Calibrating a light microscope
Measure the size of one division on the eye piece scale. This can be done with the stage micrometer or graph paper for lower magnitude microscopes. Use the size of one division to measure specimens.
A beam of electrons with a wavelength of less than 1nm is fired at the specimen to illuminate it. Electron microsopes have a magnification of up to x500,000.
TEM (transmission electron microscope) This is where a beam of electrons is fired through the specimn and focused to produce an image, similar to light microscopes but has a resolving power of 0.5 nm.
SEM (scanning electron microscope) This is where a beam of electrons is sent across the surface of the specimen and the reflected electrons are collected and an image is created. it has a lower resolving power of 3-10 nm but it creates 3d images giving information about the structure of the organisms and their appearance.
Comparison between light and electron microscopes
small Large and awkward installation
simple preparation complex preparation
no distortion of specimen Distortion of specimen
no vacuum required vacuum required
natural colours are seen artificial colouring needs to be done
up to x2000 magnification over x500,000 magnification
resolving power of 200nm resolving power of 0.5-10nm
living or dead specimens only dead specimens
Calculation for Magnification
Magnification = Size of image
Size of Specimen
Magnification and Resolution
Magnification- This is how much bigger the image is on the screen/ lense compared to the actual size of the specimen.
Resolution- This is how much the entities can be differentiated from each other. A high resolution means that you can see the different parts of the cell clearer. It is limited in light microscopes as the light from the microscopes diffracts around it.
Dry Mount- The specimen is either thin enough already, e.g a hair, or is sliced thin enough to allow light through from underneath.
Wet Mount- The specimen is placed in a fluid, e.g water or oil to be placed on the slide and a cover slip is placed over it.
Squash Slide- A wet mount is prepared but with another slide placed over the top and gently pressed down to squash the soft specimen.
Smear Slide- The edge of a slide is used to smear the liquid over the slide and a cover slide is placed over the top. A common use of this is to observe blood.
The Light Microscope
The compopund light microscope consists of two lenses (an eye pieces lens and an objective lens). The objective lens is placed near the specimin and the person looks through the eyepiece lens. This creates a high magnification that can be changed with the turret for other objective lenses or the corse and fine tuning knobs. The spcimin is illuminated by a light either under or above the spcimin depending on how opaque it is. the magnification of a light microscope is calculated by multiplying the power of the eye piece and objective lenses.
Cells do not absorb a lot of light so staining is sometimes used to colour certain parts of the cell to add greater contrast between the different cells and organelles.
Positively charged stains will be attracted to the negatively charged components in the cytosol. These include methelyene blue and crystal violet.
Negatively charged stains will be repelled by the negatively charged cytosol and will stay outside of the cell so the cell can be seen surrounded by the nigrosin or Congo red.
Differential staining: Using staining techniques to differentiate between two different organisms or different organelles within a cell of a smaple tissue.
Gram stain technique: This technique is done by adding a crystal violet stain then iodine to fix it, if it retains the stain after being washed with alcohol then it is gram positive bacteria as the gram negative only have thin cell walls so lose the stain. Then they are all given the counterstain dye, safaranin. these gram negative bacteria then appear red.
Fixing- Chemicals are added to preserve the specimens in their natural state. This is done so they can be stored for a while before they need to be used.
Sectioning- The specimens are dehydrated with alcohol and then placed in a wax so that they form a solid block that can then be procisely with a microtome.
Staining- They are treated with a stain or multiple stains so that they can be differentiated easier under the microscope.
Mounting- They are then mounted onto the slide using one of the sample preparation techniques.
They are the distortion of the specimen during the preparation stage. This includes bubbles and changes in the ultrastructure of the cell for electron microscopes. Experienced scientist can differentiate between artifacts and true structure.
Laser scanning confocal microscopy
A high intesity beam of light (laser) is shone across the specimen after hitting a dichroic mirror and passsing through an objective lense. The reflected rays reflect bach and pass through the confocal pinhole and are detected to create an image. These images are 2D but can be put with other images of the same specimen at different planes to produce 3D images. A laser is used as it has a higher illumination that normal light.
The specimen needs to be treated with a flourecent dye first for the different organelles to be seen.
This method of magnification can be used on treatments on eyes as it is non-invasive. It is also used in the develment of new drugs as it can be used to see the distribution of molecules within a cell.
Compartments for life
Metabolism is the building and breaking down reactions in the cell.
Organelles are membrane bound sections in the cytoplasm that have different envirnments for reactions to take place in the organelle as there is a membrane to allow certain materials in and out.
Some organelles are common to all eukaryotic cells:
-Cytoplasm (cytosol and cytoskeleton), Cell surface membrane /plasma membrane, Smooth endoplasmic reticulum, Rough endoplasmic reticulum, Nuceus (Nuclear envolope, nucleolus, nuclear pores), golgi apparatus, vesicles, ribosomes, centrioles, mitochondria, microtubule network.
The nucleus contains the DNA for all activities within the cell. It controls the rate of metabolism by directing the synthesis of protiens which are used for metabolism. It is the largest organelle in the cell.
It is surrounded by a double membrane known as the nuclear envolope that protects it from damage from the cytoplasm. This has pores on the edge that allow molecules in and out of the organelle. DNA is too large to leave the pores so stay inside all the time.
histones are the protein that creates the chromatin that when coils up becomes the chromosomes that can be visible before the division of a cell (mitosis).
The nucleolus is an area within the nucleus that is responsible for producing ribosomes. It is composed of RNA and proteins to produce these.
It is the organelle that contains reactions for the final stages of respiration. This produces a molecule ATP that can release energy for cell activities. Very active cells contain a lot of mitrochondria as it stores energy into usable molecules. they are more common in muscle cells ect.
The structure is two membranes, an inner and outer. The inner membrane folds to create cristae and contains the fluid interior known as the matrix. The inner membrane also contains the enzymes needed for aerobic respiration. The mitochondria also contain their own DNA known as (mt) DNA. This allows them to produce thier own enzymes and reproduce themselves.
A vesicle is a single membrane sac that carries fluids through the cell.
A lysome is a special form a vesicle that carries hydrolytic enzymes that are responsible for the breaking down of the waste materials and play a role in phagocytosis. Also they are used in cell death (apoptosis).
It is a network of fibres necessary for the shape and stability of a cell. The cytoskeleton is resonsible for cell movement and organelle movement within the cell.
It is composed of three components:
-Microtuble network- These are the tracks on which the organelles within the cell move along, especially vesicles. They are composed of the protein tubulin. Spindle fibres are composed of microtubules also. They are also used to keep the shape of the cell.
-Microfilaments- These are used in cytokenesis (the dividing of the cell into two daughter cells) and they are composed of the protein actin. They are also used for cell movement and cell contraction.
-Intermediate fibres- These give the cell mechanical strength and integrity.
They are used in the cell division as two of them position themselves at the poles of the cell (forming the centrosome) and are composed of microtubules so pull apart the chromosomes with spindle fibres. They are present in most eukaryotic cells with the exception of flowering plants and most fungi. They also are though to play a role in the positioning of cillia and flagella in cells that contain them.
Flagella and cilia
Flagella and cilia are both pretruding parts of a cell. They aren't present in all eukaryotic cells.
Flagella are longer than cilia and are used to increase the motility of the cell usually and sometimes they are used for detecting chemical changes in the environment.
Cillia are smaller but are usually present in larger numbers and can either be mobile or stationary. they are stationary in places like the nose to detect changes in the chemicals in that environment. They are mobile in places such as the trachea where they move rhymically to create a current to move particles up the trachea, away from the lungs. Also they move eggs from the ovary to the uterus similarly.
The cillia are formed in a 9+2 arrangement of microtubules. This means that they contain 2 central microtubules and 9 pairs surroundning them that slide over each other to create the movement.
The endoplasmic reticultum is composed of a membrane folded to create cistae (flattended sacs)
Smooth- This is used for the synthesis of lipids and carbohydrates and storage.
Rough- Has ribosomes bound to it that synthesis proteins. It is also responsible partly for the transport of the proteins.
They are used in protein synthesis as they create the protein itself from (m)RNA code. They are composed of RNA and are made inside the nucleolus. They can be free floating or attached to Rough ER and are not membrane bound. They can be found in the cytoplasm or in mitochondria or chloroplasts.
They have the role of packaging the proteins into vesicles, either excretary vesicles if they were made to leave teh cell or lysosomes if they were created to stay inside the cell. They have a similar stucture to the smooth ER as they have a folded membrane that creates cisternae and do not contain ribosomes.
Firstly they are sythesised in the ribosomes attached to the Rough ER.
They then move into the cisternae of the Rough ER where they are packaged into vesicles that are moved into the cis face of the golgi apparatus.
They are then processed inside the golgi apparatus and are made into transport vesicles then exit as vesicles through the trans face of the golgi apparatus.
Then they either exit the cell at the cell surface membrane as secretary vesicles and are expelled from the cell or stay in the cell as lysosomes instead.
[PLANT] Cellulose cell wall
They have a cell wall made from cellulose which is a strong complex carbohydrate that surrounds every cell of a plant. They stop pathogens from entering and create a more solid structure to support the cell and the plant itself. It is strong and rigid once the contents of the cells creates pressure inside the cell to push onto the side. This is done mostly but the vacuole of the cell.
many plant cells have large perminant vacuoles that contain the cell sap. They are surrounded my a membrane known as the tonoplast that is selectively permeable so only allows certain molecules through. They provide turgur to the cell as they have a high pressure that forces the cell membrane into the cellulose cell wall. If they appear in animal cells they only appear for a small time (transient) and are small.
They are organelles responsible for photosynthesis within the cell. They are found in the green parts of the cell, e.g stem and leaves. They have a double membrane structure with an enclosed fluid called the stroma. There are more folded membranes within the chloroplasts that create sacs known as thylakoids. These are in stacks that is collectively know as a granum. the grana are connected with membranes known as lamellae. The grana contain the chlorophyll where the photosynthesis reaction takes place. Once they create starch, the starch appears in the chloroplasts as starch grains. Chloroplasts also contain their DNA and ribosomes so can create their own proteins. The internal stucture has a large SA needed for photosynthesis.
They are the earliest forms of life on earth but still exist now in certain extreme environments and in the human body's digestive system and soil.
They are unicellular and contain slight variations on organelles that are in eukaryotic cells.
They are now known as exremophiles as they are usually found in extreme environements.
Prokaryotic cell features
The DNA is pretty much the same as in eukaryotic cells but with only one molecule of DNA supercoild to create one chromosome. The genes on the chromosome are grouped into operons that can turn on or off at any one time.
The ribosomes in prokaryotic cells are smaller (70S compared to 80S) and so are used in the formation of less complicated proteins.
Cell walls in prokaryotic cells are made from peptidoglycan (also known as murein). It is complex polymer made from sugars and amino acids.
The flagella in prokaryotic cells are not arrgaed in a 9+2 arragement. It is rotated using a molecular motar that is attached to the basal body of the cell. It uses energy from chemiosmosis, not ATP. The motor causes the hook to rotate, moving the cell.