microscopy

• microscope = an instrument employing lenses to produce a magnified image and fine detail of objects too small to see with the naked eye

  the unit for measurements of cells is usually the micrometre and for biological molecules, the nanometre (nm)

  (the metric system goes up in thousands (ignoring cm))
• magnification = how many times the size of an image is larger than the object itself, indicated by a number e.g. X40
• resolution = the degree (or smallest distance) to which it is possible to distinguish between two seperate objects which are close to each other, indicated by length e.g. 1nm

  the higher the resolution the smaller the distance that two objects can be resolved from each other and the more detail you can see
• there are 2 categories of microscope:

  • optical microscope: employ light waves
  • electron microscope: employ electron waves

  resolution of an object/feature in a microscope is limited to half of the wavelength of the wave employed. therefore the object/feature must be half the wavelength of the waves employed to be seen => light waves can only resolve objects bigger than or equal to 200nm or 0.2 micrometres
• Optical microscopes: (light microscope)

  • shines light through the speciment/object to be viewed
  • contains lenses: objective lens and eyepiece lens => object is magnified twice
  • e.g. if objective lens = X4 magnification and eyepiece lens = X10 magnification, total magnification X40
  • limit of magnification of light microscopes is 1500-2000X (dont learn specifics)
• Electron microscopes

  • same principle as light microscopes but an electron beam is used instead of light
  • lenses are magnets
  • resolution of em is much higher than a lm => far smaller detail can be seen using an em than a lm
• there are 2 types of electron microscope:

  1. transmission electron microscope (TEM)

  • tissue/cell is infiltrated with plastic resin (=> very hard) and is cut into very thin slices using a diamond knife
  • a beam of electrons is passed through exceedingly thin slices of material and produces an image on a screen. allows interior of cells to be seen
  • image only has 2 dimensional appearance
  • magnification: 500,000-2,000,000X, resolution 0.2-0.5nm (dont learn specifics)
• there are two types of electron microscope:

  2. scanning electron microscope

  • specimen is shadowed with a layer of heavy metal and covered with carbon
  • allows the surface specimens to be seen
  • except when freeze-fracture is being used where the inside of the cell is exposed)
  • it is scanned by a fine electron beam which is scattered from the surface of specimen and transmitted to a detector => image has a 3D appearance
  • magnification: 100,000-500,000X, resolution: 3-10nm (dont learn specifics)
• comparison of SEM and TEM images:

  • resolution: higher in TEM
  • magnification: higher in TEM
  • colour: black and white (both)
  • what can be viewed?: TEM: interior of cells, SEM: exterior of cells
  • appearance of image: TEM: 2D, SEM: 3D
• light microscope

  A) eyepiece

  B) course

  C) fine

  D) arm

  E) stage clips

  F) pillar

  G) base

  H) iris diaphragm

  I) iris diaphragm lever

  J) stage

  K) cover glass

  L) slide

  M) lp

  N) hp

  O) nosepiece

  P) tube

  
• preparing a temporary slide:

  1. dry mount: place coverslip to flatten object

  
• 2. a wet mount:

  
• 3. smear slides: pipitte blood into one end of slide, use second slide to spread blood across slide at an angle

  
• 4. a squash slide:

  
• preparing permanent slides:

  1. fixation: preserving material in life-like condition with minimum distortion
  2. dehydration: removing water from fixed specimen with alcohols (stop bacteria decomposing)
  3. clearing: removing dehydrating alcohols leaving specimen transparent
  4. embedding: placing in a mould with wax or resin to form a block
  5. sectioning: using a microtome to produce thin slices from the block
  6. staining colours: treating thin slices with chemical agents to 'dye' dif. structures dif. colours
  7. mounting: securing the stained slice to a slide under a coverslip
• staining for light microscopes:

  why is it necessary?

  • the interior of cells are often transparent

  staining:

  • provides contrast between components of cells e.g. organelles
  • provides contrast between a structure (e.g. cell) and its background
  • allows dif. components (e.g. organelles) of cells to be identified

  why does a stain give a dif. colour with dif. cellular structures?

  • cellular structures are composed of dif. molecules which interact differently with the specific stain
• examples of stains:

  • sudan red: lipids -> red
  • iodine: starch granules -> blue/black
  • acetic orsein: DNA/chromosomes -> red
  • toluidine blue: DNA/chromosomes -> blue
  • methylene blue: DNA/chromosomes -> blue
  • Haematoxylin: DNA/chromosomes -> blue
  • eosin: (proteins in) cytoplasm -> pink/red
• differential staining:

  • this is simply staining that uses more than one chemical stain
  • this makes differences between cells or between dif. structures within cells more visible
• calibrating a light microscope to measure leaf cells: the use of graticules and stage micrometers:

  1. a stage micrometer = a microscope slide with a scale on it measuring 10mm. each mm is divided into 10 parts. so on this stage micrometer, each small division is equal to 0.1mm = 100 micrometres (μm)
  2. line up graticule in the eyepiece lens with scale in the stage micrometer (similar to diagram on left)
• calibrating a light microscope to measure leaf cells: use of graticules and stage micrometers

  3. it is then possible to count number of divisions on the eyepiece graticule equivalent to each division on the stage micrometer and calculate length that one eyepiece division is equivalent to

  4. process should be repeated with each objective lens and you will then have a calibration factor for each lens

  • the length of 1 eyepiece graticule unit (epu) (for each lens) = distance between two divisions on the stage micrometer (100μm)/ number of eyepiece units in that distance
• use of graticules and stage micrometers (image)

  
• biological drawings:

  RULES:

  1. drawings must full more than 50% of the area given
  2. single clear lines drawn with a sharp + hard (HB, H, 2H) pencil. no sketching or broken lines
  3. complete outline of structures only, no songle lines to represent a feature
  4. no shading or colour
• biological drawings:

  RULES:

  1. correct proportions of structures: draw what you see and NOT a textbook illustration
  2. labelling: label lines in pencil, drawn with a ruler, no arrows, label lines must touch the feature, label lines must not cross each other
  3. informative title: include which lens has been used to view the slide
  4. scale or magnification must be included
  5. annotation must be included under the label (anything in physical appearance of specimen that cant be shown using rules)

  low power plan drawing of tissues and high power plan of cells:

  
• Magnification = image size / actual size