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FluoroFun > 9. class: FCS > Flashcards

9. class: FCS Flashcards

1
Q

Structure of the lecture

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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
Q
  1. SPT applications
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26
Q

Application 2: intracellular trafficking of gene complexes

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27
Q

Application 2: intracellular trafficking of gene complexes

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28
Q

Application 2: intracellular trafficking of gene complexes

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29
Q

Application 2: intracellular trafficking of gene complexes

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30
Q

Why not always just do single particle tracking ?

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31
Q

Miniaturization

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32
Q

Not noise!

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33
Q

Fluctuations

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34
Q

Fluctuations… Number fluctuations ?

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35
Q

Fluorescence correlation spectroscopy

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36
Q

Fluorescence correlation spectroscopy

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37
Q

Fluorescence correlation spectroscopy

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38
Q

From fluctuations to correlation

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39
Q

Example: Autocorrelation of freely diffusing molecules

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40
Q

Example: Autocorrelation of freely diffusing molecules

brightness

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41
Q

Nuclease activity

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42
Q

Binding studies

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43
Q

High-througput in vitro screening

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44
Q

Transcription factor (Ledgf/p75) dynamics in cells

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45
Q

Protein dynamics inside nuclei

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46
Q

Compare FCS with FRAP

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47
Q

Popularity ?

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48
Q

Principle of cross-correlation

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49
Q

Optical scheme

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50
Q

scheme

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51
Q

What does 2-color FCCS provide?

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52
Q

Example of FCCS

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53
Q

Application: LEDGF – HIV integrase

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54
Q

Application: LEDGF-IN interaction

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55
Q

Image fluctuations?

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56
Q

Image fluctuations?

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57
Q

Analyse image fluctuations?

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58
Q

Raster image correlation spectroscopy (RICS)

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59
Q

Raster image correlation spectroscopy (RICS)

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60
Q

RICS: simulations

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61
Q

RICS: diffusion

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62
Q

What if molecules move while you scan over them?

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63
Q

Raster image correlation spectroscopy (RICS)

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64
Q

See the correlations in the image… !

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65
Q

RICS or FCS ?

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66
Q

RICS or FCS ?

in vitro

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67
Q

RICS or FCS ?

in vitro

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68
Q

Raster image cross-correlation spectroscopy

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69
Q

CLSM with continuous-wave excitation

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70
Q

CLSM with pulsed interleaved excitation (PIE)

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71
Q

Application of fluctuation imaging to HIV assembly

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72
Q

RICS identifies slowly diffusing Gag in CLSM images

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73
Q

Slow interactions through CA or NC domain ?

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74
Q

Gag observed with RICS is monomeric

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75
Q

Image fluctuations?

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76
Q

Temporal image correlation spectroscopy (TICS)

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77
Q

Slow diffusion of Gag in the cytosol

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78
Q

Limitations of RICS

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79
Q

Spatial correlation in masked images

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80
Q

How to generate the mask ?

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81
Q

How to generate the mask ?

4 & 5

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82
Q

Proof of principle

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83
Q

Bacterial cells

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84
Q

Arbitrary-region RICS (ARICS)

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85
Q

RIPK1-dependent modulation of necroptosis induction and execution

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86
Q

Mapping with RICS

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87
Q

Remove crosstalk without using PIE or FLCS ?

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88
Q

Crosstalk-free multicolor RICS using statistical weighting

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89
Q

Crosstalk-free three color RICS

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90
Q

Non-spectral detectors also work! (Leica TCS SP8 X)

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91
Q

Summary (1): RICS is growing up

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FluoroFun (9 decks)

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