Microscopes

• light microscopes - summary

  • poor resolutions - due to wavelength of light
  • living samples can be examined and a colour image is obtained
• transmission electron microscope - summary

  • High magnification and resolution
  • Electrons pass through the specimen to create an image
• scanning electron microscope summary

  • High magnification and resolution 
  • Electrons bounce off the surface of the specimen to create an image 
• lazer scanning confocal microscope - summary

  • High resolution + 3D imaging 
  • Lazer light used to create an image.
• resolution 


  minimum distance between two objects in which they can still be viewed as separate
• how is resolution determined in light microscopes

  • Light/optical microscope - resolutions is determined by the wavelength of the light.
• how is resolution determined in electron microscopes 


  resolution is determined by the wavelength of the beam of electrons
• electron microscope


  • A beam of electrons have a short wavelength = high resolution
  • Image is created using electromagnetic - used to focus the beam of electrons 
  • Electrons are absorbed by air so EM must be in a vacuum
  • Image is not in colour - sample has to be stained.
• transmission electron microscope


  • Extremely THIN specimens are stained + placed in a vacuum 
  • Electron gun produces beam of electrons - passes through the specimen
  • Some parts of the specimen absorb the electrons - so makes them darker
  • Image is produced in 2D - shows detailed image of internal structure of cells
• scanning electron microscope


  • Specimen does not have to be  thin 
  •  electrons do not pass through specimen but beamed onto the surface and reflect back in different ways.
  • Produces 3D image
• lazer scanning confocal microscope


  • Type of fluorescent microscope
  • Image is created using high intensity to illuminate specimen stained w fluorescent dye
  • High resolution + depth selectivity 
  • Lets scientists view sections of tiny structures that would be hard to section off - like embryos
  • Creates 3D image
  • Microscope cans specimen point by point using focused laser beam - creating 2D/3D image - in different focal planes
  •  light is emitted from the specimen - causes fluorescence 
• dry mount

   

  when thin slices/whole specimens are viewed with just the coverslip placed on top. 
• wet mount 


  When the specimen are added to water or stain before the coverslip is mounted on top with a mounted needle to prevent and air bubbles - aquatic organism can also be viewed this way

  
• squash slide 


  are wet mounts which you push down on the coverslip to squash the sample to ensure you have a thin layer to enable light to pass through.
• smear slide 


  created using the edge of another slide to smear the sample across another slide to create a smooth thin even coated specimen - a cover slip is placed on top after smearing.

   
• sectioning


  when solid specimens are cut into very thin slices with a sharp blade
• preparing a sample for staining 


  • sample is first place on a slide and allowed to air dry.
  • This is then heat fixed by passing through a flame 
  • The specimen will adhere to the microscope slide + take up the stains 
• positively charged dyes 


  crystal violet or methylene blue - they are attracted to negatively charged materials in the cytoplasm leading to staining of cell components
• negatively charged dyes 


  Nigrosin or Congo red - repelled by the negatively charge cytosol. these dyes stay outside the cells leaving the cell unstained, only staining the background
• differential staining 


  uses many chemical stains to stain different parts of the cell in different colours
• gram staining technique 


  1. used to separate bacteria into two groups gram positive and gram negative bacteria.
  2. crystal violet is applied to a bacterial specimen on a slide then iodine, which fixes the dye. the slide is then washed with alcohol. the gram positive bacteria retain the crystal violet stain and will appear blue or purple under a microscope.
  3. gram negative bacteria have thin peptidoglycan cell wall therefore they lose the stain so they are then stained with safranin dye which is called a counter stain. these bacteria will then appear red.
• why is gram staining important 


  distinguishes within the two bacteria and helps medics to decide what to prescribe.
• why is gram staining important 


  distinguishes within the two bacteria and helps medics to decide what to prescribe.

  • gram positive bacteria are susceptible to penicillin - inhibiting the formation of cell walls
  • gram negative bacteria have much thinner walls that are not susceptible to penicillin
• acid fast technique 


  used to differential species of mycobacterium from other bacteria. a lipid solvent is used to carry carbolfuchsin dye into the cells being studies. the cells are then washed with a dilute acid alcohol solution, mycobacterium are not affected by the acid alcohol and retain the carbolfuchsin stain
• fixing 


  chemicals like formaldehyde are used to preserve specimens in as near natural state as possible
• sectioning 


  specimens are dehydrated with alcohols and then placed in a mould with wax or resin to form a hard block. this can then be sliced thinly with a knife called a microtome
• sectioning 


  specimens are dehydrated with alcohols and then placed in a mould with wax or resin to form a hard block. this can then be sliced thinly with a knife called a microtome
• magnification 


  how many times larger the image is than the actual size of the object being viewed
• how to calibrate 


   

  1. Line up the stage micrometre and eyepiece graticule whilst looking through the eyepiece 
  2. Count how many divisions on the eyepiece graticule fit onto one of the divisions on the micrometer scale
  3. Each division is on the micrometer is 10μm - can be used to calculate what one division on the eyepiece graticule is at the current magnification
• wide field microscopy

  the whole sample is illuminated at once
• diffraction 


  bending of light as it passes close to the edge of an object t
• eye piece graticule 


  • inside light microscopes the scale on a glass disc
  • used to measure the size of the object you are looking at
  • each time you change the objective lens and the magnification you have to calibrate the eyepiece
  • acts just like a ruler but measures things that are microscopic
• stage graticule 


  a scale that is needed to calibrate the eyepiece graticule
• comparison of light microscope and electron microscopes 


  m

  
• What type of microscope uses a laser beam to scan a specimen?

  Laser scanning confocal microscope
• How does a laser scanning confocal microscope generate an image?


  It uses a laser beam to scan a specimen tagged with a fluorescent dye, focusing emitted light through a pinhole onto a detector connected to a computer.
• What is the purpose of the pinhole in a laser scanning confocal microscope?


  The pinhole blocks out-of-focus light to produce clearer images.
• How does the resolution of laser scanning confocal microscopes compare to that of electron microscopes?


  Laser scanning confocal microscopes have a lower resolution than electron microscopes.
• What are the applications of laser scanning confocal microscopes?


  • Look at different depths within a specimen
  • Observe living specimens