Module 2

• Microscopes produce magnified images of objects. Light microscopes use a visible light beam (400 nm - 700 nm) to produce a 2D photomicrograph.
• Resolution in light microscopes is 200 nm so a ribosome (20nm) is not distinguishable due to magnitude of the wavelength of light - two objects can only be distinguished if light waves can pass between them.
• Magnification of Light Microscopes

  Magnification is 1500x - 2000x.
• How a light microscope works?

  Light passes from bulb under the stage, through condenser lens, through specimen and is focused via objective lens before going to the eyepiece. 4 objective lenses, usually x4, x10, x40 and x100 (an oil immersion lens). The eyepiece lens magnifies the image again, usually x10.
• • Light microscopes are:
  • Cheap
  • Easy to use
  • Portable
  • Able to view living specimens
• Total magnification =Objective lens magnification x Eyepiece lens magnification
• Laser Scanning (Confocal) Microscopes

  • Laser scanning (confocal) microscopes use lasers to scan images point by point.
  • A high-resolution, high-contrast computer image is formed from pixels.
  • Laser Scanning microscopes can be used to swiftly diagnose conditions.
  • Depth selectivity allows the microscope to view specimens at different depths.
• Electron microscopes

  Electron microscopes use a beam of electrons with wavelength 0.004nm to produce much higher resolution images. Samples must be placed in a vacuum. Produce the ultrastructure of a cell.
• Features of Transmission EM:

  • Specimen is dehydrated and stained
  • Electrons pass through specimen
  • 2D black and white micrograph
  • 2 million x magnification, 0.1 nm resolution
• Features of Scanning EM

  • Specimen often coated with a metal film
  • Electrons bounce off specimen
  • 3D image formed, can be enhanced
  • 15-200,000x magnification, 20 nm resolution
• General Advantages and Disadvantages of EM

  • Produce detailed images of organelles
  • SEM specifically producing 3D images can reveal details of cellular/tissue arrangements and contours (outlines).
  • Expensive & preparation requires skill.
  • Sample MUST be in a vacuum.
• • Most biological specimens are colourless and transparent. Unstained specimens can be observed using light interference. Other specimens are stained. Some biological specimens may be distorted when trying to cut it.
  • Stains bind to the specimen, making it visible and increasing contrast.
• Differential Staining

  Differential staining is when different stains bind specific components.
• Examples of Differential Stains

  Crystal violet or methylene blue - Positively charged and bind to negatively charged materials,

  Acetic orcein - Binds DNA, dark red colour

  Eosin - Cytoplasm stains pink

  Iodine in potassium iodide - Stains cellulose yellow, starch grains blue-black

  Nigrosin and Congo red - Negatively changed therefore cannot enter the cells as cytosol repels them, creating a stained background, making the unstained cells stand out.
• Gram Staining

  • Crystal Violet - It is added and then iodine is used to fix the stain and alcohol is used to wash away any unbound stain. Gram-positive bacteria appear blue/purple as the stain is retained due to the thick peptidoglycan cell wall later absorbing the dye.
  • Safranin - Gram-negative bacteria cannot absorb crystal violet stains as their peptidoglycan cell wall is thin, so they do not retain the stain due to thinner walls. So, Safranin is used as a counterstain, turning them red.
• General Specimen preparation:

  1. Dehydration
  2. Sectioning - Embed in wax, thin section cut,  preventing distortion (specially for soft tissue)
  3. Slicing into thin sections
• • Dry mounts - when thin slices or whole specimens are viewed, with just the coverslip placed on top e.g. plant Issue or hair 
• Wet mounts - when the specimens are water is added to the specimen before lowering the. coverslip with a mounted needle to prevent air bubbles from forming. Aquatic organisms could be viewed this way.
• Squash slides - wet mounts which you then push down on the coverslip to squash the sample to ensure you have a thin layer to enable light to pass through. This is used when creating a root tip squash sample to view the chromosomes in mitosis.
• Smear slides - created using the edge of another slide to smear the sample across another slide to create a smooth, thin, even coated specimen. A cover slip is placed on top after smearing. This is used when examining blood cells in a blood sample.
• Eyepiece graticules are used to measure object sizes in eyepiece units (epu). 

  The stage graticule is used to calibrate the eyepiece graticule.
• How to Calibrate the Eyepiece graticule

  1. Line up the stage micrometre and eyepiece graticule whilst looking through the eyepiece.
  2. Count how many divisions on the eyepiece graticule fit into one division on the micrometre scale.
  3. Each division on the micrometre is 10µm which can be used to calculate what one division on the eyepiece graticule is at that current magnification.
• Magnification - The no. of times larger an image appears compared to the real specimen. Microscopes produce linear magnification. 100x magnification means the image is 100 wider and longer.
• Resolution - The ability to distinguish two points clearly and produce fine detail. Electron microscopes have better resolution as they use electron beams with smaller wavelengths whereas an optical microscope uses the wavelength of light.
• Image size = Object size x Magnification.
• Nucleus

  • Stores chromatin (instructions for making proteins) made of DNA and histones
  • Has double membrane (nuclear envelope) with fluid between them and nuclear pores.
  • Nucleolus - dense spherical structure that makes RNA and ribosomes that pass into the cytoplasm.
  • Houses genetic material.
• Ribosomes

  • Are free floating or attached to the rough endoplasmic reticulum, each containing 2 sub-units.
  • Synthesise polypeptides using mRNA (site of protein synthesis) and act as an assembly line where mRNA is used to assemble proteins from amino acids.
  • Are 20 nm in diameter
• Mitochondria

  • Double membrane (separated by fluid-filled space) and DNA loop.
  • Inner membrane folded to form cristae; centre called the matrix.
  • Site of aerobic respiration, found in animal and plant cells
  • Produces ATP (a coenzyme that transports chemical energy for metabolism) from ADP + Pi
  • Rod/spherical/branched 2-5 μm
• Chloroplasts

  • Double membrane, starch grains and DNA loop
  • Site of photosynthesis found in the palisade cells.
  • Found in plant and algae cells only
  • Thylakoids (flattened membrane sacs), a granum (stack of thylakoid) increases SA of light for photosynthesis.
  • Stroma - fluid filling the inner membrane (light independent reactions take place).
  • 4-10 μm in length
• Rough Endoplasmic Reticulum

  • Contains membranes called cisternae that provide a large surface area for ribosomes to attach
  • Is a site for protein synthesis and helps move substances around the cell
  • Ribosomes are attached
• Smooth endoplasmic reticulum

  • Contains membranes called cistrane that provide a large surface area.
  • Is a site of carbohydrate and lipid synthesis, storage and transport.
  • Has no ribosomes attached to it.
• Golgi Apparatus

  • Series of flattened membrane-bound sacs that has membranes called cisternae.
  • Creates lysosomes; vesicles come and from the golgi.
  • Adds carbohydrates, packages and transports proteins in vesicles
  • Transports, modifies and stores lipids
• Lysosomes

  • Are vesicles (Membrane-bound sacs in cells that carry many substances around cells) of membranes
  • Created by the golgi apparatus
  • Contain hydrolytic enzymes known as lysozymes
  • Hydrolyse and break down cells in apoptosis or phagocytosed bodies
• Vacuole

  • Large permanent vacuoles only found in plant cells
  • Supports the cell when turgid
  • Acts as a store of fluid, sugars, amino acids and pigments
  • Can be filled with water and can push 
• Cell wall and membrane

  • Cell walls are found in plants (cellulose) and fungi (chitin) providing mechanical support.
  • Held rigid via turgor pressure.m
  • Membranes are partially permeable barriers.
• Cilia

  • Multiple protrusions
  • Occur in large numbers on a cell.
  • Eukaryotic and Prokaryotic cells
  • In ciliated epithelium airways moves mucus upwards
• Undulipodia/flagellum

  • Single protrusions
  • Longer than cilia and only occur in only a couple of cells.
  • Called undulipodia in eukaryotic and flagellum in prokaryotes
  • Bacterial flagellum for motility and Undulipodia in human sperm using energy from ATP.
• Cytoskeleton

  • Network of protein fibres (some called microtubules made of tubulin to move microorganisms via liquid).
  • Formed of microfilaments (actin), microtubules and intermediate filaments
  • Helps anchor  and transports organelles and provide cell shape and mechanical strength
• Centrioles

  • Small tubes of protein fibres.
  • A pair next to the nucleus in animal cells and in the cells of some protoctists.
  • Tubulin subunits, forms spindle fibres (anaphase).
  • Spindles move chromosomes in nuclear division via motor proteins
  • Forms cilia or undulipodia
• How organelles work together in cells

  Organelles work together in synthesis, transport and secretion of proteins.

  1. Transcribed mRNA leaves the nucleus via nuclear pores and attaches to the ribosome at the RER.
  2. Ribosome translates into polypeptide, which enters RER and then is ‘pinched off’ into vesicles.
  3. Vesicles from cisternae take protein to the golgi apparatus.
  4. Golgi modifies and packages protein and releases it into vesicles bound for destination, such as exocytosis.