Light microscopy This demo version only contains a sample of the full content Simplified Light Microscope Light Microscopes Light microscopes allow us to observe prokaryotic cells and identify large structures e.g. mitochondria in eukaryotic cells. X-ray diffraction Electron microscope Light microscope Bacteria, Blue-green algae Water molecule (1.7 Å) Amino acids proteins Human eye Eukaryotic cells viruses 10-7 10-6 10-5 10-4 Scale in mm (millimetres, 10-3 metres 10-3 0.01 0.1 1.0 10-4 1.0 10 100 1000 10-3 0.01 0.1 Scale in mm (millimetres, 10-6 metres, also called “microns”. The limit of resolution • The light microscope can be used to observe whole cells plus large internal structures e.g. nuclei, chloroplasts and mitochondria. • But the limit of resolution is about 0.2 mm. Temporary mounts • Temporary mounts are discarded after viewing. Specimens are transferred to a glass slide possibly in a droplet of water. • The specimen may need arrangement on the slide if tissues / cells overlap. • A glass cover-slip is placed over the specimen. • Excess water and water droplets are removed. Dry Tissue Staining • Most biological specimens in thin section are transparent making them all but invisible, so stains are used. • A selective/ differential stain may bind to molecules in e.g. only the nucleus. The outline of the nucleus is made clearer against the different colour of the background. • Selective staining works because of local differences e.g. pH variations, the presence of localized biomolecules. Artefacts • Specimens are prepared for microscopy with physical and chemical procedures e.g. tissue mashing that introduce changes – artefacts. Summary • A magnified image of an object is seen with the light microscope. • Specimens must be thin for light to pass through. • Differential staining improves visibility against a transparent, non-staining background. • Nucleus, nucleolus, chromosomes and chloroplasts are observable in the intact cell. • Prokaryotic cells can be observed after staining. Measuring and estimating magification Question: If the eye piece magnification is x10 and the objective Lens magnification is x40 What is the total image magnification ? 40 X 10 = 400x In the exam you may be shown an image e.g. an amoeba and told that the real length of the amoeba is 25mm The diagram on paper is 40 mm Q: what is the magnification ? Image size on paper magnification = Organism’s real size = = 40 mm 25 mm 40,000 mm 25 mm Make units same! (x mm by 1000) = X 1600 Calculating object size Typical image in a text book: A stained cheek cell showing the nucleus, X200 The actual object (real) size can be easily calculated. Step 1 Measure cell in mm. 30mm Step 2 Convert to mm for convenience (‘mm are smaller than mm, expect more of them so X 1000) = 30,000mm. To convert mm into mm, divide by 1000. • The image size, 30,000mm is 200X too large. • The actual length of the cell is: 30,000mm 200 = 150mm EPU calibration • The objective lens is calibrated using a special microscope slide called a stage micrometer. • The stage micrometer is simply a ruler. • Unlike the normal ruler measuring in mm the stage micrometer measures in micrometres (µm). Stage Micrometer scale 1mm 0 10 20 30 40 50 60 70 80 90 100 • The ruler on the stage micrometer is exactly 1mm • The 1mm line is divided into 100 equal divisions Estimating size under the microscope 1. Remove the eyepiece and unscrew the top lens. 2. Insert the eyepiece graticule (transparent disc with accurately spaced marks) in the eyepiece. Screw the top lens back in place and replace the eyepiece. eyepiece graticule Estmating size under the microscope 3. Place a graduated slide (the stage micrometer) on the stage and with the 10X objective in place, centre the marks of the slide in the field of view and focus. Graduated slide Accurate scale markings in central ring of graduated slide Estmating size under the microscope 4. Use the graduated slide to calibrate the graticule. The slide is marked in parts of a millimetre. By aligning the two scales as shown below you can work out how the divisions of the graticule correspond to micrometres at this magnification. Aligned eyepiece graticule scale with stage micrometer 5. By noting the length of an unknown structure in graticule divisions you can then convert this into absolute units of length, e.g. µm. 6. Each objective lens needs to be calibrated in the same way. Once calibrated objects can be measured in EPUs. EPUs are converted into absolute measurement using the figures obtained by the calibration. Summary • Magnification is the ratio of image to object size. • Magnification is achieved by eye piece and objective lens. • Magnification is image size divided by object size. • Object size is image size divided by magnification. • Resolution is the ability to discriminate two individual points. • In light microscopy resolution is determined by the quality of the lens and ultimately the wavelength of light.