Microscopy

• Describe in principle what a microscope and name 4 different types of microscope

  A microscope magnifies samples to be seen in closer detail. Compound Light MicroscopeTransmission Electron MicroscopeScanning Electron MicroscopeLaser Scanning Confocal Microscope
• Label a diagram of a light microscope and annotate with functions of component parts

  Arm - use to carryStage - put slide hereStage clips - hold slide in placeLight Source - illuminates objectBase - use to carryCoarse adjustment knobFine adjustment knobLenses: Scanning objective (x4) Low power objective (x10) High power objective (x40)

  
• State what "SEM" and "TEM" are abbreviations for

  Scanning Electron Microscope (SEM)Transmission Electron Microscope (TEM)
• Outline how an SEM works

  A beam of electrons is sent across the surface of the specimen and the reflected electrons are collected to form the image.
• Outline how a TEM works

  A beam of electrons is passed through the specimen and focussed to produce the image.
• Outline how a laser scanning confocal microscope works

  A single spot of light is moved across the sample (point illumination) causing flourescence from 'dyed' parts. Light from the specimen is filtered through a pinhole so only light from the focal plane giving the sharpest focus is detected.
• Draw a table comparing the use and properties of light, SEM, TEM and laser scanning confocal microscopes

  Light: Uses light radiation and glass lenses to focusMagnification x1500 - x2000Resolution 200nmNo vacuumSpecimens can be alive or deadStained with dyesViewed with the eyePhotomicrographsPossible artefacts but easily eliminatedElectron:Uses electrons from electron gun, focussed by electromagnetsMagnification - TEM 500000 - 2000000, SEM 100000-500000Resolution - TEM 0.2-0.5nm SEM 3-10nmNeed a vacuum - specimens must be deadStained with heavy metalsViewed by computer - detector?or flourescent screenElectron micrographs - B&WArtefacts are likely
• State the features of the images produced from light, SEM, TEM and laser scanning confocal microscopes

  Light: Usually light background, 2D, variety of colours, details larger than 200nm only. Large organelles only (nuclei, vacuole, chloroplasts, mitochondria) Usually observing small organisms/parts of organisms/whole cellsLaser Scanning: 2D but computer can construct 3D, black background, limited colour, 200nm resolution, usually observing stained structures within cells or whole cellsTEM: Always 2D, only grayscale unless false colour, V. high mag. to look at smaller organelles (high res.) so can be grainy, Usually observing organelles within cells or whole cellsSEM: Always 3D, outside surfaces/freeze fracture, 3D of inside too! (golgi bodies etc.) Greyscale/false colour, Organelles within/surfaces of cells, pollen, insects etc.
• Identify the type of microscope used when presented with a photomicrograph

  Light: Light background, colourTEM: False colour, V. high res.SEM: Black background, false colour, 3D looking, V. high res.Laser scaning confocal: Black background, limited colour
• Timeline showing development of microscope

  Romans experimented with glass lenses13th century invention of spectacles15th century telescope1609 Galileo Galilei first 'thing' to be called microscope (compound light)
• Suggest how the development of the microscope influenced scientific thinking and what the wider consequences of this might have been

  Allowed cells to be observed - disproved spontaneous generationAllows theories to be presented then later disproved and replaced as technology improves
• Explain how to use a light microscope to view a specimen at low and high powers

  Place slide on stage and secure with stage clipsView through the scanning objective lensUse the coarse adjustment to focusMove to the next lens and re-focusMove to the high power lens and use fine focus to gain a clear image
• Describe how to produce a temporary wet mount of living tissue

  Specimens are suspended in a liquid such as water or an immersion oil. A cover slip is placed on from an angle.
• Describe and explain the characteristics of a good slide preparation

  No artefacts, thin, transparent, clean slides & coverslips, good contrast, cells spread out with limited overlap, appropriate volume of liquid
• Explain why slide preparations need to be thin

  To allow light throughSo that individual cells can be identified
• Explain how to use a stage micrometer to work out the distance represented by the small divisions in an eyepiece graticule under 3 different objective lenses


  Line up eyepiece scale with stage units1 stage unit = 0.1mmDivide the number of stage units (converted into mm) by the number of eyepiece units they correspond to to find the length of 1 eye piece unitRepeat for other lensesSize of each division of the eye-piece graticule = distance on stage micrometer/number of divisions of eye-piece graticule
• Explain how to use a stage micrometer and eye-piece graticule to add a scale bar to a drawing

  Calculate the magnification of the drawing The scale bar should be the length of ...magnification x length it represents
• Explain how to use a stage micrometer and eye-piece graticule to calculate the size of a specimen

  Size of each division of eye-piece graticule x number of divisions = actual size of object
• Describe how to choose an appropriate number of significant figures, or decimal places to present data

  Round to the smallest number of original values
• Explain how an adjustment to the "plane of focus" can alter what is viewed within a cell


  The microscope only focuses on a thin optical slice at any one time. As a cell is 3D, different objects lie in different focal planes so can be brought in and out of focus, similarly, size can appear to change.
• Explain how a tissue slice might be misleading due to the very thin nature of the slice

  It is only a thin section of the whole tissue. Cells may appear to have no nucleus if it was outside the section or appear to be disconnected if the connecting pare is outside.
• Explain why staining is useful for light microscopy

  Many specimens are transparent. Staining creates a contrast in colour or intensity between objects and their surroundings, allowing them to be seen and identified. Stains selectively colour specific molecules so allow them to be identified as present or absent.Several different colours can be used on one specimen as they bind to different molecules and some stains are polychromatic; showing different colours with different molecules
• Describe the properties a stain needs to have to be useful for light microscopy

  Differential staining - must stain some objects and not others so that contrast is visibleIntense colour - only a very small amount of biological material but it must be visible
• Describe how to prepare a stained specimen for viewing for under light microscopy

  Fixing - preserve specimens in as near natural state as possibleSectioning - dehydrated with alcohols, placed in a wax/resin to form hard block - sliced very thinly by a microtomeStaining - multiple stains for different structuresMounting - secured to slide, coverslip on top, sealed to create permanent slide
• Name two common stains and the molecules they bind to

  Toluidine Blue - Phloem; red, Xylem; blue, DNA; blueIodine in potassium iodide - Starch; blue-black
• State the rules for biological drawings

  Sharp pencil, Clear continuous lines, No shading, Accuracy, More than half the available space, Correct mistakes with a good rubber, Include a title, Include a scale
• Produce a labelled and annotated low power tissue plan using a light microscope


  Do not draw individual cells - just the boundaries of different typesSharp pencil etc.Try to get proportions correct
• Produce a labelled and annotated high power drawing of a named number of cells using a light microscope

  Sharp pencil, no overlapping linesDraw detail
• State the magnification formula

  Actual size = Image size/Magnification
• Explain how to calculate the magnification of an image using the magnification formula

  Measure image sizeMake sure image and actual are in the same units
• Explain how to calculate the actual size of an object using the magnification formula

  Answer is in units of image size, may need converting
• State symbols used for millimetres, micrometres and nanometres

  millimetres - mmmicrometers - μmnanometres - nm
• Explain how to convert measurements from one unit into another

  mm x1000 -> μm x1000 -> nm
• Define resolution


  The ability to see two objects that are close together as separate objects allowing detail to be seen
• Define magnification

  The number of times larger an image is compared to the object, of the degree of enlargement of an image
• State the difference between magnification and resolution

  Magnification is enlargement but not necessarily increased detail. Magnification is just a number -no unitsResolution is stated as distance - the smallest distance between two objects where they can still be identified as separate
• State the resolution and useful maximum magnification of light microscopes, SEMs and TEMs

  Light microscope - 200nm, 1500-2000SEM - 3-10nm, 100,000 -500,000TEM - 0.2-0.5nm, 500,000 - 2,000,000