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MCB 2050 > Microscopy > Flashcards

Microscopy Flashcards

1
Q

brightfield microscopy components

A

magnification and resolution, specimen fixation/staining

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2
Q

main components of brightfield microscopy

A

light source
condenser lens
stage
objective and ocular lenses
detector

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3
Q

how is the image captured with brightfield microscopy

A

light diffracted by specimen and undiffracted light focused by objective lens - captured by video camera

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4
Q

deconvolution

A

designed to remove background and out-of-focus light

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5
Q

what is the primary purpose of microscopy

A

magnification - generate magnified high quality view of specimen

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6
Q

what is the most important aspect of todays microscope

A

resolution

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7
Q

what is resolution

A

minimum distance that can separate 2 points that still remain identifiable as separate points

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8
Q

how to calculate overall magnification

A

objective lens x ocular lens

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9
Q

what does the resolving power of microscope depend on (resolution)

A

wavelength of illumination light
numeral aperture

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10
Q

what is numerical aperture

A

light gathering qualities of objective lensed specimen mounting medium

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11
Q

how to maximize resolution

A

short wavelengths of illuminating light
increase NA

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12
Q

how to calculate resolution distance in nm

A

(0.61 x wavelength)/NA

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13
Q

what is the limit of resolution for most standard brightfield (and CLSM)

A

200nm

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14
Q

limits of resolution for human eye

A

0.1mm

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15
Q

what is the resolution limit for super resolution CLSM

A

20nm (5000X)

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16
Q

what is the resolution limit for electron microscope

A

0.2nm (500,000X)

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17
Q

why does electron microscope (EM) use electrons rather than photons

A

lesser wavelength yields higher resolution

18
Q

what is a major limitation of brightfield microscopy

A

specimens poor contrast

19
Q

what is usually done to specimen to increase contrast for brightfield microscopy

A

fixed
embedded
sectioned
stained

20
Q

formaldehyde fixation

A

cross-links amino group on adjacent proteins/nucleic acids

21
Q

what does the specimen usually get embedded in and why

A

plastic or wax for support

22
Q

what does the specimen get sectioned with

A

microtome (cuts specimen into sections)

23
Q

what does the specimen get stained with

A

molecule specific dyes

24
Q

what are the cons of fixation

A

results in cell death

25
Q

what are cons of embedding and sectioning

A

can lead to structural artifacts

26
Q

cons of staining

A

limited molecule-specific dyes can lead to poor contrast

27
Q

what is fluorescence microscopy

A

technique for visualizing fluorescent molecules in living or fixed specimens

28
Q

autofluorescence

A

endogenous fluorescence in specimen

29
Q

immunofluorescence

A

applied fluorescent dyes or dye-conjugated antibodies

30
Q

what does fluorescence microscopy rely on

A

autofluorescence
immunofluorescence
autofluorescent proteins

31
Q

pros of fluorescence microscopy

A

increased contrast and allows study of structures and (when not fixed) dynamic processes in living cells and in 3D

32
Q

cons of fluorescence microscopy

A

out-of-focus fluorescence from thick specimen results in blurred image

33
Q

what is an example of an autoflourescent protein

A

GFP - guanadenosine fluorescent protein

34
Q

what does confocal laser-scanning microscopy (CLSM) allow for

A

viewing dynamic biological/cellular processes live - specimen is usually living

35
Q

what does CLSM yield

A

individual 2D z-section (optical slice) of specimen that is less blurry than images from standard fluorescence microscopy

36
Q

what is done to the individual z-section focal planes

A

collected at different depths in sample and combined

37
Q

what does the combination of the Z-sections form

A

z-stack - generates 3D image

38
Q

limitations/cons to CLSM

A

rapid but cannot capture very dynamic cellular processes
photobleach
phototoxicity
not efficient for thick specimens/tissues
limited spatial resolution

39
Q

what is photobleach

A

when the point laser photobleaches fluorescent molecules, they are no longer fluorescent

40
Q

what is phototoxicity

A

excited fluorescent molecules react with molecular oxygen and produces free radicals which can damage/kill live cells

41
Q

is super-resolution CLSM better or worse than standard CLSM

A

10x better (20nm)

MCB 2050 (11 decks)

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