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MBB 231 > Lecture 9 - Microscopy > Flashcards

Lecture 9 - Microscopy Flashcards

1
Q

what are the 3 elements always needed for imaging in a microscope?

A
  1. a source of illumination
  2. a specimen to be examined
  3. a system of lenses to focus the illumination on the sample and form an image
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2
Q

def: illuminates the sample

A

light source

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3
Q

def: placed in front of the light source to focus the light at the desired point on the specimen

A

condenser lens

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4
Q

def: forms the primary image of the specimen, its the lens closest to the object of interest

A

objective lens

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5
Q

def: magnifies the primary image produced by the objective lens

A

ocular lens

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6
Q

def: refers to the size of the image

A

magnification

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7
Q

the smaller the limit of resolution a microscope has, the greater its _________ _______

A

resolving power

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8
Q

def: the minimum distance two points can be apart and remain apart

A

resolution

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9
Q

what 2 things does magnification consider?

A
  1. the refraction index of the lens and the medium the sample is immersed in
  2. the focal length of the lens
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10
Q

what are the 3 things resolution considers?

A
  1. the wavelength of illumination
  2. the refraction index
  3. angular aperture
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11
Q

def: measure of the change in the velocity of light as it passes from one medium to another

A

refractive index

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12
Q

what does the Abbé equation give us?

A

resolution

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13
Q

what are the 6 kinds of light microscopy?

A
  1. brightfield(unstained)
  2. phase contrast
  3. fluorescence
  4. brightfieqld(stained)
  5. differential interference contrast
  6. confocal
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14
Q

how can one improve bright field microscopy to see live cell and tissues that lack compounds that absorb light and are invisible?

A

fix specimen or stain it

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15
Q

def: preserves cells, prevents degradation, formalin and formaldehyde

A

fixation

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16
Q

def: an intact object placed on a slide

A

whole mount

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17
Q

def: fixed and embedded tissue that is cut into thin pieces and placed on a slide

A

section

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18
Q

what is the key feature of phase-contrast microscopy?

A

takes advantage of differences in refractive index and thickness to image living cells without the need to section and stain

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19
Q

what is the most useful case to use phase contrast microscopy?

A

study dynamic events, like the movement of organelles within the cell

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20
Q

what is the key feature of differential interference contrast?

A

technique has a shadow-casting effect that makes cells appear dark on one side and light on the other as a result of differences in the optical path due to phase, resulting image has a 3D effect

21
Q

what does fluorescence microscopy do?

A

detects fluorescent proteins or dyes to show locations of substances in the cell

22
Q

what does confocal scanning do?

A

uses laser beam to illuminate a single plane of fluorescently labeled specimen

23
Q

in fluorescence, the energy from an external source of light is ________ and almost immediately __________. Because energy must be conserved, the wavelength that comes out is ________ than what went in.

A

absorbed, re-emitted, longer

24
Q

def: transmits only night of a particular wavelength

A

excitation filters

25
Q

def: reflect light below a certain wavelength and transmits light above a certain wavelength

A

dichroic mirror

26
Q

def: prevents light that does not match the emission wavelength from exiting the microscope

A

emission filter

27
Q

what is fluorescent protein technology?

A

green fluorescent protein is fused to your favourite protein and then visualized in the cell

28
Q

_______ uses antibodies to locate specific molecules

A

immunostaining

29
Q

def: when the fluorescent dye is on the primary antibody

A

direct immunofluorescence

30
Q

def: when the fluorescent dye is on a secondary antibody

A

indirect immunofluorescence

31
Q

def: component of molecules which absorb light

A

chromophores

32
Q

what are the advantages of immunofluorescence microscopy?

A
  • high specificity
  • strong signal
  • identifies endogenous proteins in their native environment
33
Q

what are the disadvantages immunofluorescence microscopy?

A
  • experiments are done on dead cells (requires fixation)
  • there might not be an available antibody
34
Q

def: commonly used to visualize patterns of gene expression proteins in living cells and organisms

A

green fluorescent protein (GFP)

35
Q

what is the advantage of GFP?

A

cellular events can be observed in living cells

36
Q

what are the disadvantages of GFP?

A
  • GFP is a protein that might negatively affect your target protein
  • introducing the GFP tagged protein into cells
37
Q

what are the key features of confocal fluorescence microscopy?

A
  • produces in focus images of thick specimens (optical sectioning)
  • excludes out of focus light
  • reconstructed with computer in 3D
38
Q

what does confocal fluorescence allow?

A

allows one to distinguish structures in the middle of a cell from those at the top or bottom

39
Q

def: mathematical algorithm based process used to remove the contribution of to out-of-focus light in an image

A

deconvultion

40
Q

what are the advantages of viewing fixed cells?

A
  • organs can be viewed by sectioning thick tissue
  • easier to manage samples
  • don’t need to introduce a recombinant protein
41
Q

what are the advantages of studying live cells?

A
  • can observe the movement of biomolecules within a cell
  • provides critical context to your observations
42
Q

def: technique where you “bleach” an area of the cell and see how long it takes for unbleached molecules to return to the area (measure how fast a molecule moves)

A

fluorescence recovery after photobleaching (FRAP)

43
Q

def: technique when fluorescent molecules are close together, one can be used to excited the other (measure if two molecules are touching)

A

fluorescence resonance energy transfer (FRET)

44
Q

def: use a beam of electrons and electromagnets, rather than light and gas lenses

A

electron microscopes

45
Q

def: electron microscope - the surface of a specimen is scanned, by detecting electrons deflected from the outer surface

A

scanning electron microscopy (SEM)

46
Q

def: electron microscope - electrons are transmitted through the specimen

A

transmission electron microscopy (TEM)

47
Q

what is immuno EM?

A

when antibodies are conjugated/linked to substances that are electron dense

48
Q

def: you take multiple 2D images at different angles and using a computer, create a 3D reconstruction

A

cyro-electron tomography

49
Q

def: used for high-resolution structure determination of biomolecules in solution

A

cry-electron microscopy

MBB 231 (19 decks)

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