High.

Choosing the Right Microscope

AQA questions often ask you to justify which microscope should be used. Apply these rules:

If you need to...UseReason

View living cells or processes (e.g. cell division in real time)OpticalOnly microscope that can be used with living material

See internal organelle ultrastructure (e.g. cristae, ribosomes, nuclear pores)TEMHighest resolution (~0.1 nm); passes through specimen to show internal structure

See 3D surface features (e.g. surface of pollen grain, virus shape)SEMProduces 3D surface image; electrons reflected from surface

Measure a cell or organelle size in schoolOptical + graticulePractical and accessible; calibrated with stage micrometer

Colour imagesOpticalEM produces black and white only; colour may be added artificially

Key Synoptic Link

Cell fractionation and EM together revolutionised cell biology. Fractionation isolated organelles; EM visualised their ultrastructure. The combination of biochemical activity (fractionation) + structural evidence (EM) was needed to identify organelles as genuine, not artefacts.

DEEP Answer — Cell Fractionation Buffer

“A student homogenises liver cells to isolate mitochondria. Describe and explain the conditions required for the homogenisation solution. [6 marks]”

DEEP Framework Answer

D

DefineThe homogenisation solution must be ice-cold, isotonic, and buffered. These three conditions protect organelle integrity during the procedure. [1]

E

Explain mechanism (cold)Ice-cold prevents enzyme activity — specifically, proteases and lipases are released when cells are broken open; low temperature reduces their activity, preventing them from digesting organelle membranes and proteins. [2]

E

Explain mechanism (isotonic & buffered)Isotonic solution prevents osmotic damage: in a hypotonic solution, water would enter organelles by osmosis down the water potential gradient, causing them to swell and lyse. Buffered solution maintains a constant, appropriate pH, preventing changes in protein structure that would disrupt organelle membranes. [2]

P

Practical linkAfter homogenisation, the mixture is filtered to remove cell debris, then centrifuged at low speed (~500g) to pellet nuclei; the supernatant is centrifuged at ~10,000g to pellet mitochondria. [1]

Top Examiner Errors — Lesson 2.03

Error 1 — Confusing resolution and magnification

“Electron microscopes show more detail because they magnify more.” Wrong. The reason is: electrons have a shorter wavelength than visible light, giving greater resolution (ability to distinguish two points as separate). Magnification follows from resolution, not the other way around.

Error 2 — Vague buffer condition justifications

Saying the solution must be “cold to prevent damage” or “buffered to prevent organelles denaturing” scores zero. Precision required: cold = prevent protease/lipase activity; isotonic = prevent osmotic lysis; buffered = maintain constant pH to preserve protein structure.

Error 3 — Wrong order of pellets in centrifugation

Nuclei pellet at the lowest speed because they are the largest and densest. Ribosomes pellet at the highest speed because they are smallest. Students frequently reverse this order. Remembers: “Never Miss Ribosomes” (Nuclei → Mitochondria → Ribosomes, low → medium → high speed).

Error 4 — Missing units or wrong unit conversion

In magnification calculations, mixing mm and μm in the same equation without converting loses marks every time. Always convert to the same unit first. Forgetting to write the unit in the final answer also costs a mark.

Practice Question

A student uses cell fractionation to isolate chloroplasts from leaf cells. The cells are homogenised in a solution and the homogenate is centrifuged three times at increasing speeds.

(a) State three properties of the homogenisation solution and explain why each is necessary. [6 marks]

(b) The student centrifuges the filtered homogenate at low speed and collects the pellet. They claim this pellet contains mostly chloroplasts. Evaluate this claim. [2 marks]

(c) An image of a chloroplast taken using a TEM measures 45 mm in length. The actual length of the chloroplast is 5 μm. Calculate the magnification. Show your working and give your answer to the nearest whole number. [2 marks]

D:

(a) Ice-cold — to prevent enzyme (protease/lipase) activity; enzymes released on cell breakage would digest organelle proteins and membranes. [2] Isotonic — to prevent osmotic damage; in a hypotonic solution, water would enter organelles by osmosis, causing lysis. [2] Buffered — to maintain a constant, appropriate pH; prevents changes in protein structure and preserves organelle membrane integrity. [2]

E:

(b) The claim is incorrect [1]. At low speed (~500g), the largest and densest organelles — nuclei — pellet first. Chloroplasts (and mitochondria) require a medium speed (~10,000g) to pellet; they would still be in the supernatant after low-speed centrifugation. [1]

P:

(c) Convert 45 mm to μm: 45 mm = 45,000 μm. Magnification = 45,000 ÷ 5 = ×9,000. [2 — 1 for correct conversion, 1 for correct answer]

Summary

Microscope Comparison — Key Facts

TypeMax MagnificationResolutionImageLiving material?

Optical×1,500~200 nm2D; colourYes

TEM×500,000~0.1 nm2D; B&WNo

SEM×100,000~1–20 nm3D surface; B&WNo

Fractionation Buffer Conditions

Cold — inhibit proteases/lipases

Isotonic — prevent osmotic lysis

Buffered — maintain constant pH

Pellet Order

Low speed: Nuclei

Medium speed: Mitochondria (+ chloroplasts)

High speed: Ribosomes + ER fragments

Magnification Formula

M = Image size ÷ Actual size | Convert to same units first | 1 mm = 1,000 μm = 1,000,000 nm

Three Most Dropped Marks

(1) Writing “EM has higher magnification so shows more detail” — correct reason is shorter electron wavelength → better resolution. (2) Vague buffer justifications — must state specific consequences (protease activity; osmotic lysis; pH change). (3) Wrong pellet order — nuclei pellet FIRST at LOW speed.