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Cellulaire Oncologie > HC10 - Visualizing cells > Flashcards

HC10 - Visualizing cells Flashcards

1
Q

electron microscopy in

A

dead, fixed cells

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2
Q

transmission light microscopy

A

brightfield microscopy

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3
Q

due to low contrast

A

organelles are hardly visble

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4
Q

histological stains

A

to visualize proteins, fats, DNA, organelles

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5
Q

dyes for histologicals stains can

A

absorb a certain colour but don’t emit

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6
Q

light (3)

A
  • electromagnatic radiation
  • quantified in protons
  • can act as both a wave and particle
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7
Q

enhancing contrast

A

in phase = bright, out of phase= dim

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8
Q

dark field microscopy

A

light enters at an angle > only scattered light rays will enter the objective > thicker pieces will light up

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9
Q

phase contrast/DIC microscopy

A

waves out of phase create contrast when combined > difference in density/thickness detected

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10
Q

resolution (r)

A

minimal distance where one can still distinguish 2 points of equal intensity from each other

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11
Q

smaller r

A

more details observed

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12
Q

objective lens

A

collects a cone of light rays to create an image

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13
Q

condensor lens

A

focuses a cone of light onto each point of the specimen

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14
Q

a higher numerical aperture leads to

A

greater resolution

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15
Q

microtome

A

creates thin sample slices (10-50 um) of specimen embedded in wax or resin

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16
Q

hematoxylin

A

ECM

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17
Q

eosin

A

nucleus

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18
Q

dast green

A

cellulose of cell wall

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19
Q

safarin

A

xylem

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20
Q

RNA in sity hybridization

A

tracking activities of genes

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21
Q

electron microscopy

A

highly detail in dead cells

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22
Q

dead cells electron microscopy

A

high energy electron beam

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23
Q

fluorescence microscopy can occur

A

in living cells

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24
Q

fluorescence

A

a dye molecule absorbs energy and can send out light afterwards

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25
Q

red-shifted colour

A

emission light has a lower energy and higher wavelength

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26
Q

altering of GFP DNA sequence creates

A

different variants fluophores

27
Q

beam-splitting mirror

A

selects the right wavelength for fluorescence

28
Q

emission filter

A

filters out unwanted fluorescent signals

29
Q

quantum dots (selenium, cadnium) charachteristics (3)

A
  • have very sharp emission bands > only emits one colour
  • photostable > take images without losing colour
  • disadvantage = coating needed > hydrophilic and big in size (10 nm)
30
Q

immunofluorescence (in fixed cells)

A

fluorescent antibodies for specific proteins

31
Q

diffraction limitations > blur

A

Point Spread Function (PSF)

32
Q

improving resolution (2)

A
  • smaller lambda
  • larger theta and n
33
Q

deconvoluting images

A

with the use of computer software

34
Q

wide field fluorescence microscopy

A

in-focus and out-of-focus plane

35
Q

multi-point detector

A

camera

36
Q

confocal microscopy uses a

A

laser

37
Q

objective confocal microscopy

A

focused light = point of focus > molecules in out-of-focus will also be excited

38
Q

confocal pinholes

A

emitted fluorescent ligh from in focus point is focused at the pinhole and reaches detector > more contrast in z-direction

39
Q

obtaining 3D images

A

moving the illumination spot over the sample using scanners > Galvanometer mirror

40
Q

for 3D imaging

A

multiple images are obtained at different heights = slower

41
Q

calcium waves fertilized egg cel

A

will activate the injected Aequorin protein that luminesces in blue light

42
Q

time dynamics of spliceosome proteins

A

moving Cajal bodies in plant nucleus

43
Q

Annexin A4 > repair of plasma membrane

A

clustering of Annexin A4-sGFP in HeLa cells induced by adding calcium usin 1uM ionomycin

44
Q

breakage of plasma membrane

A

influx calcium > positive protein bound

45
Q

Fröster Resonance Energy Transfer (FRET)

A

for visualizing interactions > close proximity = interaction

46
Q

FRET calcium biosensor

A

FRET between CFP and YFP > cadmodulin calcium binding sites > conformational change> shortens distance > FRET
- low CFP and high YFP

47
Q

visualization of movement

A

photoactivation of dark GFPs

48
Q

photoactivation

A

fluorescence in selected region > diffusion of proteins

49
Q

Fluorescence Recovery After Photobleaching (FRAP)

A

visualizing movement by destroying fluorescent dyes > replenishment from other regions

50
Q

example FRAP

A

galactosyl transferase in Golgi and ER

51
Q

Total Interal Reflection Microscopy (TIRM)

A

visualization of structures at/near membrane

52
Q

critical angle for total internal reflection

A

no light through the sample > only molecules in evanescent field fluoresce

53
Q

fixing cells EM

A

glutaraldehude

54
Q

contrast dye EM

A

osmium tetoxide = metals

55
Q

copper grid EM

A

covered with carbon and/or plastic film

56
Q

Transmission EM (TEM)

A

electron bundle through sample

57
Q

Scanning EM (SEM)

A

3D imaging and reconstruction > different angles and movement of detector

58
Q

Super Resolution Microscopy (SRM)

A

to overcome the resolution limitation of ~200 nm

59
Q

types of SRM (4)

A
  • Structered Illumination Microscopy (SIM)
  • Expansion Microscopy
  • Stimulated Emission by depletion (STED)
  • Stochastic localization techniques
60
Q

Stimulated Emission by Depletion (STED)

A

SRM for live cells > 5-7 nm

61
Q

STED beam (red light)

A

causes excited molecules to return to resting state > only molecules not in STED beam emit light = effective fluorescent spot

62
Q

Stochastic localization techniques

A

PALM, STORM, GSDIM

63
Q

mechanism stochastic localization techniques

A

the position of a fluorphore can be determined with higher accuracy than the resolution of a confocal microscope > determination of center blurred spot

64
Q

succesive cycles of activation and bleaching for

A

well-separated single fluorescent molecules

Cellulaire Oncologie (14 decks)

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