microscopy , chapter 2

Question 1

magnification equasion

Answer 1

magnification = size of image/actual size

Question 2

resolution

Answer 2

the ability of a microscope to distinguish details, it determines the amount of detail that can be seen.

Question 3

how is resolution limited ?

Answer 3

by the diffraction of light as it passes through samples

Question 4

what is diffraction?

Answer 4

the spreading of waves as the pass through/ around something

Question 5

what happens when resolution is limited by diffraction?

Answer 5

the structures are no longer seen as seperate entities.

Question 6

features of electron microscopy

Answer 6

• a beam of electrons with a wavelength of less than 1nm.
• cells can be seen in more detail because electrons have a much smaller wavelenght than light microscopes.
• they can produce images with magnifications of up to X500,000.

Question 7

disadvantages of electron microscopy

Answer 7

• expensive.
• can only be used in controlled and dedicated spaces.
• specimens can be damaged by the electron beam.
• preparation is complex.

Question 8

the negative stain technique

Answer 8

dyes (such as nigrosin or congo red) are negatively charged dyes and are repelled by materials in the cytoplasm, leaving the cells unstained which then stand out against the stained backround.

Question 9

how are cells stained?

Answer 9

dyes (such as mythelene blue and crystal violet) are positively charged dyes and are which are attracted to negatively charged cytoplasm leading to staining of components in the cell.

Question 10

the gram stain technique.

Answer 10

• used to seperate gram-negative and gram-positive bacteria.
• crystal violet is applied to a bacteria on a slide, then iodine.
• the slide is then washed with alcohole.
• the gram-positive bacteria retain the crystal violet stain and will appear blue/purple. Gram-negative bacteria have thinner walls and therefore lose the stain.
• The gram-negative bacteria is then stained by a counter stain and appears red.

Question 11

acid-fast technique

Answer 11

• used to differenciate mycrobacterium from other bacteria.
• a lipid solvent is used to carry dye into the cells being studied.
• the cells are then washe with a dilute acid-alcohol solution,
• mycrobacterium are not affected by the acid-alcohol solution and retain the dye which is bright red.
• other bacteria lose the dye and are exposed to mythalene blue stain.

Question 12

sample preparation : DRY MOUNT

Answer 12

• specimens are viewed whole, cut into very thin slices(sectioning).
• the specimen is placed in the centre of the slide and a cover slip is placed on top of the sample. (hair, pollen, dust, muscle tissue, plants..)

Question 13

sample preparation : WET MOUNT

Answer 13

• specimens are suspended in a liquid such as water or immersion oil. A cover slip is placed over the specimen and the liquid. (aquatic samples, or other living organisms)

Question 14

sample preparation : SQUASH SLIDES

Answer 14

• a wet mount is prepared and then a lens tissure is used to gently press down of the cover slip.
• squash slides are good for soft sampled (root tip squashes are used to look at cell division)

Question 15

sample preparation : SMEAR SLIDES

Answer 15

• the edge of a slide is used to smear the sample accross another slide. A cover is then placed over the sample.
  -(e.g. a sample of blood)

Question 16

transmission electron microscope (TEM)

Answer 16

A beam of electrons is transmitted through a specimen and focused to produce an imagine. This has the best resolution.

Question 17

scanning electron microscope (SEM)

Answer 17

A beam of electrons is sent accross the surface of a specimen and the reflected electrons are collected. The resolving power is from 3 - 10nm, so the resolution is not as good as the TEM but three-dimesnional images of surfaces are made.

Question 18

sample preparation

Answer 18

double fixation, steralise it and stop it from decomposing
dehydration, water would boil in a vacuum and damage the sample
epoxy resin embedding, to make it rigid
ultrathin sectioning, to allow electrons to pass through the sample
staining eith heavy metals, heavy metals will attatch to the different structures and create contrast

Question 19

laser scanning confocal microscopy

Answer 19

• moves a single spot of laser light along the specimen.
• specimen components labelled with flouresent dye emit light.
• filtered through pinhole.
• only light close to focal plane is detected.
• unwanted radiation does not pass through pinhole.