Microscopy

• what and when where the first microscopes developed first developed

  The first type of microscopes to be developed were light microscopes in the 16th to 17th century
• when was cell theory developed

  19th century, before then, microscopes didn't have a high enough level of magnification to allow them to see individual cells
• what does cell theory state?

  states that: -both plants and animal tissue is composed of cells -cells are the basic unit of all life -cells only develop from existing cells
• Why are microscopes important?

  Allows you to magnify objects to see individual cells in greater detail. Doing so allows us to discover how the cells structures relate to their functions
• whats a dry mount sample?

  when solid specimens are viewed whole, or cut into thin slices, this is called sectioning.
• whats are some examples of dry mount sampling?

  hair, pollen, dust and insect parts
• what is wet mount sampling?

  when specimens are suspended in a liquid like water/oil. the cover slip is placed on top from an angle to avoid air bubbles. Aquatic organisms can be viewed this way
• what is squash slide sampling?

  first a wet mount slide is prepared, then a lens tissue is used to gently press down the cover slip. Good technique to test soft samples
• 
  

  whats an example of something that can be viewed in squash sides? root tip squashes
• what are smear slides?

  an edge of a slide is used to smear the sample, creating a thin, even coating on another slide. a cover slip is then placed on top
• wht can be tested on a smear slide?

  a blood sample
• why must specimens be thin?


  so light can pass through them so details can be seen
• why must a cover slip be placed onto a wet mount at an angle?

  to prevent air bubbles from being trapped in
• whats diffraction


  the bending fo light as it passes close to the edge of an object
• which dyes are positively charged?

  Crystal violet/ methylene blue. They're attracted to negatively charged materials in cytoplasm leading to staining of cell components
• which dyes are negatively charged?

  Nigrosin/ Congo red. They're repelled by the negatively charged cytosol and so stay outside of the cell, not staining the cell which then stand out against the stained background. this is a negative stain technique
• what does differential staining do?


  Can distinguish between 2 types of organisms that would otherwise be hard to identify
• What's the Gram Staining Technique?

  Used to separate bacteria into 1. gram positive and 2. gram negative bacteria
• gram stain technique

  1. crystal violet is first applied to a bacterial specimen on a slide, then iodine, which fixes the slide 2. the slide is then washed with alcohol 3. the gram positive bacteria retain the crystal violet stain and appear blue/purple under a microscope 4. gram negative bacteria have thinner cell walls and so lose the stain
• gram staining technique 


  gram negative bacteria are then stained with safranin dye which is called a counter stain. These bacteria will then appear red
• stages in producing slides

  1. fixing- chemicals are sued to preserve specimens 2. sectioning- specimens are sliced thinly 3. staining-specimens stained to show different features 4. Mounting- a cover slip is placed on top of a microscope slide
• what's magnification?


  how many times larger the image is than the actual size of the object being viewed
• what's resolution?


  the ability to see individual objects as separate entities
• how can resolution be increased?

  By using beams of electrons which have a wavelength thousands of times shorter than light
• magnification calculation 


  Magnification= image size/actual size of object
• 1m=1000mm 1mm=1000μm 1μm=1000nm
• whats the limiting factor in light microscopy?

  Resolution
• advantages of electron microscopes

  1. more detail of cell ultra structure can be seen as electrons have a much smaller wavelength than light waves 2. tehy can produce images with magnification up to x500 000 and still have a clear resolution
• disadvantages of electron microscopes

  1. expensive equipment 2. can only be used in a carefully controlled environment 3. specimens can be damaged by the electron beams and because the preparation process is complex (artefacts)
• what are the 2 types of electron microscopes?

  Tramsmission electron microscope (TEM) and Scanning electron microscope (SEM)
• how do transmission electron microscopes work?

  a beam of electrons are transmitted through a specimen and focused to produce an image. They have the best resolving power of 0.5nm
• how do scanning electron microscopes work?

  a beam of electrons are sent across the surface of the specimen and the reflected electrons are collected. The resolving power is around 3-10nm. 3D images are produced.
• Light microscope


  • Inexpensive to buy and operate
  • Small and portable
  • Simple sample preparation
  • Sample preparation does not usually lead to distortion
  • Vacuum is not required
  • Natural colour of sample is seen (or stains are used)
  • Up to 2000 magnification
  • Resolving power is 200 nm
  • Specimens can be living or dead
• Electron microscope


  • Expensive to buy and operate
  • Large and needs to be installed
  • Complex sample preparation
  • Sample preparation often distorts material
  • Vacuum is required
  • Black and white images produced (but can be coloured digitally)
  • Over x500 000 magnification
  • Resolving power of transmission electron microscope is 0.5 nm and a scanning electron microscope is 3-10 nm
  • Specimens are dead
• what's an artefact?


  a visible structural detail caused by processing the specimen and not a feature of the actual specimen
• Laser Scanning Confocal Microscope

  A type of microscope that uses a laser to produce optical sections, allowing for high-resolution imaging of thin sections within a thick specimen
• advantages of LSCM's

  1. very high resolution images can be obtained 2. a 3D image can be produced 3. Non-invasive 4.
• disadvantages of LSCM's 


  expensive