Module 2 2.1.1 Cell Structure By Ms Cullen Organisms • All living organisms are made of cells they are either: Unicellular – for example bacteria, which only consist of 1 cell or Multicellular – where the organism is made up of more than 1 cell, could be as many as millions of cells. Cells under the • Identify the 2 types of generalsied cell as seen light microscope under the light microscope. • Annotate diagrams in your handbook with the function of each of the organelles. • Note differences between the 2 types of generalised cells. Cells under the light microscope How the light microscope works 1. Light passes from the bulb under the stage, through a condenser lens, then through the specimen. 2. The beam of light is focussed through the objective lens and then through the eyepiece lens. 3. There are usually 3 or 4 objective lenses with different magnifications x4, x10, x40 and x100 in an oil immersion lens. 4. The eyepiece lens is usually x10. Magnification • Magnification – to work out magnification multiply the magnification of the two lenses together. • eg. x 10 eyepiece lens and x 20 objective lens: 10 x 20 = 200 x magnification. • What would the magnification be using a x100 objective lens with a x 10 eyepiece lens? • Light microscopes can magnify up to x 1400. Resolution Resolution is how clear an image is. • Two objects which are close together viewed under the light microscope may appear as a single image and magnifying can only make this single image larger, not increase the resolution. • The maximum resolution of a light microscope is 200nm. Objects closer together than this are not seen as separate. Preparation of specimens • Staining – coloured stains are chemicals used to bind to chemicals on or in the specimen, allowing the specimen to be seen more easily. Stain Use Colours produced Methylene blue Staining living cells Dark blue nucleus, light blue cytoplasm Iodine solution Staining living plant cells Very dark blue starch grains Acidified phloroglucinol Staining lignin (substance in cell Bright Red walls of xylem vessels) Acetic orcein Staining nuclei and chromosomes Red Eosin Only stains dead cells Pink Methylene green Staining plant cell walls Green Preparation of specimens • Sectioning – process in which specimens are embedded in wax and thin sections are cut without distorting the structure of the specimen. • This is particularly useful when making sections of soft tissue eg. brain. Activity 2.1 Prepare and view specimens under a light microscope. 1. Use methylene green and iodine solution to look at onion cells 2. Prepare an unstained temporary mount of Elodea 3. Use methylene blue stain to look at your cheek cells 4. Use potato and iodine solution to prepare a slide showing graduation of staining Units of measurement To put things in perspective……….. …..a pin head is 1,000,000 nm across …. a human hair is about 80,000 nm (or 80μm) in diameter! Unit Symbol Equivalent in metres Fraction of a metre Metre m 1 one Decimetre dm 0.1 one tenth Centimetre cm 0.01 one hundredth Millimetre mm 0.001 one thousandth Micrometre μm 0.000 001 one millionth (10-6) nanometre nm 0.000 000 001 one thousand millionth (10-9) Eyepiece graticules • These are used to measure the size of a specimen. • These are microscopic rulers, 1mm long and divided into 100 divisions. • Each division is 0.01mm or 10μm. Eyepiece lens magnification Objective lens magnification Total magnification Value of 1 eyepiece division (μm) X 10 X4 X 40 25 X10 X 10 X 100 10 X 10 X 40 X 400 2.5 X 10 X 100 (oil immersion lens) X 1000 1.0 Activity 2 Use worked example on P.7 Heinemann OCR AS Biology book to work out size of amoeba Figure 1 P.6. Use the graticules and previous table (also P.7 in textbooks) to work out the size of the nucleus in your cells under the microscope. Magnification and micrographs Look at example on P. 7 in textbook Actual size = image size magnification See example fig 4 P.7 I A x M • Now have a go at Exam Q5 on blood cells Activity 2.2 Magnification and Calibration 1. Calibrate eyepiece graticule measurements with a stage micrometer’s μm 2. Calculate the size of a specimen 3. Estimate the size of microscopic structures 4. Answer Qs at end of activity and on follow up sheet 1. Count the number of divisions on the stage micrometer, that overlap with the eyepiece graticule scale. 2. The 1mm stage micrometer has 100 divisions. Therefore each division is worth 10 μm. 3. Calculate the size of the overlapping region of the stage micrometer by multiplying the number of divisions by 10 μm. Add to table. 4. Record the number of eyepiece graticule units (EPGU). 5. Divide the stage micrometer measurement by no of EPGU and this will give you the value of each of the EPGU. Objective lens used Sizes of overlapping regions Conversion Calculation of the two scales number/µ (µm ÷ Stage Number of m per EPGUs) micrometer/µm EPGUs EPGU (no. of divisions x 10µm) x4 10 40 Microscope and Biological Drawing Skills • Clear, continuous lines for your sketch, use a SHARP pencil! • NO SHADING! • Diagram should cover at least 50% of space given. • All label lines should be straight – use a ruler. • Label lines should touch the structure you are identifying. • Label lines should not have arrows on the end. The Electron Microscope Microscopy: Electron microscope • The filament acts as an electron gun emitting electrons as it is heated. • They are attracted to a positively charged electrode (anode). • The condenser & objective lenses are electromagnets that straighten the beam focusing it on the specimen Microscopy: Electron microscope • The projector lenses focus an image onto the viewing screen. • A vacuum is needed inside the chamber to avoid scattering the electrons. • Specimens must be dead. • Resolution is typically 1nm, but up to 0.20nm. The Electron Microscope Microscopy: Electron microscope • There are two types of electron microscope: • Transmission (TEM): The electron beam passes through a very carefully prepared extremely thin slice of specimen. • Scanning (SEM): The electrons are reflected from the surface of specimen. 3D images are produced. Transmission electron micrographs Scanning electron micrographs Light & electron microscopes Light Radiation used Magnification Resolution Focusing by Biological material (dead or alive?) Size Appearance Preparation of material Cost of unit Electron The development of the light microscope • Light microscopes have continued to develop with new technologies. • Fluorescent microscopes use a higher light intensity and specimens which have been treated with fluorescent chemicals or ‘dyes’. • Fluorescence is the absorption and re-radiation of light. • Light of a lower wavelength and lower energy is emitted and used to produce a magnified image. Laser Scanning Confocal Microscopy • This moves a single spot of light across a specimen, causing it to fluoresce where organelles have been treated with dye. • The emitted light is filtered through a pinhole aperture. • Only light radiated from very close to the focal plane is detected. • The focal plane is the distance that gives the sharpest focus. • Very thin sections of specimens are examined. Light from elsewhere is removed. Giving very high resolution images. • 2D images are produced but a 3D image can be produced by creating images at different focal planes. Laser Scanning Confocal Microscopy • The beam splitter is a dichroic mirror, which only reflects one wavelength of light from the laser and allows other wavelengths to pass through. • The position of the 2 apertures means the light waves from the laser follow the same path as the light waves radiated when the sample fluoresces. • This means they both have the same focal plane, hence the name, confocal. Laser Scanning Confocal Microscopy - Advantages • • • • Non-invasive. Used in diagnosis of eye diseases. Being developed for endoscopic procedures. Can be used to see distribution of molecules within cells and is therefore being used in development of new drugs. • In the future it may be used to perform virtual biopsies. https://www.youtube.com/watch?v=g5U-n4Toq60 Generalised Animal and Plant Cells under the Electron Microscope • List the organelles that can be seen in the cells under the electron microscope that could not be seen under the light microscope. Comparison of plant and animal cell ultra structure Eukaryotic cells • Eukaryote means ‘true nucleus’. • They contain a number of distinct organelles, which have membranes that allow various processes to take place inside of them. • DNA is enclosed by a nuclear membrane. • Their ribosomes have a diameter of approx 22nm and are known as 80S ribosomes. • Divided into plant cells and animal cells. • Larger than prokaryotic cells. Diameter of cell is usually around 20-40 μm. Electron micrograph: Animal cell (liver cell or hepatocyte) Electron micrograph: Plant cell (leaf palisade cell + stomata, right) Cell ultra structure • Using ‘OCR Biology’ P.12-13 and ‘Advanced Biology for You’P.32-7 - make notes & diagrams on the structure and function of the following cell organelles: NUCLEUS, NUCLEOLUS, ROUGH ENDOPLASMIC RETICULUM, SMOOTH ENDOPLASMIC RETICULUM, GOLGI APPARATUS, LYSOSOMES, CHLOROPLASTS, MITOCHONDRIA, RIBOSOMES, CENTRIOLES AND MICROTUBULES, CELLULOSE CELL WALL, PLANT VACUOLES. • Divide these into those found in both plant and animal cells and those unique to each. Cytoskeleton - Function • Provide mechanical strength to cells • Aid transport within cells • Enables cell movement Cytoskeleton • The cytoskeleton is a network of protein fibres that give the cell it’s shape and stability. • Some of the fibres known as actin filaments, like the fibres found in muscle cells, can move past each other. This allows some cells, eg white blood cells, and some organelles to move. • Other fibres known as microtubules can be used to move microorganisms through liquid, or waft liquid past a cell. Cytoskeleton • Other proteins are able to move organelles along fibres. • This is how chromosomes are moved during mitosis and how vesicles move from ER to golgi apparatus. • The proteins are called microtubule motors • They use ATP during these movements. http://thomson.fosterscience.com/Biology/UnitCellsAndCellProcesses/InteractiveCell.htm Flagella (undulipodia) and Cilia • In eukaryotes flagella (undulipodia) and cilia are the same, although undulipodia are longer. • They are hair like projections from the surface of the cell • Each one consists of a cylinder containing 9 microtubules in a circle and 2 microtubules in a central bundle. • Undulipodia and cilia move because the microtubules use ATP. Flagella (undulipodia) and Cilia • In sperm the undulipodia form the tail, allowing the whole cell to ‘swim’. • In ciliated epithelial tissue, the beating movement if the cilia moves substances across the surface of the cells eg mucus Flagella (undulipodia) and Cilia • Undulipodia usually occur in ones or twos on a cell, whereas cilia occur in large numbers on a cell. • Cilia are less than 10 μm long Prokaryotic cells • Make up prokaryotic organisms: Bacteria & Blue-Green Bacteria. • Simple cells without a nucleus or distinct (true) cell organelles. • Prokaryote means ‘before-nucleus’. • They were probably the first organisms to evolve on Earth • Contain about 0.2% of the number of genes found in eukaryotes. Bacteria: Escherichia Coli Structure of Prokaryotic Cells • They are 1-5 μm, so much smaller than eukaryote cells. • Rigid MUREIN (sometimes called peptidoglycan) strengthened cell wall (not cellulose), which maintains shape & prevents osmotic damage. • Potentially Flagella for movement. They are simple compared with eukaryotic flagella being composed of a single cylinder of protein subunits. Structure of Prokaryotic Cells • Potentially inner extensions of the cell surface membrane called MESOSOMES. These are sites of respiration by enzymes as there are no mitochondria. • Ribosomes are smaller than those found in eukaryotic cells (approx 18nm) and are known as 70S ribosomes. • A single bacterial chromosome of about 2000 genes (no nucleus) in the nucleoid. • DNA rings which can replicate separately from the main chromosome called PLASMIDS. These have been utilised in recombinant DNA technology, for example producing human Insulin. Bacterial structure Comparison of prokaryotes & eukaryotes Prokaryotes Organisms Size Nucleus? Cell walls Organelles Flagella Eukaryotes Complete end of unit Qs on worksheet.