Microscopes

Question 1

What is the formula to work out magnification?

Answer 1

magnification = image size / actual size

Question 2

How are samples prepared to be viewed through a light microscope?

Answer 2

stains bind to to chemicals in the specimen, this allows the specimen to be seen

some stains bind to specific cell features

to prevent distorting the structure, the specimen is embedded in wax before being cut

Question 3

What is resolution? And what is the resolution for: the eye, a light microscope and a electron microscope?

Answer 3

the degree to which it is possible to clearly distinguish clearly between objects that are close together

eye - 100 μm

light microscope - 200nm

electron microscope - 0.2nm

Question 4

How do electron microscopes work?

Answer 4

fire a beam of electrons use magnets instead of lenses to focus the beam of electrons onto the specimen projected onto a screen (grey-scale image)

Question 5

What are the two types of electron microscopes and what are the differences between them?

Answer 5

Scanning electron microscope:

• electrons are reflected off the sample to produce a 3D image
• x100,000 magnification
• 3-10fm resolving power

Transmission electron microscope:

• electron pass through a thin prepared sample
• electrons don’t pass through denser parts as easily, this produces contrast x500,000 magnification
• resolving power of 0.5fm

Question 6

What are the advantages of using an electron microscope?

Answer 6

1000x higher resolution than a light microscope

produce detailed images of organelles inside a cell

SEM produces 3D images that reveal the detail of contour in cellular arrangements

Question 7

What are the disadvantages of using an electron microscope?

Answer 7

has to be in a vacuum (yknow why)

expensive preparing samples

and using it requires skill

colour has to be added and guessed

specimens are dead

Question 8

How do light microscopes work?

Answer 8

there is 2 lenses, the objective (close to sample) and the eyepiece which magnify the image twice

light underneat the stage turret adjusts which objective lens is used

there are to focusing knobs (coarse and fine)

resolving power of 200nm

Question 9

What is the difference between an eyepiece graticule and a stage graticule?

Answer 9

eyepiece graticule has regular division, these need to be calibrated for each magnification

the stage graticule shows true length - remains constant, no matter what magnification the cells are viewed at

Question 10

How is scale worked out from the stage micrometer?

Answer 10

• put the stage micrometer on the stage of the microscope and align the eyepiece graticule
  
  • so that the divsion of the eyepiece graticule align with those on the stage micromeer
• count and record the number of line in the graticule that correspond with 0.1mm (0.01mm) on the stage micrometer
• calc the actual distance between the divisions on the eyepiece graticule, express answer in standard form
• when measuring the width of the object, such as a cell, position the graticul over the object and count the number of graticule divisions
  
  • record l;ength in eyepiece units (EPU)
• calc actual length uysing this formula:
  
  • actual length = length of structure in EPUs / number of graticule divisions equal to 0.1mm

Question 11

What is an artefact? Give the examples

Answer 11

• visible structural detail caused by processing the specimen and not a feature of the specimen
• artefacts appear in both light and electron microscopy
• bulbbles that get trapped under the cover slip as you prepare a slide for light microscopy are artefacts
• when preparing samples changes in ultrastructure of cells or inveviatable
  
  • loss of continuity in membranes
  • distortion of organelles
  • empty spaces in the cyotplasm of cell

Question 12

What are fluorescent microscopes? How do they work?

Answer 12

• higher intensity light used to illuminate a specimen that has been treated with a fluorecent chemical (fluorescent ‘dye’)
• fluorescence is the absorbtion and re-radiation of light
• light of a longer wavelength isw emitted and used to proudce a magnified image

Question 13

What is a laser scanning confocal microscope?

Answer 13

• technique for fluorescent microscopes
• moves a single spot of focused light across a specimen
  
  • point illumination
• this causes fluorescence from the components labelled with a ‘dye’
  *

Question 14

How does a laser scanning confocal microscope work?

Answer 14

• the emitted light from the specimen is filtered through a pinhole aperture
• only light radiated from very close to the focal plane (distance that gives the sharpest focus) is detected
• light emitted from the other parts of the specimen would reduce the resolution and cause blurring
  
  • this unwanted radiation does not pass through the pinhole and is not detected
  • a laser is used instead of light to get higher intensities, which improves the illumination
• as very thing section of specimen are examined and light from elsewhere is removed, very hgih res images can be obtained

Question 15

How are 2D and 3D images produced by laser scanning confocal microscope?

Answer 15

• 2D
  
  • spot illuminating the specimen is moved across the specimen and a 2D image is produced
• 3D
  
  • 3D image can be produced by creating images at different focal planes

Question 16

How is laser scanning confocal microscopy used?

Answer 16

• non-invasive
• used in diagnosis of diseases in the eye and being developed to be used in endoscopic procedures
• used to see the distribution of molc within cells means it can be used in the development of new drugs
• future used
  
  • viral biopsies

Question 17

What is the beamsplitter?

Answer 17

• dichoric mirror
• only reflects one wavelength from the laser
• allows other wavelengths produced by the sample to pass through

Question 18

How is the LSCM confocal?

Answer 18

• positions of the two pinholes means the light waves from the laser (illuminating the sample) follow the same path as the light waves radiated when the sample fluoresces
• same focal plane
  
  • hence the term confocal

Question 19

How is a graticule used to calibrate a light microscope?

Answer 19

• put stage micrometer in place and the eyepiece graticule in the eye piece
• get the scale on the micrometer slide in clear focus
• align the micrometer scale with the scale in the eyepiece, take a reading from the two scales
  *

Question 20

Why can’t the details of a mitrochondria be seen with a light microscope?

Answer 20

• mitochondria too small
• resolution not high enough
• wavelength of light too long

Question 21

What are the advantages of staining specimens?

Answer 21

• easier to see
• increases contrast
• recognise different cell types or organelles
• recognise different molecules
  
  • e.g. organelles