microscopy

Question 1

what are the three principles of cell theory?

Answer 1

1- basic unit of life is the cell
2- all animals and plants are made of cells
3-all cells are derived from pre-existing cells

Question 2

what are the uses of microscopy?

Answer 2

• study cell structure

- study the intracellular localisation, dynamics and interactions of specific cellular components in living cells

Question 3

how was mitosis first observed in a living cell?

Answer 3

drosophila embryos do not immediately undergo cytokinesis. first few divisions are nuclear. can observe e synchronous mitosis of nuclei in one cell

Question 4

what is meant by amplitude?

Answer 4

‘height’ of the light wave, determines intensity of the light

Question 5

why is wavelength important?

Answer 5

limits the size of the objects that can be seen

Question 6

what happens when light interacts with an object?

Answer 6

there is a change in the phase relationship of the light waves

Question 7

in what ways can the phase relationship of light waves be changed?

Answer 7

-if the light waves are in phase after passing through an object, this is constructive interference.
amplitude increases and object appears brighter
–light waves become out of phase - destructive interference. amplitude is smaller and the object appears lighter

Question 8

what are the principle requirements of a light microscope?

Answer 8

1- magnification
2- resolution
3- contrast formation
4- illumination

Question 9

what does the condenser of light microscope do?

Answer 9

focuses light from the light source onto the specimen

Question 10

what is magnification determined by?

Answer 10

objective lens and eyepiece lens

total mag = eyepiece mag x objective mag

Question 11

what is resolution defined as?

Answer 11

the ability to see two very small and closely spaced objects as separate entities

Question 12

what is Abbe’s law used for?

Answer 12

for determination of the resolving power of the lens

Question 13

what does numerical aperture of the lens measure?

Answer 13

light collecting ability

Question 14

how does placing oil between the sample and the objective increase resolution?

Answer 14

increases the amount of light collected by the objective lens

Question 15

in which two ways can contrast be increased?

Answer 15

1- biological stains

2- using light microscopes with special optical systems

Question 16

give an example of a biological stain used to increase contrast

Answer 16

hematoxylin - has affinity for negatively charged molecules and will stain DNA, RNA and acidic proteins

Question 17

how do biological stains increase contrast?

Answer 17

by reducing amplitude of light waves of a certain wavelength

Question 18

what is meant by fluorescence?

Answer 18

a three stage process that occurs in molecules called fluorophores

Question 19

how does fluorescence occur?

Answer 19

fluorophores are excited to a higher energy state, in this state, there are conformational changes and collisions which cause it to lose energy. when the molecule returns to the ground state, it has a much longer wavelength

Question 20

which property of fluorescent dyes permit them to be used in microscopy?

Answer 20

each fluorescent dye has a characteristic excitation wavelength and emission spectrum

Question 21

how are fluorophores excited in a fluorescent microscope?

Answer 21

using a halogen lamp

Question 22

what is the function of the first barrier filter in a fluorescent microscope?

Answer 22

lets through only blue light of wavelength 450-490nm

Question 23

what is the purpose of the dichroic mirror in a fluorescent microscope?

Answer 23

reflects light below 510nm but transmits light above 510nm

Question 24

what is the purpose of the second barrier filter in a fluorescent microscope?

Answer 24

cuts off unwanted fluorescent signals, passing the emission to the eyepiece

Question 25

what is immunofluorescent microscopy used for?

Answer 25

detecting specific molecules inside fixed cells

Question 26

what is the difference between a monoclonal and polyclonal antibody?

Answer 26

polyclonal antibodies bind to and recognise several epitopes on one protein, whereas monoclonal antibodies bind and recognise only a single epitope

Question 27

what is a hybridoma?

Answer 27

refers to the process of fusing a B-lymphocyte to a cancer cell, generating a single type of antibody

Question 28

what is direct immunofluorescence microscopy used for?

Answer 28

high abundance proteins

Question 29

why is BSA used as part of immunofluorescence microscopy?

Answer 29

prevents proteins from binding non-specifically via hydrogen bonding, hydrophobic effect etc.

Question 30

what is different about indirect immunofluorescence microscopy?

Answer 30

you use a primary (unlabelled) antibody and aa secondary antibody to amplify the signal from the first antibody

Question 31

what is useful about GFP?

Answer 31

can be used to study the intracellular location of a protein in living cells

Question 32

what is the structure of GFP?

Answer 32

monomeric form is made up of 11 antiparallel B-strands in a beta barrel with a single a-helix in the centre. the a-helix contains 3 residues which make up the GFP fluorophore

Question 33

what 3 amino acids make up the GFP fluorophore?

Answer 33

Gly-67, Ser-65 and Tyr-66

Question 34

why are confocal laser scanning microscopes useful?

Answer 34

complex three dimensional objects are difficult to image using a conventional light microscope

Question 35

how is a sample prepared for use in a transmission electron microscope?

Answer 35

1 - specimen is fixed
2- specimen is dried
3- specimen is embedded in a plastic resin
4- the stained sample is placed on a copper grid in a microscope

Question 36

what is the resolution of an electron microscope?

Answer 36

0.2nm