define resolution and magnification and explain the differences between these terms, with reference to light microscopy and electron microscopy

Microscopy in Cell Studies (Cambridge IGCSE / A‑Level)

Learning objective

Define resolution and magnification, explain how they differ, and apply these concepts to both light microscopy and electron microscopy. In addition, develop the practical skills required by the syllabus: preparing temporary mounts, calculating magnification, using an eyepiece graticule, and interpreting photomicrographs.

1. Key definitions

• Magnification (M) – the ratio of the apparent size of the image to the true size of the object.

  \$M=\frac{\text{Image size}}{\text{Object size}}\$

  In practice, total magnification = eyepiece power × objective power.

• Resolution (R) – the smallest distance between two points that can be distinguished as separate.

  For a diffraction‑limited light microscope (circular aperture):

  \$R=\frac{0.61\,\lambda}{\text{NA}}\$

  where λ is the wavelength of the illumination (≈ 550 nm for green light) and NA is the numerical aperture of the objective.

  Note: the formula assumes ideal optics; real instruments are also limited by lens quality, illumination uniformity and specimen preparation.

2. How magnification and resolution relate

• Resolution determines the finest detail that can be distinguished.
• Magnification merely enlarges whatever detail has already been resolved.
• When magnification exceeds the resolving power of the instrument the image contains no new information – this is called empty (or useless) magnification.

2.1 Realistic limits for empty magnification

For a well‑aligned school light microscope the practical resolving power is ≈ 0.2 µm. The useful magnification is roughly 1000 × resolving power, giving a maximum useful total magnification of about 200 × 1000 ≈ 200 000× in theory. In practice, most classroom microscopes are limited to 1 000–2 000× because of:

• Finite eyepiece field of view
• Aberrations in the objective lenses
• Eye‑comfort and depth‑of‑field constraints

3. Numerical aperture (NA)

• NA = n sin θ, where n is the refractive index of the medium between specimen and objective and θ is the half‑angle of the light cone collected.
• Higher NA → larger light‑gathering cone → better resolution.

4. Calculating magnification – worked example

Suppose a microscope has a 10× eyepiece and a 40× objective.

1. Total magnification = 10 × 40 = 400×.
2. If a stage micrometer has 0.01 mm divisions and, under the same objective, 5 divisions span 1 cm on the ocular grid, then:

   • Actual distance represented by 1 cm on the grid = 5 × 0.01 mm = 0.05 mm.
   • Magnification = (1 cm = 10 mm) ÷ 0.05 mm = 200×, confirming the instrument’s calibration.

5. Preparing temporary mounts (syllabus requirement 1.1)

1. Wet‑mount (live specimen)

   • Place a drop of water (or suitable liquid medium) on a clean glass slide.
   • Transfer a small piece of the specimen (e.g., onion epidermis) onto the drop.
   • Gently lower a cover‑slip, avoiding air bubbles.
   • Observe immediately – no staining required.

2. Stained temporary mount (fixed specimen)

   • Place a drop of stain (e.g., iodine, methylene blue) on the slide.
   • Add a tiny fragment of the specimen and allow the stain to act for 30 s–1 min.
   • Rinse briefly with water, add a drop of water, then cover with a cover‑slip.

6. Contrast‑enhancing techniques in light microscopy

• Bright‑field with staining – conventional method; stains absorb light, increasing contrast.
• Phase‑contrast microscopy – converts phase shifts of transparent specimens into intensity differences; ideal for live, unstained cells.
• Fluorescence microscopy – fluorophores emit light of a longer wavelength; allows specific labelling of organelles or proteins.

7. Light microscope vs. electron microscope

8. Cells as the basic units of living organisms (syllabus 1.2)

Viruses are not cells because they lack a self‑contained metabolic system, cannot grow or reproduce independently, and are composed of nucleic acid surrounded by a protein capsid (sometimes with a lipid envelope). In the syllabus they are treated separately from cellular organisms, but electron microscopy is the technique most often used to visualise viral particles (≈ 20–300 nm).

9. Interpreting photomicrographs – classroom activity

1. Provide students with a labelled photomicrograph of a stained animal cell (scale bar = 20 µm) and a plant cell (scale bar = 50 µm).
2. Task:

   • Identify and label nucleus, mitochondria, chloroplasts (plant), vacuole, cell wall, and any visible granules.
   • Measure the apparent length of the nucleus (e.g., 1.5 cm on the printed image).
   • Calculate the real size using the scale bar:

     Real size = (measured length ÷ scale‑bar length) × scale‑bar value.

   • Discuss which organelles could be seen only because the microscope’s resolution was sufficient.

10. Connecting microscopy to later topics

11. Summary

• Magnification tells how much larger an image appears; resolution tells the smallest distance that can be distinguished.
• In a light microscope, resolution is limited by the wavelength of visible light and the NA of the objective (≈ 0.2 µm); practical magnification is therefore limited to about 1 000–2 000×.
• Electron microscopes use electrons with wavelengths <0.01 nm, giving sub‑nanometre resolution and magnifications up to 10 M×, but require extensive sample preparation and specialised facilities.
• Understanding these concepts, together with slide‑preparation techniques, NA, and contrast methods, equips students to choose the appropriate microscope for a given cellular structure and to interpret the resulting images throughout the biology syllabus.