5. class single molecules Flashcards by Thomas Hasiweder

Q

Why doing single molecule detection ?

bulk experiment

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Q

Why doing single molecule detection ?

answer

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Q

Getting rid of ensemble averaging

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No need for synchronization

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Q

The samples you want to look at:

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How to do single molecule spectroscopy?

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Technical considerations for SMD

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How to do single molecule spectroscopy?

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Scanning confocal vs wide field microscopy

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Scanning confocal fluorescence microscopy

setup

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Scanning confocal fluorescence microscopy

signal

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Q

Blinking of single molecules

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Q

Blinking of single molecules

jablonski

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Two-color detection scheme: FRET

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Two-color detection scheme: FRET

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Spectral measurements

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Life-time measurements

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macro micro time

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Single molecule lifetime measurements

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Single molecule lifetime measurements

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Sequential laser excitation schemes: PIE

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Wide-field microscopy

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Wide field microscopy

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Wide-field microscopy

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Wide-field microscopy

Single particle tracking

1. SPT set-up

1.1 SPT set-up: controlling laser exposure

1.1 SPT set-up: controlling laser exposure

1.1 SPT set-up: controlling laser exposure

1.2 SPT set-up: matching image magnification to CCD pixel size

1.2 SPT set-up: matching image magnification to CCD pixel size

1.3 SPT set-up: Dual color SPT

1.3 SPT set-up: Dual color SPT

2. Locating the particles by image processing

2.1 Intensity thresholding - Examples

2.2 Background removal

2.3 Intensity thresholding - algorithms

2.4 Object detection & selection

2.5 Object properties

2.7 SPT software – automated particle detection

3. Calculation of trajectories

3.1 Nearest neighbour: caveats

3.1 Nearest Neighbour: caveats

3.2 Calculation of trajectories: examples

4.1 Trajectory analysis - Theory

Diffusion constants from the MSD ?

4.2 Trajectory analysis – modes of motion

4.3 Trajectory classification

4.4 Localisation precision (Gaussian fit)

4.5 Localisation precision determines lower mobility limit

4.6 Trajectory analysis: example

5. SPT applications

Application: intracellular trafficking of gene complexe

Application: intracellular trafficking of gene complexes

Advanced SPT

Advanced orbital tracking

Conclusions: SPT

Single molecule enzymatics

Single molecule enzymatics dynamics

Time dependent kcat

The lipase B from the yeast candida antarctica

Non fluorescent substrate conversion

Long observation time possible

Several conformations are involved

How many conformations are involved?

The ‘lazy’ enzyme

Size of the fluorogenic substrate

Size of the substrate

Single molecule enzymatics

Single molecule enzymatics

Single molecule enzymatics

Phospholipases

Phospholipases

Single molecule enzymatics

Single molecule enzymatics first step

+ skript!

Single molecule enzymatics?

Dual color excitation, simultaneous detection

Water soluble perylenediimide dye (PDI)

Dual color excitation, simultaneous detection

Active PLA1 – High concentration

Active PLA1 – Low concentration

Active PLA1 vs inactive

Single enzyme trajectories

Image analysis

Wide field microscopy

Wide-field microscopy

Image analysis

Cummulative distribution functions

Enzyme mobility

Model of mode of operation

Getting closer to cell membranes....

Getting closer to nature.... Hydrolysis of multi-component layers: POPC and DPG

Hydrolysis of giant unilamellar vesicles (GUV’s)

Hydrolysis of giant unilamellar vesicles (GUV’s)

Cytoplasma ER

YFP-PLCβ1 – Ca2+ response