Cell theory and microscopy

Unit 2 · Chemical and Cellular Basis of Life

The cell theory, light and electron microscopes, magnification, resolution, calculating actual size and cell fractionation.

The cell theory

Robert Hooke (1665) named "cells" when he saw the walls of dead cork cells. Leeuwenhoek saw living cells, and in the 1830s Schleiden (plants) and Schwann (animals) proposed that all living things consist of cells; Virchow (1855) added that new cells come only from existing cells. The modern cell theory:

1. All living organisms are made of one or more cells.
2. The cell is the basic unit of structure and function of life.
3. All cells arise from pre-existing cells (by cell division).

Exceptions that challenge parts of the theory include skeletal muscle fibres (multinucleate), fungal hyphae without cross-walls (coenocytic), and viruses (not cellular, and showing signs of life only inside a host). Cells are small because the surface area to volume ratio falls as a cell grows: a large cell cannot exchange materials fast enough across its surface to supply its volume.

Magnification and resolution

• Magnification is how many times larger the image is than the real object: $M = \dfrac{\text{image size}}{\text{actual size}}$.
• Resolution (resolving power) is the minimum distance between two points at which they are seen as two. Better resolution reveals more detail, and magnifying a poorly resolved image only makes it larger and blurred.

To calculate an actual size, use the formula in the form $\text{actual size} = \dfrac{\text{image size}}{M}$. Measure the image, convert units, then divide. A scale bar gives the magnification directly. Units: $1\ \text{mm} = 1000\ \mu\text{m}$ and $1\ \mu\text{m} = 1000\ \text{nm}$.

Example. A cell in a photomicrograph measures $36\ \text{mm}$ across at $\times 600$. Actual size $= 36 / 600 = 0.06\ \text{mm} = 60\ \mu\text{m}$.

Using an eyepiece graticule: the graticule scale is calibrated against a stage micrometer for each objective lens, and then it measures objects directly.

Types of microscope

	Light microscope	Transmission electron microscope (TEM)	Scanning electron microscope (SEM)
Radiation	Visible light	Electron beam	Electron beam
Maximum resolution	about $0.2\ \mu\text{m}$	about $0.1\ \text{nm}$ (practically 1 nm)	about $3-10\ \text{nm}$
Useful magnification	up to $\times 1500$	up to $\times 500,000$	up to $\times 100,000$
Specimen	Living or dead, thin	Dead, ultra-thin sections in a vacuum, stained with heavy metals	Dead, coated with gold, in a vacuum
Image	2D, colour	2D, black and white, internal detail	3D surface, black and white

The shorter wavelength of electrons (about $0.005\ \text{nm}$) compared with light (400 to 700 nm) is why electron microscopes resolve more. Their drawbacks are cost, size, the need for a vacuum (so no living specimens) and artefacts produced by preparation. Light microscopes use stains (methylene blue, iodine, safranin) to give contrast.

Cell fractionation

Organelles can be separated to study them:

1. Homogenise the tissue (blender) in a cold, buffered, isotonic solution: cold slows enzymes that would damage organelles, the buffer keeps the pH constant, and isotonic prevents osmotic bursting or shrinking.
2. Filter to remove large debris.
3. Differential centrifugation: spin at increasing speeds. Each time, the densest organelles form a pellet and the supernatant is respun at higher speed. The order of sedimentation is: nuclei, then chloroplasts/mitochondria, then lysosomes, then ER fragments and ribosomes.