Microscopy

• Microscopy


  Instruments for viewing and studying very small structures

  
• Most cells are microscopic and thus, instruments for viewing and studying these very small structures have been developed
• Individual cells were first described using light microscopy and light microscopy has continued to play a major role in biological research
• The development of electron microscopes greatly extended the ability to resolve subcellular particles and has yielded new information on the organisation of plant and animal tissues
• Each microscopy technique is useful because it is designed to emphasise particular structural features of the cell

  
• Micrometre (µm)


  1 x 10-6 m
• Nanometre (nm)


  1 x 10-9 m
• 1m= 1,000,000 µm
• 1 µm = 1 x 10-4 cm
• 1cm= 10,000 µm
• Light microscope


  Uses light as the source of radiation
• Compound microscope


  The most common light microscope in use today, contains several lenses that magnify the image of a specimen under study

  
• Compound microscope


  • A real magnified image produced by one lens called the objective is further magnified by another- the eyepiece- which forms a final, magnified virtual image for observation by the user's eye

  
• Objective lens


  The lens used to vary the magnifying power of the microscope

  
• Eyepiece


  Lens through which the observer looks at specimen, used to project the image into the human eye
• Stage


  The surface onto which the slide containing the specimen under investigation is placed
• Diaphragm


  Found under the stage, important for adjusting the circumference of the light source
• Fine focus knob/Fine adjustment


  Used to make the image more clear
• Coarse focus knob/Coarse adjustment

  Used to find the specimen
• Condenser


  Focuses light through the specimen thus increasing illumination of the specimen
• Criteria for useful image in compound microscope

  • Adequate resolution
  • Sufficient contrast
  • Sufficient total magnification

  
• Magnification


  The amount by which the image size is greater than the object size

  
• In the compound microscope, the total magnification is the magnification of the objective lens multiplied by the magnification of the eyepiece

  
• Scale bars


  Used to determine the actual size of objects in microscope images

  
• Stereomicroscope


  Light is reflected from the surface of a specimen (rather than transmitted through specimen), two slightly different images are produced and enter each eye enabling the viewer to view specimens in 3D, usually lower magnification
• Uses of stereomicroscope

  • When specimen is too large or thick (opaque = not transparent)
  • Specimen handling is required e.g. identification of small animals
  • Dissections

  
• Resolution


  The ability of a microscope to distinguish between two very closely positioned objects

  
• The resolution of the electron microscope is much greater than that of the light microscope
• The electron microscope is not better than the light microscope, they are used for different jobs
• Staining


  Using dyes/coloured pigments to stain specific parts of the cell individually
• Advantages of light microscope

  • Preparation of specimens is quick and simple
  • Both living and non-living specimens can be observed
  • The specimen remains intact when viewed so once a glass slide is prepared it can be kept and viewed over again
  • It is less expensive to buy and to run than the electron microscope
• Preparing temporary slides for light microscopy

  1. Fixation

  2. Staining

  3. Mounting

  
• Fixation


  Killing the specimen rapidly using a fixative to preserve the specimen in a life-like condition, maintaining the original shape and structure
• Wet mount


  Specimen is mounted in a drop of water
• Steps for viewing a specimen under the microscope

  1. Check low power objective is in position

  2. Check diaphragm is fully open

  3. Check condenser is completely racked up

  4. Place slide on stage

  5. Use low power objective to find specimen

  6. Switch to high power objective and use fine focus to obtain clear image

  
• Micrometry


  Measurement of microscopic objects by means of a microscope using a stage micrometer and eyepiece graticule

  
• Calibrating eyepiece graticule with stage micrometer

  1. Eyepiece lens is unscrewed and eyepiece graticule is inserted

  2. Stage micrometer is placed on stage and focused

  3. Eyepiece lens is rotated to match scales

  4. Number of micrometer divisions that fit in eyepiece units is counted

  5. Value of one division in eyepiece graticule is calculated

  
• The resolving power of a light microscope is limited by the wavelength of light
• Electron microscopes use a beam of electrons instead of light, providing much greater resolution power
• Electron microscopes can achieve a resolution of about 0.5nm, 500 times better than a light microscope