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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
red-shifted colour
emission light has a lower energy and higher wavelength
26
altering of GFP DNA sequence creates
different variants fluophores
27
beam-splitting mirror
selects the right wavelength for fluorescence
28
emission filter
filters out unwanted fluorescent signals
29
quantum dots (selenium, cadnium) charachteristics (3)
- 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
immunofluorescence (in fixed cells)
fluorescent antibodies for specific proteins
31
diffraction limitations > blur
Point Spread Function (PSF)
32
improving resolution (2)
- smaller lambda - larger theta and n
33
deconvoluting images
with the use of computer software
34
wide field fluorescence microscopy
in-focus and out-of-focus plane
35
multi-point detector
camera
36
confocal microscopy uses a
laser
37
objective confocal microscopy
focused light = point of focus > molecules in out-of-focus will also be excited
38
confocal pinholes
emitted fluorescent ligh from in focus point is focused at the pinhole and reaches detector > more contrast in z-direction
39
obtaining 3D images
moving the illumination spot over the sample using scanners > Galvanometer mirror
40
for 3D imaging
multiple images are obtained at different heights = slower
41
calcium waves fertilized egg cel
will activate the injected Aequorin protein that luminesces in blue light
42
time dynamics of spliceosome proteins
moving Cajal bodies in plant nucleus
43
Annexin A4 > repair of plasma membrane
clustering of Annexin A4-sGFP in HeLa cells induced by adding calcium usin 1uM ionomycin
44
breakage of plasma membrane
influx calcium > positive protein bound
45
Fröster Resonance Energy Transfer (FRET)
for visualizing interactions > close proximity = interaction
46
FRET calcium biosensor
FRET between CFP and YFP > cadmodulin calcium binding sites > conformational change> shortens distance > FRET - low CFP and high YFP
47
visualization of movement
photoactivation of dark GFPs
48
photoactivation
fluorescence in selected region > diffusion of proteins
49
Fluorescence Recovery After Photobleaching (FRAP)
visualizing movement by destroying fluorescent dyes > replenishment from other regions
50
example FRAP
galactosyl transferase in Golgi and ER
51
Total Interal Reflection Microscopy (TIRM)
visualization of structures at/near membrane
52
critical angle for total internal reflection
no light through the sample > only molecules in evanescent field fluoresce
53
fixing cells EM
glutaraldehude
54
contrast dye EM
osmium tetoxide = metals
55
copper grid EM
covered with carbon and/or plastic film
56
Transmission EM (TEM)
electron bundle through sample
57
Scanning EM (SEM)
3D imaging and reconstruction > different angles and movement of detector
58
Super Resolution Microscopy (SRM)
to overcome the resolution limitation of ~200 nm
59
types of SRM (4)
- Structered Illumination Microscopy (SIM) - Expansion Microscopy - Stimulated Emission by depletion (STED) - Stochastic localization techniques
60
Stimulated Emission by Depletion (STED)
SRM for live cells > 5-7 nm
61
STED beam (red light)
causes excited molecules to return to resting state > only molecules not in STED beam emit light = effective fluorescent spot
62
Stochastic localization techniques
PALM, STORM, GSDIM
63
mechanism stochastic localization techniques
the position of a fluorphore can be determined with higher accuracy than the resolution of a confocal microscope > determination of center blurred spot
64
succesive cycles of activation and bleaching for
well-separated single fluorescent molecules

Cellulaire Oncologie (14 decks)

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