Types of Microscopy

Microscopy is the foundation of cell biology. Without the ability to magnify and resolve cellular structures, the detailed understanding of the ultrastructure of cells that you are required to know for OCR A-Level Biology A (module 2.1.1) would be impossible. This lesson examines the main types of microscope used in modern biology: light (optical) microscopy, transmission electron microscopy (TEM), scanning electron microscopy (SEM), and laser scanning confocal microscopy. You must understand the principles behind each technique, know the differences in their resolving power and magnification, and be able to justify which microscope is most appropriate for a given investigation.

Key Definitions:

• Magnification — how many times larger an image appears compared to the actual object.
• Resolution — the minimum distance between two points that can be distinguished as separate. A higher resolution allows finer detail to be seen.

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Why We Need Microscopes

The average eukaryotic cell is approximately 10–100 µm across. A typical mitochondrion is around 1–2 µm long, and a ribosome is roughly 20 nm in diameter. The human eye, by contrast, has a resolution of only about 0.1 mm (100 µm). This means we cannot distinguish most organelles, and certainly not individual macromolecules, without the aid of a microscope.

The limit of resolution of any microscope is determined by the wavelength of the radiation used to illuminate the specimen. This is given by the Abbe diffraction limit, approximately:

$d = \frac{\lambda}{2 \cdot NA}$d=2⋅NAλ​

where $d$d is the minimum resolvable distance, $\lambda$λ is the wavelength, and $NA$NA is the numerical aperture of the lens. The shorter the wavelength, the smaller $d$d becomes, and the higher the resolution.

• Visible light: wavelength 400–700 nm → resolution limit around 200 nm (0.2 µm).
• Electron beams: effective wavelength around 0.004 nm → theoretical resolution around 0.1 nm, though in practice around 0.2 nm for TEM.

This is why electron microscopes can resolve structures that light microscopes cannot.

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Light (Optical) Microscopy

Light microscopes pass visible light through (or reflect it from) a specimen, and glass lenses focus the rays to form a magnified image.

Key Features

• Illumination: visible light from a bulb or mirror.
• Lenses: glass convex lenses.
• Maximum magnification: around ×1,500 to ×2,000.
• Maximum resolution: around 200 nm, limited by the wavelength of visible light.
• Specimen: can be living or dead. Living tissue can be observed, making time-lapse studies of processes such as mitosis and cytoplasmic streaming possible.
• Colour: produces colour images, particularly useful when staining is employed.
• Cost: relatively inexpensive; widely available in schools, colleges, and universities.

What Can Be Seen

Light microscopy reveals the overall shape of cells, the nucleus, chloroplasts (in plant cells), large vacuoles, and in some cases mitochondria as small rods. It is not sufficient to resolve the ribosomes, endoplasmic reticulum, Golgi apparatus, lysosomes, or the cristae of mitochondria.

Exam Tip: Be explicit about the resolution of a light microscope in exam answers. If the question asks why ribosomes cannot be seen, state: "Ribosomes are approximately 20 nm in diameter, which is smaller than the resolution limit of a light microscope (around 200 nm). Therefore, they cannot be resolved."

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Transmission Electron Microscopy (TEM)

TEM was developed in the 1930s and revolutionised cell biology. A beam of electrons is accelerated through a vacuum and directed at a very thin specimen. Electrons that pass through the specimen are focused by electromagnets (which act as lenses) onto a fluorescent screen or a digital detector.

Key Features

• Illumination: a beam of electrons at around 100 kV.
• Lenses: electromagnetic lenses.
• Maximum magnification: up to around ×500,000 (some modern instruments exceed this).
• Maximum resolution: around 0.2 nm, sufficient to resolve individual organelles and even some macromolecules.
• Specimen: must be dead (placed in a vacuum), chemically fixed, dehydrated, embedded in resin, and sliced into ultra-thin sections (50–100 nm thick).
• Image: 2D, black-and-white. False colour may be added digitally afterwards.

How It Works

1. Electrons are produced by a heated tungsten filament (the cathode).
2. They are accelerated towards the anode through a potential difference.
3. The beam passes through a condenser electromagnet which focuses it onto the specimen.
4. Denser parts of the specimen (typically areas stained with heavy metals such as lead or osmium) absorb or scatter more electrons, appearing darker on the final image.
5. Electrons passing through the specimen are focused by the objective and projector electromagnets onto a fluorescent screen, where they are recorded as a photomicrograph (or electron micrograph).

Advantages and Disadvantages

Advantage	Disadvantage
Very high resolution (0.2 nm)	Cannot view living cells — must be in vacuum
Reveals ultrastructure of organelles	Complex and lengthy preparation; risk of artefacts
Very high magnification	Only thin sections can be used
Detailed internal 2D images	Image is black and white
	Extremely expensive; large, immobile equipment

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Scanning Electron Microscopy (SEM)

SEM produces detailed 3D-like images of the surface of a specimen. Instead of passing through the specimen, the electron beam is scanned across its surface in a raster pattern.

Key Features

• Illumination: a fine beam of electrons scans the specimen.
• Maximum magnification: around ×100,000 to ×200,000.
• Maximum resolution: around 3–10 nm (lower than TEM).
• Specimen: dead, dehydrated, and coated in a thin layer of a heavy metal (e.g., gold or platinum).
• Image: 3D-appearance, black-and-white (can be false-coloured digitally).

How It Works

When the primary electron beam strikes the specimen, secondary electrons are emitted from the surface. These are collected by a detector, and the number of electrons detected from each point on the surface is used to build up a greyscale image. Because the intensity varies with surface topography, the resulting image has a distinctly three-dimensional appearance.

Uses

SEM is ideal for studying the external form of cells (e.g., pollen grains, insect eyes, the surface of leaves, red blood cells) where surface texture and shape are the focus of interest.

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Laser Scanning Confocal Microscopy

Confocal microscopy is a specialised form of light microscopy that uses laser light and fluorescence to produce very sharp, high-contrast images, often of thick biological specimens.

Key Features

• Illumination: a single wavelength of laser light.
• Specimen: living or dead. The specimen is typically labelled with fluorescent dyes (fluorophores) that emit light at a particular wavelength when excited.
• Resolution: slightly better than conventional light microscopy (around 180 nm), because out-of-focus light is excluded.

How It Works

1. A laser beam is focused to a single point on the specimen.
2. Fluorescent molecules at that point are excited and re-emit light at a longer wavelength.
3. The emitted light passes back through the objective lens and through a pinhole aperture (confocal pinhole) positioned at the conjugate focal plane.
4. Only light from the exact point of focus passes through the pinhole; out-of-focus light is blocked.
5. The laser is scanned across the specimen (and through different focal depths), building up a sharp image slice by slice.
6. A computer can reconstruct a full 3D image from these optical sections.

Advantages

• Produces very high-contrast, high-resolution images compared with ordinary light microscopy.
• Allows 3D reconstruction of thick specimens (e.g., whole living cells, tissue biopsies).
• Living cells can be observed, so dynamic processes (ion movements, protein trafficking, neuronal activity) can be studied in real time.
• Multiple fluorophores can be used simultaneously to label different structures in contrasting colours.

Limitations

• Still limited by the wavelength of light — cannot resolve structures below around 180 nm.
• Fluorescent dyes can bleach (lose fluorescence) with prolonged illumination.
• Lasers can damage living cells (phototoxicity).
• Expensive and requires skilled operators.

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Comparison Tree of Microscopes

graph TD
    A[Microscopes] --> B[Light microscopes]
    A --> C[Electron microscopes]
    B --> B1[Compound light microscope]
    B --> B2[Laser scanning confocal microscope]
    C --> C1[TEM: transmission]
    C --> C2[SEM: scanning]
    B1 --> B1a[Living/dead specimens]
    B1 --> B1b[Resolution 200 nm]
    B1 --> B1c[Mag up to 1500x]
    B2 --> B2a[Living or dead]
    B2 --> B2b[Uses fluorescence and pinhole]
    B2 --> B2c[3D optical sectioning]
    C1 --> C1a[Dead, thin sections]
    C1 --> C1b[Resolution 0.2 nm]
    C1 --> C1c[2D internal ultrastructure]
    C2 --> C2a[Dead, surface-coated]
    C2 --> C2b[Resolution 3-10 nm]
    C2 --> C2c[3D surface images]

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Comparison Table of Microscope Types

Feature	Light microscope	TEM	SEM	Confocal (laser)
Illumination	Visible light	Electron beam	Electron beam	Laser (visible)
Max magnification	~×1,500	~×500,000	~×200,000	~×1,500
Max resolution	~200 nm	~0.2 nm	~3–10 nm	~180 nm
Specimen condition	Living or dead	Dead, thin section	Dead, coated	Living or dead
Image type	2D, colour	2D, black & white	3D-like, black & white	2D/3D, fluorescent colours
Cost and size	Low / small	Very high / large	Very high / large	High / large
Typical use	Basic cell morphology	Internal ultrastructure	Surface topography	3D fluorescent imaging of live cells

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Common Exam Mistakes

• Confusing magnification with resolution. A photograph can be magnified endlessly, but if resolution is exceeded, no new detail appears — it just becomes blurred. This is called empty magnification.
• Stating that TEM has higher magnification than light microscopy without mentioning resolution. Magnification alone is meaningless; it is the combination of higher magnification and higher resolution that allows TEM to reveal organelles.
• Forgetting that TEM specimens must be dead. A common error is to describe TEM being used to observe "living mitochondria". The vacuum required for the electron beam kills any cell instantly.
• Confusing SEM and TEM images. TEM images show internal cross-sections (slices), whereas SEM images show external surfaces with a 3D appearance.
• Saying that confocal microscopes use electrons. They do not — they use laser light and work on fluorescence.

Exam Tip: When asked to suggest the most appropriate microscope for a study, always consider: (i) whether the specimen needs to be alive; (ii) whether surface or internal detail is required; (iii) the size of structures to be resolved. Justify your choice using these criteria.

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Quick Recap

• Light microscopes are limited by the wavelength of visible light (resolution ~200 nm) but can observe living cells.
• TEM gives the highest resolution (~0.2 nm) and reveals internal organelle ultrastructure, but specimens must be dead and very thin.
• SEM produces detailed 3D-like surface images using secondary electrons, with a resolution around 3–10 nm.
• Laser scanning confocal microscopy uses a pinhole to exclude out-of-focus light, producing sharp 3D fluorescent images of living or dead specimens.
• Always justify microscope choice by referring to magnification, resolution, and whether the specimen can be living.

Reference: OCR A-Level Biology A (H420) specification 2.1.1 (a)–(b).