9. class: FCS Flashcards
(91 cards)
1
Q
Structure of the lecture
A
2
Q
Brownian diffusion - basics
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3
Q
Why D ?
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4
Q
Not only Brownian diffusion makes Life dynamic
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5
Q
Not only Brownian diffusion makes Life dynamic
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6
Q
Quantifying mobility: two ensemble methods
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7
Q
Quantifying mobility: one single-molecule method
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8
Q
FRAP
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9
Q
Transcription factor (Ledgf/p75) dynamics in cells
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10
Q
HIV-1 integrase enzyme locks LEDGF/p75 on chromatin
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11
Q
Example: mobility of xylanase on substrates
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12
Q
Example: endolysin peptidoglycan binding domain
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13
Q
Variations on FRAP: FLIP, CP, PA
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14
Q
FRAP – old … but not worn out!
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15
Q
FRAP analysis
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16
Q
What’s wrong with FRAP ?
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17
Q
Why single molecules ?
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18
Q
Single particle tracking (SPT)
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19
Q
Dual color SPT
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20
Q
Who’s who?
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21
Q
Who’s who?
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22
Q
Calculation of trajectories: examples
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23
Q
Trajectory classification
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24
Q
4.6 Trajectory analysis: example
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25
5. SPT applications
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Application 2: intracellular trafficking of gene complexes
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Application 2: intracellular trafficking of gene complexes
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Application 2: intracellular trafficking of gene complexes
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Application 2: intracellular trafficking of gene complexes
30
Why not always just do single particle tracking ?
31
Miniaturization
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Not noise!
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Fluctuations
34
Fluctuations... Number fluctuations ?
35
Fluorescence correlation spectroscopy
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Fluorescence correlation spectroscopy
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Fluorescence correlation spectroscopy
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From fluctuations to correlation
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Example: Autocorrelation of freely diffusing molecules
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Example: Autocorrelation of freely diffusing molecules
brightness
41
Nuclease activity
42
Binding studies
43
High-througput in vitro screening
44
Transcription factor (Ledgf/p75) dynamics in cells
45
Protein dynamics inside nuclei
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Compare FCS with FRAP
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Popularity ?
48
Principle of cross-correlation
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Optical scheme
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scheme
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What does 2-color FCCS provide?
52
Example of FCCS
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Application: LEDGF – HIV integrase
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Application: LEDGF-IN interaction
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Image fluctuations?
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Image fluctuations?
57
Analyse image fluctuations?
58
Raster image correlation spectroscopy (RICS)
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Raster image correlation spectroscopy (RICS)
60
RICS: simulations
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RICS: diffusion
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What if molecules move while you scan over them?
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Raster image correlation spectroscopy (RICS)
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See the correlations in the image... !
65
RICS or FCS ?
66
RICS or FCS ?
in vitro
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RICS or FCS ?
in vitro
68
Raster image cross-correlation spectroscopy
69
CLSM with continuous-wave excitation
70
CLSM with pulsed interleaved excitation (PIE)
71
Application of fluctuation imaging to HIV assembly
72
RICS identifies slowly diffusing Gag in CLSM images
73
Slow interactions through CA or NC domain ?
74
Gag observed with RICS is monomeric
75
Image fluctuations?
76
Temporal image correlation spectroscopy (TICS)
77
Slow diffusion of Gag in the cytosol
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Limitations of RICS
79
Spatial correlation in masked images
80
How to generate the mask ?
81
How to generate the mask ?
4 & 5
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Proof of principle
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Bacterial cells
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Arbitrary-region RICS (ARICS)
85
RIPK1-dependent modulation of necroptosis induction and execution
86
Mapping with RICS
87
Remove crosstalk without using PIE or FLCS ?
88
Crosstalk-free multicolor RICS using statistical weighting
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Crosstalk-free three color RICS
90
Non-spectral detectors also work! (Leica TCS SP8 X)
91
Summary (1): RICS is growing up
