Cell Structure

The first life forms on Earth existed as single cells which were essentially a string of DNA enclosed in a cell wall, not dissimilar to modern bacterial cells (prokaryotes). After millions of years, multicellular organisms started to form containing organelles which specific functions. In fact, scientists believe that organelles such as mitochondria and chloroplasts arose when eukaryotic cells engulfed a bacteria. The bacteria remained in the cell, providing the organism with a handy supply of energy.

Microscopy

The microscope was first invented over four hundred years ago and it revolutionised science. For the first time scientists were able to visualise and confirm the existence of bacteria, laying the groundwork for the germ theory of disease which saved millions of lives. The first ever microscope was essentially two lenses inside a tube which resembled an empty loo roll but since then microscopes have come a long way with incredible powers of magnification.

The light microscope is the type of microscope you’ll have used in school. It uses light to magnify objects up to 1,500x their actual size. They have a resolution of approximately 0.2 μm which isn’t large enough to visualise any of the smaller organelles, such as ribosomes and lysosomes. They are more commonly used for visualising whole cells or tissues. An advantage of light microscopy is that it can visualise living cells so we can watch behaviours such as cell division in real time.

Laser-scanning confocal microscopes are an advanced type of light microscope which use an intense beam of light (a laser) to scan samples tagged with a fluorescent dye. When the laser hits the dye, the dye emits light which can be used to form an image.

The transmission electron microscope (TEM) is more powerful than a light microscope and has a high enough resolution (around 0.0002 μm) to visualise individual organelles. A TEM uses electromagnets to focus a beam of electrons at a sample. Electrons have a much shorter wavelength compared to visible light which means higher-resolution, detailed images can be produced. A disadvantage of TEM is that the sample needs to be fixed and placed in a vacuum, which means that live cells cannot be used.

The scanning electron microscope (SEM) has a lower resolution (around 0.002 μm) than the TEM but they can produce 3D images of cells and organelles. They emit a beam of electrons towards a sample, knocking electrons off it which are used to build an image. Like TEMs, SEMs cannot be used with live cells. Both types of electron microscope are pretty big and expensive so you’ll only find them in specialised research facilities and hospitals.

Preparation of a microscope slide

• Cut a really thin layer from your sample. If the specimen is too thick, light will not be able to pass through it.

• Place a drop of water onto the microscope slide.

• Using tweezers, place your specimen onto the drop of water and place a coverslip on top.

• Add a stain if needed - place a droplet next to the coverslip and allow it to absorb across the sample.

Calibrating the eyepiece graticule and the stage micrometer

If you wanted to measure the size of your specimen, you’ll first need to align the eyepiece graticule and the stage micrometer which are little rulers which are found on the lens and the stage respectively. To do the calibration you need to carry out the following steps:

• Place the stage micrometer on the stage and focus the lens so that you can clearly see the divisions.

• Align the eyepiece graticule with the stage micrometer.

• Each division of the stage micrometer is 0.1 mm. If the eyepiece graticule spans a total of three divisions, then we know that the total length of the eyepiece graticule is 0.2 mm.

• The graticule is divided by a scale from 0 to 100, which means that each individual division is a length of 0.002 mm.

• Now we can take away the stage micrometer and add our sample, using the eyepiece graticule to measure its size.

Staining

Most biological samples are transparent and need to be stained to increase the contrast between different organelles so that they can be easily seen under the microscope. Different stains are used for different organelles: methylene blue is used to visualise DNA whereas eosin stains the cytoplasm. Iodine is often used for staining plant tissues.

Magnification

Make sure you understand the difference between magnification and resolution. Magnification is how enlarged the image is compared to the original object. Resolution is defined as how well a microscope distinguishes between two points that are close together (i.e. how much detail it can make out). Light microscopes have a much lower resolution, so produce less detailed images, compared to electron microscopes.

You can work out the magnification of a specimen viewed under a microscope using the equation:

Let’s say we magnify a 2 μm bacterial cell to form an image which is 16 cm long. The magnification we must have used is:

• Convert both into the same units. 16 cm = 160 mm = 160,000 μm

• 160,000 / 2 = 80,000 x magnification

Ultrastructure of eukaryotic cells

All organisms are divided into two different domains: eukaryotes and prokaryotes. More complex, multicellular organisms are classed as eukaryotes whereas single-celled bacteria are prokaryotes. Eukaryotic cells, such as the cells of animals, plants and fungi may contain the following organelles:

Nucleus - contains DNA which controls the activities of the cell by containing the base sequences (the ‘instructions’ needed to make proteins. The DNA is associated with histone proteins and referred to as chromatin which is wound into structures called chromosomes.

Nucleolus - this is a region within the nucleus where ribosomes are made.

Nuclear envelope - a double membrane which surrounds the nucleus. It contains pores which allows small molecules (like single stranded RNA) to pass into the cytoplasm but keeps hefty chromosomes safely inside its walls.

Rough endoplasmic reticulum (RER) - the RER is an extension of the nuclear envelope and is coated with ribosomes. It facilitates protein synthesis by providing a large surface area for ribosomes. It then transports the newly synthesised proteins to the Golgi apparatus for modification.

Smooth endoplasmic reticulum (SER) - synthesises lipids including cholesterol and steroid hormones (such as estrogen).

Golgi apparatus - made up of a group of fluid-filled membrane-bound flattened sacs surrounded by vesicles. It receives proteins from the RER and lipids from the SER. It modifies the proteins and lipids and repackages them into vesicles. The Golgi apparatus is also the site of lysosome synthesis.

Ribosomes - ribosomes are responsible for the translation of RNA into protein (protein synthesis). They either float freely in the cytoplasm or are stuck onto the rough endoplasmic reticulum.

Mitochondria - site of ATP production during aerobic respiration. It is self-replicating so can become numerous in cells with high energy requirements. It contains a double membrane with folds called cristae, which provides a large surface area for respiration.

Lysosomes - phospholipid rings which contain digestive enzymes separate from the rest of the cytoplasm. Lysosomes engulf and destroy old organelles or foreign material.

Chloroplasts - the site of photosynthesis. It is enclosed by a double membrane and has internal thylakoid membranes arranged in stacks to form grana linked by lamellae. These structures are found only in plants and certain types of photosynthesising bacteria or protoctists.

Plasma membrane - consists of a phospholipid bilayer with additional proteins to serve as carriers. It also contains cholesterol to regulate membrane fluidity. The plasma membrane contains the cell contents and holds the cell together, whilst controlling the movement of substances into and out of the cell.

Centrioles - these are bundles of microtubules which form spindle fibres during mitosis in order to pull sister chromatids apart. They are also important for the formation of cilia and flagella. They are not found in plant and bacterial cells.

Cell wall - a rigid structure made of cellulose (in plants), chitin (in fungi) and murein (in prokaryotes) which provides support to the cell.

Flagella - a tail-like structure which are made up of bundles of microtubules. The microtubules contract to make the flagellum move and propel the cell forward. Cells with a flagellum include sperm cells, which use it to swim up the fallopian tubes to fertilise the egg cell.

Cilia - finger-like projections found on the surface of some cells. These also contain bundles of microtubules which contract to make the cilia move. Cilia are found on epithelial cells lining the trachea and move to sweep mucus up the windpipe.

Middle lamella - the middle lamella is found outside of the cell walls in plant cells. It is responsible for sticking plant cells together and providing stability. It is mostly made of a substance called calcium pectate.

Amyoplasts - these are plant storage granules which contain starch and are mostly found in bulbs and tubers. Amyoplasts can convert the starch back into glucose when the plant cell needs more glucose for respiration.

Vacuole - the vacuole is an organelle which stores cell sap and may also store nutrients and proteins. It helps to keep plant cells turgid. Some vacuoles can perform a similar function to lysosomes and digest large molecules.

Tonoplast - the tonoplast is a membrane which surrounds the vacuole which functions to separate the vacuole from the rest of the cell.

Plasmodesmata - plasmodesmata are narrow channels of cytoplasm within the cell walls of plants. It allows two neighbouring plant cells to transport substances between them and to communicate.

Pits - pits are regions of a plant cell where the cell wall becomes very thin. Pits are arranged in pairs so that the pit of one plant cell is aligned with the pit of another plant cell. Like plasmodesmata, pits allow neighbouring plant cells to exchange substances.

Protein production

During the production of proteins, different organelles function like an assembly line in a factory, each tweeking the protein a little until it ends up neatly packaged in a vesicle and released from the factory floor and ready to do its job in the cell. Proteins are first made on ribosomes which are either floating alone in the cytoplasm or attached to the rough ER. The long polypeptide chain is folded at the rough ER and transported to the Golgi apparatus inside vesicles. At the Golgi, they are modified and processed by various enzymes. The protein may have a carbohydrate chain stuck onto its surface, or the addition of a sulfate or phosphate group. The dolled up protein is then placed into another vesicle which travels to the part of the cell where the protein is needed. If the protein is a carrier protein, the vesicle will deliver the protein to the plasma membrane where it will be incorporated.

Cytoskeleton

Cells contain an important structure within its cytoplasm called the cytoskeleton. It’s a miniature skeleton that gives the cell shape and keeps organelles in position. It is made up of small tubes of protein called microtubules which form a network throughout the cell. The main functions of the cytoskeleton are:

1. Provides mechanical strength to cells

2. Allows the movement of organelles within the cell

3. Enables movement of the cell

Comparing eukaryotic and prokaryotic cells

Prokaryotes have some organelles that are absent from eukaryotic cells. These include:

Pili - pili are hair-like structures which stick out from the plasma membrane. They are used to communicate with other cells (including the transfer of plasmids between bacteria).

Mesosomes - the mesosome is a folded portion of the inner membrane. While some scientists believe that it plays a role in chemical reactions, such as respiration, other scientists doubt whether it even exists and think that it may just be an artefact produced during the preparation of bacterial samples for microscopy.

Plasmids - plasmids are small, circular rings of DNA which are separate from the main chromosome. They house genes which are not crucial for survival but might prove useful - such as antibiotic-resistance genes, for example. Plasmids can replicate independently from the main chromosomal DNA.

Slime capsule - in addition to a cell wall, some bacteria also have a capsule which is made of slime. The main function of the capsule is to protect the bacterium against an immune system attack.

Cell fractionation

Cell fractionation is a technique which separates organelles according to their density - you might want to do this if you want to visualise certain organelles under the microscope separately. It involves bursting the cell surface membrane to release the organelles and spinning the cell solution at really high speeds.

Homogenisation - the first step of cell fractionation is homogenisation. This is where you break apart the plasma membrane to release the organelles. This can be done by vibrating the cells or by breaking them apart in a blender. It is important that this cells are placed into a solution which is ice-cold, isotonic and buffered.

• Ice-cold - the solution needs to be ice-cold to slow down the activity of enzymes. This is important because some enzymes will degrade organelles (such as the enzymes found inside lysosomes) so we need to reduce their activity to preserve the cell’s organelles.

• Isotonic - the solute concentration (and therefore water potential) of the solution needs to be the same as the cells that have been broken down, otherwise water would move into the organelles by osmosis, resulting in damage

• Buffered - adding a buffer to a solution ensures the pH stays constant. This is important because proteins are denatured by changes in pH - remember that proteins are a key component of various organelles.

Filtration - the homogenised solution is filtered to remove any tissue debris. The organelles are small enough to pass through the holes of the filter paper so will be present in the filtrate.

Ultracentrifugation - this is where we spin the filtrate at increasing speeds. The heaviest organelles will sink to the bottom of the test-tube, forming a pellet. We can transfer the remaining solution (the supernatant) to a separate test tube, which will be spun at a slightly higher speed. This is repeated until you obtain the organelle that you want. Remember that the organelles will be separated from the solution from the heaviest to the lightest. Nuclei will come out of the solution first, followed by mitochondria, then lysosomes, then the endoplasmic reticulum. Ribosomes will be the last organelles to form a pellet, since these are the lightest organelles in a cell.