1.1.1 Viewing cells with light microscopes

Parts of a light microscope

1. A light microscope shines visible light up through a thin specimen and uses curved glass lenses to bend that light outwards, so the image reaching your eye is far larger than the object on the slide.
2. The lamp sits in the base beneath the stage and supplies the beam of light that travels upwards through the specimen.
3. The condenser is a lens under the stage that gathers light from the lamp and concentrates it onto the small area where the specimen lies.
4. The iris diaphragm is an adjustable ring in the condenser that changes how much of that light passes through, which sets the contrast of the image.
5. The stage is the flat platform the slide rests on, and it has a hole in the middle so light can pass straight through the specimen.
6. Stage clips hold the slide firmly in place so the specimen does not slip out of view while you move it.
7. The objective lenses are mounted on a rotating turret directly above the specimen, and a school microscope normally carries three, marked $\times 4$, $\times 10$ and $\times 40$.
8. The eyepiece lens sits at the top of the tube and is the lens you look down, and it is usually $\times 10$.
9. The coarse focus knob moves the stage or the tube through a large distance and brings the specimen roughly into view at low power.
10. The fine focus knob moves it through a very small distance and sharpens the image once the specimen is almost in focus.
11. Light therefore travels from the lamp, through the condenser, through the specimen, into the objective lens and then into the eyepiece lens, and each of the two lenses enlarges the image in turn.

Preparing and staining a slide

1. Light has to pass through the specimen to reach your eye, so the specimen must be thin enough to be almost transparent, ideally a single layer of cells.
2. The specimen is laid flat in the centre of a clean glass microscope slide, the rigid strip of glass that carries it under the lenses.
3. A drop of water or stain is added on top, which stops the specimen drying out and shrivelling while you look at it.
4. A cover slip is the thin square of glass placed over the specimen, and it flattens the specimen into an even layer and keeps the liquid away from the objective lens.
5. The cover slip is held at roughly $45^{\circ }$ on a mounted needle and lowered slowly, so the liquid spreads sideways and drives the air out ahead of it.
6. Air trapped under the cover slip appears as a circle with a thick dark rim, and it is easy to mistake for a cell until you notice that nothing lies inside it.
7. Most plant and animal cells are colourless and nearly transparent, so under plain light they show little more than faint grey outlines.
8. A stain is a coloured chemical that binds strongly to some structures and weakly to others, so different parts of the cell take on different depths of colour and become visible.
9. Iodine solution stains plant cytoplasm pale yellow and stains the nucleus a much darker orange-brown, which is why the nucleus of an onion cell stands out against the rest of the cell.
10. Methylene blue stains the nucleus of an animal cell dark blue, which makes the nucleus easy to pick out in a cheek cell that is otherwise almost invisible.
11. Crystal violet and eosin work in the same way and are chosen when a different structure or a different tissue needs to be picked out.
12. Staining kills the cells, so a stained slide shows the structure of a cell but never shows it carrying out a living process.

Calculating magnification

1. Magnification compares the size of the image with the size of the real object, and it has no unit because it is one length divided by another.
2. The equation is $magnification=\frac{size\; of\; image}{size\; of\; real\; object}$.
3. In symbols this is $M=\frac{I}{A}$, where $I$ is the measured size of the image and $A$ is the actual size of the object.
4. Rearranging gives $I=M\times A$ for the image size and $A=\frac{I}{M}$ for the real size.
5. The total magnification of a microscope is the eyepiece magnification multiplied by the objective magnification, so a $\times 10$ eyepiece used with a $\times 40$ objective gives $10\times 40=\times 400$.
6. Both lengths must be converted into the same unit before dividing, because a mismatch of units changes the answer by a factor of a thousand or more.
7. The conversions needed are $1mm=1000\mu m$ and $1\mu m=1000nm$.
8. Cells are normally measured in micrometres, so a typical animal cell is roughly $20\mu m$ across and a bacterial cell roughly $2\mu m$ long.
9. Very small measurements are written in standard form to keep them manageable, so $0.002mm$ is written as $2\times 10^{-3}mm$.
10. A scale bar printed beside a photograph is a line of known real length, so measuring the bar with a ruler and dividing by the length it stands for gives the magnification of that photograph.
11. Once the magnification of a photograph is known, the real size of anything else in the same photograph is found by measuring it and dividing by that magnification.

1.1.2 Sub-cellular structures and functions

Eukaryotic and prokaryotic cells

1. Every living organism is built from cells, and all cells fall into one of two groups according to where the genetic material is kept.
2. A eukaryotic cell keeps its genetic material inside a nucleus, a compartment sealed off by its own membrane, and animals, plants and fungi are all built from eukaryotic cells.
3. A prokaryotic cell has no nucleus, so its genetic material lies loose in the cytoplasm, and bacteria are prokaryotic.
4. Eukaryotic cells are much the larger of the two, typically $10\mu m$ to $100\mu m$ across, while prokaryotic cells are typically $1\mu m$ to $5\mu m$ long.
5. A eukaryotic cell contains many membrane-bound organelles, each a separate compartment specialised for one job.
6. Dividing the cell into compartments lets different reactions run at the same time without interfering with one another, because each organelle holds the particular enzymes its own reaction needs.
7. A prokaryotic cell has no membrane-bound organelles at all, so its reactions take place in the cytoplasm or at the cell membrane.
8. Both types of cell have cytoplasm, a cell membrane and ribosomes, because every cell has to hold its contents together, control what crosses its surface and make its own proteins.

Where the genetic material is kept

1. The nucleus of a eukaryotic cell is enclosed by its own membrane and is usually the largest structure that can be picked out under a light microscope.
2. Inside the nucleus the DNA is coiled into long structures called chromosomes, and a human body cell holds $46$ of them arranged in $23$ pairs.
3. Each chromosome carries many genes, and a gene is a length of DNA that codes for one protein.
4. The nucleus controls the activities of the cell because the proteins built from those instructions include the enzymes that decide which reactions the cell can carry out.
5. Enclosing the DNA in a membrane keeps it apart from the reactions going on in the surrounding cytoplasm, which protects it from damage.
6. A prokaryotic cell instead has a single circular loop of DNA lying free in the cytoplasm, and this loop does the job the chromosomes do in a eukaryotic cell.
7. Many prokaryotic cells also carry plasmids, which are small extra rings of DNA kept separate from the main loop.
8. A plasmid usually carries genes that are only useful in particular conditions, such as genes giving resistance to an antibiotic.
9. Plasmids can be copied and passed from one bacterium to another, so a useful gene can spread through a population of bacteria quickly.

Mitochondria, chloroplasts and ribosomes

1. Mitochondria are rod-shaped organelles with a double membrane, and the inner one is deeply folded.
2. They hold the enzymes that catalyse the reactions of aerobic respiration, which is the process that transfers energy from glucose for the cell to use.
3. The folds of the inner membrane give a large surface area for those enzymes, so more respiration can take place inside the same small volume.
4. Cells that use a great deal of energy contain far more mitochondria, so a muscle cell holds thousands while a fat-storage cell holds very few.
5. The number of mitochondria in a cell is therefore a direct clue to how much energy that cell demands.
6. Chloroplasts are found in plant and algal cells and are the site of photosynthesis.
7. Each chloroplast contains chlorophyll, the green pigment that absorbs the light energy used to build glucose from carbon dioxide and water.
8. The chlorophyll is held on stacks of internal membranes, which spreads it out so that as much light as possible is absorbed.
9. Chloroplasts are found only in the plant cells that receive light, so leaf cells are packed with them and root cells have none.
10. Ribosomes are the smallest of these organelles and are the site of protein synthesis.
11. At a ribosome, amino acids are joined together in the order coded by a gene, and the chain that results is a protein.
12. Ribosomes are found in every cell, prokaryotic cells included, because every cell has to build its own enzymes and other proteins.
13. Some ribosomes float free in the cytoplasm while others are attached to membranes, and a cell that secretes a lot of protein carries very large numbers of them.

The cell membrane and its receptors

1. The cell membrane surrounds every cell, holding the cytoplasm and organelles inside and separating them from the surroundings.
2. It is built from a double layer of lipid molecules with proteins set into it, which is why it is flexible rather than rigid.
3. The membrane acts as a selective barrier because it is partially permeable, letting some substances through while holding others back.
4. Small molecules such as oxygen, carbon dioxide and water cross it easily, while large molecules such as starch and proteins, and charged ions, do not cross freely.
5. Controlling what crosses the surface lets the cell hold its internal conditions steady even when the surroundings change, and steady conditions are what keep its enzymes working.
6. The membrane also carries receptor molecules, which are proteins that project from its outer surface.
7. Each receptor has a shape that matches one particular molecule, so only that molecule can bind to it.
8. When the matching molecule binds, the receptor sets off a change inside the cell, which is how a cell detects a chemical signal and responds to it.
9. Different cells carry different receptors, so a hormone carried in the blood past every cell in the body changes the behaviour of only the cells whose receptors match it.

Structures found only in plant cells

1. Cytoplasm fills both plant and animal cells, holds the organelles in place and is where most of the cell's chemical reactions happen.
2. A plant cell has three structures that an animal cell never has: a cell wall, a permanent vacuole and chloroplasts.
3. The cell wall lies outside the cell membrane and is made of cellulose, a tough carbohydrate laid down as criss-crossing fibres.
4. Those crossed fibres make the wall strong in every direction, so it stops the cell bursting when water enters and holds the plant upright.
5. The wall is fully permeable, so it plays no part in choosing what enters the cell, and that job is left entirely to the membrane beneath it.
6. The permanent vacuole is a large sac filled with cell sap, a solution of sugars and mineral ions.
7. A full vacuole pushes the cytoplasm outwards against the wall and keeps the cell firm, and a plant whose cells lose that pressure wilts.
8. The vacuole also stores the sugars and ions the cell is not using at that moment.
9. Fungal cells also have a cell wall, but it is made of chitin rather than cellulose, and a fungal cell has no chloroplasts because it does not photosynthesise.

1.1.3 Electron microscopy and resolution

Resolution and the limit of light

1. Resolution is the smallest distance between two points at which they can still be seen as two separate points instead of merging into one blur.
2. Resolution is measured as a distance, so a smaller number means a better resolution and finer detail.
3. Magnification and resolution do different jobs: magnification sets how large the image is, and resolution sets how much detail that image can hold.
4. Once two structures lie closer together than the resolution of the instrument, no extra magnification will separate them and the image simply becomes a larger blur.
5. The resolution of a light microscope is limited by the wavelength of visible light, because two points closer together than about half a wavelength cannot be told apart.
6. Visible light has wavelengths of roughly $400nm$ to $700nm$, which fixes the best resolution of a light microscope at about $200nm$.
7. That limit also caps the useful magnification, which is why a light microscope is not taken far beyond about $\times 1500$.
8. A structure smaller than $200nm$, such as a ribosome at roughly $20nm$ across, is therefore invisible to light no matter how the microscope is set up.

How an electron microscope works

1. An electron microscope replaces the beam of light with a beam of electrons.
2. The electrons are released from a heated filament called the electron gun at the top of the column and are accelerated downwards by a high voltage.
3. Electrons carry a negative charge, so the beam is focused by electromagnets rather than glass lenses, and changing the current through a magnet changes how strongly the beam is bent.
4. The whole column is held under a vacuum, because electrons collide with air particles and would be scattered off course long before reaching the specimen.
5. In a transmission electron microscope, the electrons pass straight through a specimen that has been cut into an extremely thin slice.
6. The specimen is treated with heavy metal salts, which gather on particular structures and scatter electrons strongly, in the same way that a stain builds contrast on a light microscope slide.
7. Dense parts of the specimen scatter more electrons and appear dark, while thinner or less dense parts let more electrons through and appear pale, and this difference is what forms the image.
8. The electrons that get through strike a fluorescent screen or an electronic detector, producing an image in shades of grey because electrons carry no colour information.
9. A beam of electrons behaves as though it has a far shorter wavelength than visible light, and that shorter wavelength is the reason the resolution is so much better.
10. A transmission electron microscope resolves detail down to about $0.2nm$ and magnifies usefully to well over $\times 1000000$.
11. A scanning electron microscope instead sweeps the beam back and forth across the surface of a whole specimen and builds a three-dimensional picture of that surface, at a lower resolution than a transmission instrument reaches.

What electron microscopy revealed

1. Under a light microscope a cell showed a nucleus, cytoplasm, a membrane, chloroplasts and the outlines of mitochondria, and nothing finer than that.
2. Mitochondria appeared as featureless rods, so their internal arrangement could only be guessed at.
3. The far higher resolution of the electron microscope revealed the ultrastructure of the cell, the fine internal detail that light could never separate.
4. It showed that a mitochondrion has two membranes and that the inner one is folded into shelves, which explained how the organelle packs a large surface area of respiration enzymes into a tiny volume.
5. It revealed ribosomes for the first time, as tiny dense granules lying free in the cytoplasm and attached to membranes, which showed where proteins are assembled.
6. It showed that a chloroplast contains stacks of flattened membrane sacs carrying the chlorophyll, which explained how the pigment is spread out so that as much light as possible is absorbed.
7. It showed the cell membrane as two distinct dark lines rather than one, which supported the idea of a membrane built from a double layer of lipid.
8. It revealed folded internal membrane systems that light had never shown at all, so the cytoplasm turned out to be organised rather than a featureless jelly.
9. Each of these discoveries let biologists match a structure to the job it does, and it is that link between structure and function that transformed how cells were understood.