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Cells and microscopy

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One of the unifying concept in biology, that is the cell, has been arrived upon in big part thanks to microscopy. Additionally, microscopes have enabled the accumulation of knowledge about the cellular components i.e. organelles. Microscopes use visible light, as well as electrons and lasers to produce images of very small specimens prepared on slides, often dissected in a specific manner and stained with dyes, some fluorescent.


You will need to know about the difference between light, transmission electron and scanning electron microscopes – LM, TEM and SEM. Both the latter (as the name suggests) use a beam of electrons, rather than light, to produce an image of the sample.


TEM uses electrons which pass through the sample, so the resulting micrograph (image) shows everything within the sample in black and white, for example organelles in a cell. SEM uses electrons which scan the sample in 3D, resulting in a coloured micrograph with 3D detail, but no components from within the sample.


In light microscopy, light does go through the sample, but the outcome depends on the thickness of the sample. For example, the plant root slice in the diagram (LM) is thin enough to be able to see through the thickness of the sample. Light would also travel freely through air but not various materials of high opacity.



When talking about microscopes, differentiating between resolution and magnification is important. In principle, it’s not hard to understand. Imagine zooming in a photo to try to see a detail. That is magnification. Now imagine the photo has a low resolution, and if you magnify it, you can only see annoying pixels. If the image had a high resolution, you would be able to see the detail clearly after zooming in. So magnifying is zooming in, while resolution is the focus power. You will need to be able to calculate actual sizes and magnifications of various drawings. The equation for that is Image size on paper = Magnification x Actual size. This gives magnification = image size on paper / actual size. “I AM” summarises it nicely in a triangle.



Staining is a key precursor to microscopy. Most samples would not register well under a microscope without some form of staining. This can also be critical to the experiment carried out. For example, we might need a stain for the cell nucleus as well as a stain for the cell fibres.

For microscopes with fluorescent wavelength filters, fluorescent stains are used. These can be bound to very specific antibodies to target specific cell types or cell organelles.



Muscle cells can be stained for their nuclei (blue) or one of the constituent proteins like actin (red). Stains of different colours and hence wavelengths are used to differentiate the various parts that we want to visualise. Advanced microscopes such as confocal microscopes that use lasers can focus on multiple points in the sample, and relay multiple wavelengths at the same time to create an impression of a section through the sample, add together the data through the multiple layers in the sample, and create complex images of specimens.



The structure of eukaryotic (having a cell nucleus) versus prokaryotic (no nucleus), animal versus plant, and of viruses differs as seen with microscopy. Certain unique features such as cell walls, chloroplasts, the presence of a nucleus or lack thereof, and size amongst other features serves in distinguishing types of cell and organelles.


For example, a chloroplast would not be expected in a cell sample of an animal that does not photosynthesise. On the other hand, both being eukaryotes, we would expect them to have a nucleus. Compared to a prokaryote such as a bacterium, this is different because prokaryotes don’t have a nucleus.


What a prokaryote like a bacterium might have in common with a eukaryote such as a plant is a cell wall. They both have one, however a fellow eukaryote to the plant cell, such as an animal cell would not have a cell wall.


A virus is much simpler and only has a few distinguishable features under electron microscopy, such as its outer protein coat, the genetic material it contains within, and attachment proteins that might resemble legs.


Investigating cells in blood smears


Simple light microscopes can be used to look at the different cell types found in blood.


Blood is fun! Blood is to body as the Thames is to London, although I sure hope slightly cleaner…


Blood is roughly split into the plasma and blood cells including erythrocytes and leucocytes (neutrophils, eosinophils, monocytes, lymphocytes). Plasma is the solution that blood cells are found in, and as such acts as their extracellular matrix. For skin cells for example, the extracellular matrix is formed of collagen, so it’s different to have it essentially a liquid like plasma. Plasma is a water solution containing proteins, sugars, clotting factors (as well as platelets involved in clotting), hormones, electrolytes, carbon dioxide and oxygen.



A blood smear a.k.a. film can be prepared for investigation by using a tiny drop of blood and smearing a microscope slide with it slowly, to form a thin layer. This is then allowed to air dry, then stained to easily identify blood cells using a microscope. Differential staining targets certain blood cells such as leucocytes. This can also identify malaria parasites, and uses a special dye called Leishman’s stain.


The prevalent erythrocytes are red blood cells/RBC (and also the most common blood cells) carrying haemoglobin around the body. Haemoglobin can bind and release oxygen and is central to aerobic respiration.



Leucocytes of varying types are white blood cells/WBC, colourless, and act in defence against infection and disease.



There are many types of white blood cell, many of which are very large compared to RBCs. Neutrophils are the most common and, alongside monocytes, digest invading cells of bacteria and fungi by engulfing them in a process called phagocytosis. The invader is engulfed, isolated in a lysosome that contains digestive enzymes, and its remains disposed off and recycled or excreted.



Monocytes also live longer and present antigens of invaders to a type of lymphocyte called a T cell for later reference should the same invader come back later in the future. Moreover, monocytes eventually leave the bloodstream to settle in a different tissue and become macrophages in charge of clearing up cell debris and further immune function.


Platelets have no nucleus and aid blood clotting. They appear as small dark spots and are much smaller than red blood cells.



Finally, lymphocytes are relatively abundant in blood but much more prevalent in the lymphatic system and take part in the adaptive immune response. Phagocytosis as carried out by neutrophils and monocytes is part of the innate immune response and as such is more generic. Lymphocytes include B cells, T cells and natural killer cells (NK cells are part of the innate immune response however).


They can make antibodies against various pathogens or abnormal cells in the body such as those in tumours, present them on their surface and find target cells, and destroy any cells which do not present the expected antigens on their surface.


Each cell in the blood carries out specific functions that are related to its structure i.e. its size, shape and components such as the presence or absence of a nucleus.


Using a haemocytometer

Counting cells is a key quantitative task e.g. establishing the concentration of red blood cells in a blood sample.


These cells can be quantified by using a special marking device called a haemocytometer under a microscope. A very small volume of the sample is pipetted under a glass slide over a marked area (the grid), and cells in a small section of the grid are counted to work out the total.



For example, if there were 9 cells in the 10x objective square visible from the grid, it could be multiplied by 16 to obtain the total number of cells found in the top right-hand square of the grid, and again by 9 to obtain the total of the whole grid. Then, the total could be further multiplied to get the total cells in the whole cultured volume. If this was a 10 microlitre cell culture sample used in the haemocytometer, we would multiply the total number of cells by, e.g. 1,500 to get the total number of cells for a 15,000 microlitre (15 ml) cell culture.

In this case, that would be 9 x 16 x 9 x 1,500 = 1,944,000 cells in the 15 ml culture. This could be expressed as (1,944,000 / 15 = 129,600) 129,600 cells per ml. An even further shorthand for this is 129,600 cells ml-1.


Flow cytometry

Automated ways of counting and characterising blood cells are critical to diagnosing conditions such as blood cancers. Flow cytometry involves the high-throughput, automated technique of placing cells from a sample in a stream of fluid and sorting them one by one to be electronically detected e.g. via a laser and recorded.



Fluorescent labels are used to facilitate the detection of blood cells. This ensures that only the target cells are counted, as they have the label.


Eukaryotic and prokaryotic cell ultrastructure


Prokaryotic cells

Prokaryotes do not have a nucleus like eukaryotes do. Their DNA is not membrane-bound, just free in the cytoplasm. The extra features of prokaryotic cells vs. eukaryotic cells you must learn are:


-the cytoplasm overall does not contain membrane-bound organelles such as mitochondria and endoplasmic reticulum


-prokaryotic ribosomes are smaller than their eukaryotic counterparts; due to their size (and the centrifugation level they separate from the cell at) they are termed 70S ribosomes; the bigger eukaryotic ribosomes are 80S


-as previously covered, and their primary defining element, they lack a nucleus; instead, their DNA is a single circular molecule freely present in the cytoplasm and not associated with any proteins such as histones in eukaryotes; however, the general area where the genetic material hangs out is termed a nucleoid


-they have a cell wall which contains a special glycoprotein called murein (also known as peptidoglycan)



Some prokaryotes also go further to have some specialised parts, some seen in the diagram:


-one or more plasmids which are also circular DNA loops but much smaller; these can be exchanged between cells or even between different species as they can carry genes for antibiotic resistance


-a capsule made of polysaccharides as their outermost layer (on top of the cell wall on top of the plasma membrane)


-one or more flagella which aid in locomotion


pilli enable cell adhesion to surfaces such as hosts or surfaces in the envrironment


-the mesosome is an indent in the cell membrane that has been controversial. It is observed under the microscope following cell fixing to the slide, but not in cells that have been frozen. Several functions were associated with it, such as DNA replication, but over time it has been shown to be an artefact of preparing the cells via fixing for microscopy. Artefacts are things that are observed and appear significant and real, but turn out to be due to the process of handling the item, and are thus not real.


Eukaryotic cells

The core components of cells are the outer membrane, the cytoplasm (substance inside which contains all other stuff) and the nucleus (contains DNA). All the other stuff is made up of various components with specific functions – these are called organelles.



The ones you must know about are:

1. Centriole
2. Nucleus
3. Mitochondria
4. Chloroplasts (plants)
5. Golgi apparatus and Golgi vesicles
6. Lysosomes
7. Ribosomes
8. Rough endoplasmic reticulum and smooth endoplasmic reticulum
9. Cell wall (plants)
10. Cell vacuole (plants)


Some organelles such as the nucleus, cell membrane, cell wall, chloroplasts and endoplasmic reticulum can be seen using light or electron microscopes. Ribosomes and other organelles are too small to be visible except with the most powerful microscopes.



The linear size of a cell can be measured under a microscope using a tiny scale called a graticule. Details on this key technique are covered in the practical skills topic.


In complex multicellular organisms, eukaryotic cells are specialised and therefore organised accordingly into tissues, organs and systems. For example, a small intestine epithelial cell which absorbs nutrients from food is part of the epithelium tissue of the small intestine organ of the digestive system.


So, what are we waiting for? Let’s delve right into these organelles.


1. Centrioles and the cytoskeleton

Cell division requires the active remodelling of the cell’s cytoskeleton. The cytoskeleton is made of multiple kinds of protein that offer it the right structure and aid during the process of division.


The centriole is a tubey spaghetti thing that aids in cell division when the duplicated chromosomes need to move into their subsequent new offspring cells from the parent cell (during mitosis).



They’re made of a special protein called tubulin because they’re tubeeeeeeeeeeeees. Why didn’t they call it spaghettulin? I guess spaghetti aren’t hollow but…


2. Nucleus

Usually it is the large rounded organelle in a cell. It has a double membrane with many pores through which materials can pass. Each cell normally has one nucleus. The main functions are cell division, replication and protein synthesis. These can be achieved due to the presence of chromosomes in the nucleus, which are made of linear DNA tightly wound up with the help of special proteins such as histones, as well as one or more nucleoli.


DNA and chromosomes may seem like completely separate things. Well, they’re not. In fact, all chromosomes are individual DNA molecules coiled and twisted around, because DNA is huge. At least in eukaryotes it is. That’s one of the first differences between eukaryotes and prokaryotes in their DNA – prokaryotes have less DNA.


Eukaryotic DNA is stored within the nucleus of each cell (apart from cells without one, e.g. red blood cells). Because of its sheer size, it must be organised well. Proteins called histones help do just that:



The nucleolus is a part of the nucleus that has many special functions including creating ribosomes.


3. Mitochondria (mitochondrion, singular)

This is basically the easily identifiable sausage-like organelle with the cool inner membrane that forms the cristae. It is the site of aerobic respiration, where most ATP is made. Look:

Mitochondria has its own DNA because back in ancient times it used to be its own simple organism before it got engulfed and started chilling with a bigger cell and then all the multicellular business happened (or was that before?) and then BAM! complex beings and stuff. ATP synthase is the enzyme that catalyses the formation of ATP, while the cristae ensure a large surface area for all these energy-producing reactions to occur.


It’s the powerhouse of the cell and mutations in its DNA can cause severe illness. So much so, that for the inheritable variety, a genetic engineering intervention has been introduced. It was covered in the news as the “three-parent” affair and involves replacing the biological mother’s faulty egg mitochondria with a donor egg with healthy mitochondria.


The main chromosomal DNA in the child’s nucleus is of the biological parents, but because the mitochondrial DNA is sourced elsewhere, it became exaggeratedly dubbed the three-parent technique. The first child to be born of this technique was a boy. As it happens, mitochondrial DNA is passed on the maternal line (that’s how genetic connections can be traced back to “mitochondrial Eve”) so the boy won’t have children with the “foreign” DNA, just in case anything might’ve gone wrong.


4. Chloroplasts (plants)

Chloroplasts contain all the substances and machinery necessary for photosynthesis. Without photosynthesis, you and I would not be here right now. Impressive. Take a closer look at this intricate organelle, the chloroplast, to whom we owe our lives:



This gooey mess whose constituent components have slightly uncommon and incredibly hard to memorise names is a chloroplast. Let’s crunch them one by one.

Thylakoid, a disc-shaped organelle which contains chlorophyll (the lovely green pigment) rhymes with Kayla+Droid. Trust me, once you get the hang of this little word you will love it. Chlorophyll is involved in capturing sunlight (and the light dependent reaction). Multiple thylakoids stack together like towers within the chloroplast. A tower is called a granum, pl. grana. This arrangement enhances the surface area available.


The stroma is the fluid-filled space which is the site of the light-independent reaction.


The outer membrane and inner membrane are selectively permeable to allow O2, CO2, glucose and certain ions through.


Features which make chloroplasts well adapted to serve their function:


1. Chloroplasts are relatively flat and so ease the diffusion of molecules coming in and going out. This is achieved by a shorter diffusion pathway.

2. Plenty of available surface area for the reaction between chlorophyll and light to take place.
Now that wasn’t so bad!


5. Golgi apparatus and Golgi vesicles

The apparatus is a stack of flattened membrane discs which receives packages of protein from the rough ER, and is involved in synthesising chemicals before they are secreted from the cell. There are 3 types of vesicle the Golgi apparatus produces:


a) exocytotic vesicles which are routed to leave the cell; upon packaging their content, they bud off and approach the plasma membrane where they fuse and release the content into the extracellular space; an example of this is antibody release

b) secretory vesicles also head out of the cell; the difference here is that they stand by until a signal arrives for them to move towards the membrane and release their content e.g. neurones secreting neurotransmitters

c) lysosomal vesicles contain proteins and ribosomes headed for lysosomes which degrade them; read on to learn more about the lysosome


6. Lysosomes

These are small vesicles of membrane that contain enzymes which take part in digestion. They look like tiny balls. Its enzymes are thus called lysozymes for example acid hydrolases or proteases which break down its waste products. The cargo includes both digestive enzymes and membrane proteins.



7. Ribosomes

My personal favourite ? Ribosomes are made of a small subunit (30S) and a large subunit (50S). Together, they form the whole ribosome which in eukaryotes is 80S. They are found on the rough ER and free within the cytoplasm, and they are the site of translation where the genetic code is used to build protein. Under the microscope within a cell, they appear as mere dots. But remember, awesome comes in small packages!



8. Rough endoplasmic reticulum and smooth endoplasmic reticulum

There are two different kinds of endoplasmic reticulum – rough and smooth endoplasmic reticulum, hence their short names rough ER and smooth ER. The roughness and smoothness business is down to ribosomes attached to the rough ER, but not to smooth ER.


Rough ERtransport system: collects, stores, packages and transports the proteins made on the ribosomes


Smooth ER – synthesis of lipids and some steroids; detoxification e.g. alcohol breakdown.
What does it look like? Well, imagine this is a bit like the inner membrane of the mitochondria, but more tightly packed.



9. Cell wall (plants)

A leaf cell is surrounded by its cell wall which is made of cellulose. The role of the cell wall is multi-fold:


1. Provides the plant with strength

2. Prevents the cell from bursting due to water flooding in by exerting pressure against the water flow

3. Gives tissues mechanical strength e.g. plants that rise high above the ground

4. Maintains the cell’s specific shape.



10. Cell vacuole (plants)

The vacuole is a very large vesicle which contains a solution of various organic and inorganic compounds. The size of the vacuole changes upon cell requirements, as the compounds within are used up or stored. Some of the functions of the vacuole, depending on cell type, include:


-storing small molecules and water
-isolating waste products or harmful substances
-maintaining cell rigidity by keeping in check hydrostatic pressure



The membrane surrounding the vacuole is called tonoplast. It controls the transport of things across it between the vacuole and the rest of the cell, via many transporter proteins. These also determine the reshaping of the vacuole as needed.


Cell plasma membranes



Membranes are made of phospholipids, which are made of lipids. Lipids are the stuff of oils, fats and waxes. They’re not water soluble which makes them good for energy storage as their presence doesn’t interfere with the amount of solutes in the cell’s water (water potential) and their structure holds a lot of chemical energy (hence why fats have more than double the amount of calories when consumed compared to either carbohydrates or proteins). Their insolubility to water also comes in handy for waterproofing purposes such as the top side of plant leaves. In animals, fat can serve as insulation to conserve heat.


Unlike proteins and carbohydrates, lipids are not polymers. Lipids which store energy are triglycerides, while those which form membranes are phospholipids. Triglycerides are formed by a molecule of glycerol with three fatty acids attached. The reaction which results in triglycerides is condensation.


Glycerol (green) + 3x fatty acids (red)
The fatty acids can be simplified in drawing:



The bonds formed (C-O) are called ester bonds. Fatty acids can be saturated or unsaturated (monounsaturated; polyunsaturated). Saturated fatty acids have all their carbon (C) atoms linked to hydrogen (H) atoms, hence saturated with hydrogen. If there is a carbon atom with a double bond to its neighbour carbon atom, then it will only have one bond to a hydrogen atom, hence it is unsaturated. If there is one double bond present, the fatty acid is monounsaturated. If there are multiple double bonds present, the fatty acid is polyunsaturated.


In phospholipids one of the fatty acids is replaced by a phosphate group.



Phospholipids have a hydrophilic (water loving) head, and hydrophobic (water repelling) tails. This results in the formation of a phospholipid bilayer (double layer), which forms the basis for the plasma membrane.



These membranes are what separate cells in the body, and enable the transfer of different chemicals between cells and with their environment. Since most chemicals in cells are water soluble, lipid membranes act as good barriers that can control the movement of different substances between cells. Only certain small molecules would be able to cross the membrane freely. Other compounds would require carriers or special channels to enable them to overcome the repelling effect between the membrane and their particular chemical state e.g. chemical charge, size, etc.


The test for lipids is the emulsion test. This test takes advantage of the property of lipids of not dissolving in water, but dissolving in ethanol (alcohol). You dissolve the sample into ethanol by shaking, then pour it into water. If milky white droplets are formed, the sample is positive for lipids.


Plasma membranes

Armed with this knowledge of lipids, as well as carbohydrates and proteins, we can now explore the structure of plasma membranes, specifically in the context of the fluid-mosaic model. Phospholipids have a hydrophilic (water loving) head, and hydrophobic (water repelling) tails. This results in the formation of a phospholipid bilayer (double layer), which forms the basis for the plasma membrane.


The name of fluid-mosaic model comes from:


Fluid = the arrangement of proteins contained in the membrane is always changing

Mosaic = the proteins present are spread around in a mosaic-like fashion.



It’s pretty isn’t it? The proteins are crucial to cell communication as well as the selective permeability of the membrane. The glycoproteins’ (sugars/carbohydrates attached to a protein) side chains act as receptors. Lipid soluble stuff such as vitamins A, D and K, as well as oxygen and carbon dioxide, can pass freely though the membrane. Cholesterol can be part of the membrane to restrict the movement of other components.


Intrinsic proteins and extrinsic proteins differ in whether they span the thickness of the plasma membrane. Intrinsic proteins a.k.a. integral proteins span the whole width, while extrinsic proteins only cover part of the membrane thickness and the rest faces either inside or outside the cell.


The main properties of molecules that determine how they may be transported across a membrane are solubility, size and charge.



Large molecules can’t cross the membrane, charged molecules also can’t, and naturally, lipid-repelling (or water-attracting) molecules can’t. Conversely, small molecules can cross the membrane barrier, alongside molecules with no charge (nonionised) as well as lipophilic (hydrophobic) molecules.
It’s important to understand the role of microvilli. These are elongations of plasma membrane which increase the surface area available for reaction or absorption.


The plasma membrane interacts a lot with other organelles including other membrane-bound organelles such as the nucleus and Golgi apparatus. The production and secretion of proteins is central to these interactions.



Proteins are synthesised based on the genetic information present in DNA in the nucleus, and manufactured by the ribosomes. At the endoplasmic reticulum site, proteins are packaged in vesicles and taken to the Golgi apparatus. The inner face is its cis face while the outer face is its trans face. Vesicles travel across the folded membranes and are secreted back into secretory vesicles to be expelled via the plasma membrane out of the cell.



This movement is enabled by motor proteins that interact with the cytoskeleton to organise things and facilitate transport.


Passive transport

Diffusion = the spread of particles from a region of higher concentration to a region of lower concentration, until the particles are evenly spread out.


Diffusion takes place when you use a spray in a room, for example. The particles in the spray move randomly, knocking each other, which results in them spreading throughout the room gradually, from high concentration to low concentration. Therefore, diffusion acts down (or along) a concentration gradient.


It is important to know what affects the rate of diffusion. These are:


1. Surface area – the greater the surface area, the faster diffusion will occur

2. Difference in concentration – the higher the difference (the steeper the gradient), the faster diffusion will take place

3. The thickness of the exchange surface – the thicker the exchange surface, the slower the rate of diffusion.


Of course there are other factors such as temperature (increased kinetic energy results in faster diffusion) and the diffusion pathway (distance). The latter is a side effect of (3.) The thickness of the exchange surface, in some respects.


In some cases, diffusion is aided by certain proteins. This is called facilitated diffusion. The responsible proteins speed up diffusion of substances which would otherwise take longer to pass through the plasma membrane. The key points about facilitated diffusion which differentiate it from active transport (which also uses proteins):


-it occurs down a concentration gradient
-it uses no metabolic energy


Two kinds of protein achieve facilitated diffusion: carrier proteins and ion channels. Carrier proteins transport substances from one side of the membrane to the other, usually by co-transport. For example, glucose is transported along with an Na+ ion.



Ion channels are proteins with gates that can be open or closed to allow or stop certain ions from entering, e.g. Na+ (sodium) and K+ (potassium) ions.



Experiments to investigate the factors that affect diffusion involve carrying out the same observable diffusion experiment under different conditions. Temperature strongly affects the rate of diffusion, increasing it as it goes up. A diffusion experiment may be carried out at room temperature, after reagents have been heated up, and after reagents have been cooled e.g. in a fridge; or using a water bath; microwave, etc. depending on reagent.


In cells, diffusion rates are affected by temperature, diffusion path length, diffusion surface area and others. For example, gas diffusion is more efficient in leaves that are thin and have a large surface area – hence many leaves do.


Active transport

A lot of molecules essential to life are too large to simply cross the plasma membrane, or even pass through protein channels embedded within. The way these are transported is by being enveloped in lipid bubbles that join with the main membrane and open up to release the content to the other side of the membrane (endocytosis). Conversely, a bubble, called vesicle, already in the cytoplasm can merge with the plasma membrane and release its content on the outside (exocytosis). This process does use energy (ATP, see next topic).



Endocytosis and exocytosis are reverse processes, involve the fusion of vesicles with the plasma membrane, and transport large amounts, hence being methods of bulk transport.


Unlike diffusion and facilitated diffusion, active transport requires energy in the form of ATP (adenosine triphosphate), and moves substances against a concentration gradient (from a lower concentration to a higher concentration). This process is essential in removing of all toxins from the body, as well as the movement of rare chemicals.


Active transport is achieved by specific carrier proteins in the plasma membrane, and relies on adequate oxygen supply (which results in ATP being available). Here’s a quick video that shows the process:



There are certain cells which carry our active transport more than others, for example in the kidney. These cells have special adaptations, such as microvilli for increased surface area, hence more carrier proteins available, as well as many mitochondria for the production of ATP.





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