lecture 2

Question 1

Anton von leeuwen hoek

Answer 1

• first person to see bacteria
• built many single lens microscopes
• achieved magnification of 200x

Question 2

what did AVL call the living protozoa and bacteria he observed through his miscropcope

Answer 2

• animalcules

Question 3

what was the structure of AVLs microscope

Answer 3

• finely polished glass lens between brass plates

Question 4

modern compound micorscop

Answer 4

• has a light source
• has a condenser that focuses the light on the specimen that is on a slide
• there is magnification from the objective lens that collects light after it has passed through the specimen
• reaches the ocular lens that focus’s the light on the eye

Question 5

what is the magnification of a typical compound micrscope

Answer 5

40 to 1000x

Question 6

what is the max magnification of a compound microscope

Answer 6

• 1000x

Question 7

refractive structures and the compound microscope

Answer 7

• higher refractive index = darker
• lower refractive index = just white light, it is just passing through

Question 7

resolution

Answer 7

• ablity to distinguish between two very closely positions obejctes as separate entities
• minimum distance between two separate objects at which they are still discernible as two seperate
• we want a really small resolution

Question 8

what can be seen with a light microscope

Answer 8

• plant and animal cells, bacteria
• NOT viruses, globular proteins, small molecules, or atoms

Question 9

what is the max resolutiionof a light microscope

Answer 9

• usually cannot observe objects that are less than 0.2 micrometers apart, o. r 200 nm, this is the limit of resolution

Question 10

resolution equation

Answer 10

d = 0.61(lambda)/ Nsinalpha

Question 11

N

Answer 11

refractive index between the specimen and the objective lens
- you can add things like water or oil that will have higher refractive indexes
- sin alpha isa half angle of light entering the objcetive
- a specimen that is close tot he objective wilhave a wide half angle which is optimal

Question 12

how to get a low resolution

Answer 12

• reduce the numerator by using a shorter wavelength of light
• increase the denominator by using oil or water or liquid with a higher refractive index or increase alpha (how long or round the lens is)

Question 13

numerical aperture

Answer 13

• Nsinalpha (higher = better, that reduces the denominator which makes the overall resolution smaller)

Question 14

contrasts in light microscopy

Answer 14

• when a part of the cell (i.e. the nucleus) refracts more light, the portion of light that passes through that part of the cell will be slowed down a bit (a quarter wavelength) and that causes a bit of interferenceop

Question 15

optimal interference in light microscopy

Answer 15

• when the two waves are completely out of phase, tan d there is interference that causes the production of dim lighght

Question 16

by how much do cellular constituents with high refractive properties slow the passage of a light beam

Answer 16

• a quarter wavelength

Question 17

phase contrast microscopy

Answer 17

• still light microscopy
• used to examine live, unstained cells
• small differences of refractive indexes in cell and thicknesses are exploited and converted into contrast visible to th eye

Question 18

process of phase contrast microscopy

Answer 18

• light from lamp
• annular diaphragm turns light into a cone of light, the big beam iss separated into a cone
• the cone is focused via a condenser lens
• part of cell with higher refractive properties will bend the light a little
• this causes the 1/4 wavelength slow down
• that light is passed through a phase plate
• the diffracted quarter slowed light is diffracted towards the centre part of the palte
• this slight is slowed down another quarter length
• ## refracted light is directed towards the center

Question 19

what does the total 1/2 wavelength slowed do?

Answer 19

• gives more detail of intracellular components

Question 20

issue with phase contrast microscopy

Answer 20

• for rounded cells, at periphery, refracted light can be accelerated instead of slowed
• ## that doubles the amplitude of the peaks

Question 21

DIC microscopy

Answer 21

• examine live unstained cells
• small differences in refractive index and thickness within the cell ar converted into contrast that is visible to the eye.
• this uses polarized light, the microscope has polarizers
• more defined outline of large organelles such as nucleus and vacuole = better detail of cell edge

Question 22

fluorescence microscopy

Answer 22

• uses fluorescence

Question 23

fluoresce

Answer 23

emit visible light when absorb light at a specific wavelength

Question 24

benefits of fluorescence microscopy

Answer 24

- visualize more than one protein or cell strucutre

Question 25

fluorochromes

Answer 25

- usd in fluorescent micrsocopy - absorb electrons to cause the outer oribital to be excited to a higher state - when the electrons drop to its normal orbital, visible light released, fluorescenee

Question 26

stokesshigt

Answer 26

- different between excitation and emission optima - different fluorochromes have different optimal = doesn't use a light bulb, uses an intensee light source like a laser

Question 27

fluorochrome colours

Answer 27

- wide vareity - have different excitation and emission wavelentghs - allows for labelling of more than one protein or organelle at the same time

Question 28

examples of chemicals linked to flurocochromes that are able to stain cell structures nd organelles

Answer 28

DAPI mitrotracker Red Rhodmaine labelled phalloidin

Question 29

DAPI

Answer 29

- stain nuclei blue - high affinity to DNA -fluroeces blue - binds well to nucleo

Question 30

mitotracker red

Answer 30

- gets caught in michondiral inter membrane space

Question 31

rhodamine labelled phalloidin

Answer 31

- stain mitochondria or actin filaments red

Question 32

immunofluorescent staining

Answer 32

- conjugating a dye with antibodies to localize molecules of interest

Question 33

fluorescent imaging in live cells