Chapter 2


Light Microscopy and Sample Prep

Compound light microscope: 2 lenses, objective lens near sample and eyepiece lens that specimen is viewed through, objective magnifies, and eyepiece magnifies again, illumination is provided by light under sample.

Dry mount: Solid specimen viewed or cut into thin slices, put on centre of slide and cover slip placed over.

Wet mount: Specimen suspended in liquid, cover slip placed on at an angle, can view aquatic samples.

Squash slides: Wet mount prepared, lens tissue used to press down the cover slip, good for soft samples, make sure to not break cover slip when pressing down.

Smear slides: Edge of slide used to smear the sample creating thin even coating then place cover slip over, used for blood cells.

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In light microscopy the whole sample is illuminated so images have low contrast and areas are often transparent, stains increase contrast between different components as different parts take up the stain at different levels. To prepare a sample for staining you must air and then oven dry it. Crystal violet or mythylene blue are + charged dyes which are attracted to the - charged materials in the cytoplasm. Dyes like congo red are are repelled by - charged cytosol so stay outside the cell so  the cell stands out against the stained background - negative staining. 

Differential staining is used to distinguish between two types of organisms that would be difficult to otherwise. Gram staining is used to separate bacteria into two groups gram negative and gram positive. Crystal violet is first added to the bacterial specimen, then iodine to fix the dye, gram positive retain the stain so will appear blue or purple, gram negative have thinner walls so will lose the stain, and are then stained with safranin dye which will make them red. Gram positive are susceptible to penicillin whereas gram negative are not. 

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Magnification: How many times larger the image is than the actual size of the object being viewed, interchangable ojective lenses can adjust this. However magnifiying an object doesn't increase the amount of detail that can be seen, also need to increase the resolution.

Resolution: Ability to see induvidual objects as seperate entities, resolution is limited by the diffraction of light as it passes through samples, diffraction causes the light waves to spread as they pass close to physical structures, and the refracted light can overlap so the structures are no longer seen as seperate entities, resolution can be increased by using breams of electrons as their wavelength is 1000 times shorter than light. 

Magnification = size of image/actual size of object

Actual size of object = size of image/magnification

size of image = magnfication x actual sixe of object

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To measure the size of a sample under a microscope you use an yepiece graticule which is like a transparent ruler with numbes but no units and a stage micrometer which is a microscopic slide with an accurate scale and units used to work out the value of each of the divisions on the eyepiece graticule at a specific magnification. So you linr up the eyepiece graticule with the stage micrometer (each division on the micrometer is 1mm long), work out how many divisions on the graticule is the same as 1 on the micrometer and then divide those two numers to get the size of each division in mm, then you can look at the specimen to see how many divisions long it is so you can work out its actual size. 

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Electron Microscopy and Artifacts

In electron microscopy, a beam of electrons with a wavelength of under 1 nm is used to illuminmate the the cell ultrastructure and can produce images with magnifications up to x500 000 and still have a clear resolution.

TEM: Beam of electrons transmittd through a specimen and focused to produce a 2D image, resolving power of 0.5 nm.

SEM: Beam of electrons sent across surface of a specimen and the reflected electrons are collected, resolving power of 3-10 nm and produces accurate 3D images. 

The main disadvantages of electron micrsocopy are that it is very expensive, not mobile, a vacuum is needed, and sample preparation can often create artifacts as the preparation process is very complex. An artifact is a visible structural detail missing due to processing of a specimen as changes to ultrastructure are inevitable, but as techniques improve lots of these artifactst can be eliminated.

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Laser scanning confocal microscopy and Fluorescent

A laser scanning confocal microscope moves a single pinpoint spot of light across a specimen which causes fluorescence from the dyed regions, the light emiited is filtered through a pinhole aperture so only light from that specific region is deteced. Light emitted from other regions doesn't pass through the pinhole so is not detected, a lser is used for higher intensity to get better ilumination, the pinspot is moved across the specimen to produce a detailed 2D image.

By using antibodies with fluorescent tags specific features can be targeted and studied with confocal microscopy and they can be genetically modified to fluoresce different colours fro the different compononents being examined. 

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Atomic force microscopy

AFM gathers information about a specimen by 'feeling' its surface, a sharp tip on a cantilever is used to scan the surface of a specimen and when brought very close to it thr forces between the atoms cause the cantilever to deflect, which can be measured using a laser beam reflected from the top of a cantilever into a detector, fixation and staining aren't required so no damage to the specimens occurs, living systems can aslo be examined and AFM has a resolution of 0.1 nm. It can be used to identify new chemical compounds from nature and drug targets on cellular proteins in the pharmaceutical industry. 

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The Nucleus and Nucleolus

The nucleus contains the coded genetic information in the form of a DNA molecule, Dna directs the synthesis of all proteins needed for the cell, so DNA controls metabolism as it controls the synthesis of the enzymes needed for metabolic function. The nucleus is usually the largest organelle, the DNA is contained within a double membrane called a nuclear envelope to protect from damage in the cytoplasm, the envelope has nuclear pores to allow molecules to move in and out of the nucleus. DNA associates with proteins called histones to form a complex called chromatin which coils and condenses to form chromosomes (which are only present before cellular division).

The nucleolus is an area in the nuvleus respoinsible for prodcuing ribosomes and is made of proteins and RNA used to produce rRNA used to form ribosomes. 

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Mitochondria are eseential organelles in almost all eukaryotic cells that are the site of the final stages of cellular respiration where the energy stored in the bonds of ATP is made available, no. of these organelle is a reflection of its metabolic activity. Mitochondrion have a a diuble membrane structure, the inner one is highly folded to form cristae and the fluid interior is the matrix. The membrane forming the cristae contain the enzymes used in anaerobic respiration. The mitochondria also have a small amount of mtDNA meaning that they can produce thier own enzymes and reproduce themselves. 

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Vesicles and lysosomes

Vesicles are membranous sacs that have storage and transport roles and consist just of a single membrane with fluid inside, vesicles are used to transport materials inside the the cell. 

Lysosomes are specialised forms of vesicles that contain hydrolytic enzymes responsible for breaking down waste materials in cells and also play a role in breaking down pathogens that enter the cell. 

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Cytoskeleton and centrioles

The cytoskeleton is a network of fibres used for shape and stability through the cytoplasm of all eukaryotic cells, organelles are held in place by these fibres and they control cell movement. The cytoskeleton has 3 components:

Microfilaments: contractile fibres formed from the protein actin and are responsible for cell movement and cell contraction during cytokinesis.The movement of cells is determined by the rate tjat monomer subunits are attached or removed at each end of the microfilaments and whether they are added or removed is determined by the concentration of subunits at each end, and due to the different rates of addition at eother end, at specific concentrations subunits will be added at one end and removed at another - treadmilling.  

Microtubules: globular tubulin protiens that polymerise to form tubes used as scaffolding that determine the shape of the cell and aslo act as tracks for organelles like vesicles to move in the cytoplasm. 

Intermediate fibres: Give mechanical strength to cells

Centrioles are a component of the cytoskeleton (composed of microtubules) and two together formed a centrosome which is involved in spindle assembly in cell division. 

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Flagella and Cilia

Both flagella (whip like) and cilia (hair like) are extensions. Flagellaare used for cell motility or a sensory organelle, cilia can be obile or stationary. Stationary ones are used in sensory organs, moblie ones beat in a rhythmic manner creating a current to move fluids or objects adjacent to the cells. Each cilium has 2 central microtubules surrounded by nine pairs like a wheel - the 9 + 2 arrangement, pairs of microtubules slide over each other causing the cilia to beat. 

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Organelles of protein synthesis

Endoplasmic reticulum: Network of membranes enclosing flattened sacs called cisternae, smooth ER is responsible for lipid and carbohydrate synthesis and storage, rough ER has ribosomes bound to the surface and is responsible for the synthesis and trasnport of proteins.

Ribosomes: The site of protein synthesis, they can be free floating or attached to the rough ER, they aren't surrounded by a membrane and are made of rRNA made in the nucleolus.

Golgi apparatus: Similar strucutre to smooth ER, compact strucutre formed of cisternae and has a role in modifying proteins and packaging them into vesicles to either leave or stay in the cell.

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Protein production

1. Proteins are synthesised on the ribosomes bound to the rough ER.

2. They pass into its cisternae and are packaged into transport vesicles.

3. The vesicles move towards the Golgi apparatus via transport function of cytoskeleton.

4. The vesicles fuse with the cis face of the Golgi apparatus and the proteins enter and are then strucuturally modified before leaving the Golgi in vesicles from its trans face.

5. Secretory vesicles carry proteins to the cell surface membrane and fuse with it releasing their contents via exocytosis. Some vesicles form lysosomes which remain in the cell. 

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The ultrastructure of plant cells

Cellulose cell wall: plant cells have a rigid structure and have a cell wall surrounding their cell surface membrane, this wall is made of cellulose which is freely permeable but gives the cell its shape. The contents of the cell press against the cell wall making it rigid and supports the inudvidual cell and the whole plant, it also acts a defense mechanism.

Plant cell organelles:

Vacuoles are membrane lined sacs in the cytoplasm that contain the cell sap, many plant cells have a permanent vacuole which helps maintain turgor to maintain a ridig framework. The membrane  of a vacuole is called a tonoplast and is selectively permeable.

Chloroplasts are the organelles responsible for photosynthesis. They have a double membrane structure, the fluid enclosed in the chloroplasts is called the stroma, they also have an internal network of membranes which form flattened sacs called thylakoids which stack together to make a granum. The grana are joined by lamellae membranes and contain the chlorophyll pigments and are the site of light dependant reactions. Starch produced by photosynthesis are stored as starch grains. The internal membrane provides the large surface area needed for the components of photosynthesis.

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Prokaryotic cells

 Prokaryotic cells may have been the earliest form of life on earth, they are also known as extremophiles as are adapted to extreme enviroments and are mainly in the Archae domain, prokaryotic organisms are always unicellular with a simple structrue. Their DNA is supercoiled to form one chromosome. Their ribosomes are smaller (70s) and create less complex proteins. Prokaryotes have a cell wall mmade of peptidoglycan which is made of amino acids and sugars. Their flagella are thinner and don't follow the 9 + 2 arrangements, the energy used to rotate them is supplied from chemiosmosis. 

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