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Cell biology > lecture 2 > Flashcards

lecture 2 Flashcards

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1
Q

Anton von leeuwen hoek

A
  • first person to see bacteria
  • built many single lens microscopes
  • achieved magnification of 200x
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2
Q

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

A
  • animalcules
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3
Q

what was the structure of AVLs microscope

A
  • finely polished glass lens between brass plates
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4
Q

modern compound micorscop

A
  • 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
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5
Q

what is the magnification of a typical compound micrscope

A

40 to 1000x

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6
Q

what is the max magnification of a compound microscope

A
  • 1000x
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7
Q

refractive structures and the compound microscope

A
  • higher refractive index = darker
  • lower refractive index = just white light, it is just passing through
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7
Q

resolution

A
  • 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
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8
Q

what can be seen with a light microscope

A
  • plant and animal cells, bacteria
  • NOT viruses, globular proteins, small molecules, or atoms
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9
Q

what is the max resolutiionof a light microscope

A
  • usually cannot observe objects that are less than 0.2 micrometers apart, o. r 200 nm, this is the limit of resolution
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10
Q

resolution equation

A

d = 0.61(lambda)/ Nsinalpha

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11
Q

N

A

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

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12
Q

how to get a low resolution

A
  • 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)
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13
Q

numerical aperture

A
  • Nsinalpha (higher = better, that reduces the denominator which makes the overall resolution smaller)
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14
Q

contrasts in light microscopy

A
  • 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
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15
Q

optimal interference in light microscopy

A
  • when the two waves are completely out of phase, tan d there is interference that causes the production of dim lighght
16
Q

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

A
  • a quarter wavelength
17
Q

phase contrast microscopy

A
  • 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
18
Q

process of phase contrast microscopy

A
  • 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
19
Q

what does the total 1/2 wavelength slowed do?

A
  • gives more detail of intracellular components
20
Q

issue with phase contrast microscopy

A
  • for rounded cells, at periphery, refracted light can be accelerated instead of slowed
  • ## that doubles the amplitude of the peaks
21
Q

DIC microscopy

A
  • 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
22
Q

fluorescence microscopy

A
  • uses fluorescence
23
Q

fluoresce

A

emit visible light when absorb light at a specific wavelength

24
Q

benefits of fluorescence microscopy

A
  • visualize more than one protein or cell strucutre
25
Q

fluorochromes

A
  • 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
26
Q

stokesshigt

A
  • 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
27
Q

fluorochrome colours

A
  • wide vareity
  • have different excitation and emission wavelentghs
  • allows for labelling of more than one protein or organelle at the same time
28
Q

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

A

DAPI
mitrotracker Red
Rhodmaine labelled phalloidin

29
Q

DAPI

A
  • stain nuclei blue
  • high affinity to DNA
    -fluroeces blue
  • binds well to nucleo
30
Q

mitotracker red

A
  • gets caught in michondiral inter membrane space
31
Q

rhodamine labelled phalloidin

A
  • stain mitochondria or actin filaments red
32
Q

immunofluorescent staining

A
  • conjugating a dye with antibodies to localize molecules of interest
33
Q

fluorescent imaging in live cells

A
34
Q
A
35
Q
A

Cell biology (15 decks)

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