Literature review on the use of fluorescence recovery after photobleaching (FRAP) in different mater...
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Literature review on the use of fluorescence recovery after photobleaching (FRAP) in different materials. Joel Hagman Supervisors Anne-Marie Hermansson Niklas Lorén. Outline. Principles of CLSM Principles of FRAP Brief History of FRAP Bleaching modes Important parameters for FRAP

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Literature review on the use of fluorescence recovery after photobleaching (FRAP) in different materials

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Literature review on the use of fluorescence recovery after photobleaching frap in different materials

Literature review on the use of fluorescence recovery after photobleaching (FRAP) in different materials

Joel Hagman

Supervisors

Anne-Marie Hermansson

Niklas Lorén


Outline

Outline

  • Principles of CLSM

  • Principles of FRAP

  • Brief History of FRAP

  • Bleaching modes

  • Important parameters for FRAP

  • FRAP in different materials

  • Conclusions


Principles of confocal laser scanning microscopy clsm

Principles of Confocal Laser Scanning Microscopy (CLSM)


Principles of frap

Principles of FRAP

Braeckmans PhD thesis


Brief history of frap

1974 - Peters et al.

1976 - Axelrod et al.

1978 - Smith & McConnel

1982 - Davoust et al.

1983 - Soumpasis

[…]

1993 - Blonk et al.

1997 - Tinland et al.

2003 - Braeckmans et al.

2007 - Braeckmans et al.

Features

First attempt

Stationary beam

First tries of fringe pattern

Enhanced fringe pattern

Theory enhancement

[…]

FRAP in CLSM

FRAP & electrophoresis

Bleached disc

Bleached line

Brief history of FRAP

Time


Bleaching modes

Bleaching modes

Stationary beam

Fringe pattern

Huyck

Huyck

Bleached disk

Bleached line


Stationary beam

Stationary beam

  • Axelrod, 1976

  • 2D diffusion

  • Easy

  • Does not account for homo/heterogenety

  • “Far from correct”


Stationary beam1

Stationary beam

Soumpasis 1983


Fringe pattern

Fringe pattern

  • Davoust, 1982

  • 2D diffusion

  • “Solves some issues from stationary beam”

    • Removes the dependency of the beam width

    • May allow a minor flow

  • More complex equipment needed

  • None CLSM technique


Fringe pattern1

Fringe pattern

Tinland 2000


Bleached disk

Bleached disk

  • Braeckmans, 2003

  • 2D/3D diffusion

  • Eliminates beam edge effects

  • Requires a large bleach area

  • Sensitive for heterogeneity

Braeckmans 2006


Bleached disk1

Bleached disk

Huyck

Braeckmans 2003


Bleached line

Bleached line

  • Braeckmans, 2007

  • 1D diffusion

  • Less accurate then disk model

  • Can be performed in more heterogene areas

  • Faster then disk model


Bleached line1

Bleached line

Huyck

Braeckmans 2007


Important parameters for frap

Important parameters for FRAP

Effect of inter-line distance (Δy)

ω = radial Gaussian resolution

N = l/Δy+1 number of scans

l = 20 w

Braeckmans PhD thesis


Important parameters for frap1

Important parameters for FRAP

Effect of Numerical Aperture (NA)

Effect of bleach intensity distribution

Braeckmans PhD thesis

Blonk 1993


Frap in different materials

FRAP in different materials

  • Gels (some material)

  • Solutions (a lot of material)

  • Cells (a lot of material)

  • Emulsions (none)

  • Lipids (plenty of material)

  • Gel electrophoresis (some material)


Literature review on the use of fluorescence recovery after photobleaching frap in different materials

Gels

Probe molecular weight (Stokes radius) combined with

gelconcentration and dynamic viscosity

Mw decreases

Mw decreases

Dynamic viscosity

de Smedt 1997


Literature review on the use of fluorescence recovery after photobleaching frap in different materials

Gels

Enzymatic treatment

Burke 2000

Enzyme modification time (min)

β-mannanase creates backbone cleaving

α-galactosidase creates side chain cleaving


Solutions

Solutions

Enzymatic treatment

Mw and gel concentration

Increasing gel concentration

Mw

Bu 1994

Burke 2000


Cells

Cells

“FRAP revealed rapid dynamics of actin in spines”

Star 2002


Cells1

Cells

Nuclear dynamics of AML1 fusion proteins in HeLa cells

Qiu 2006


During electrophoresis

During electrophoresis

Effect of electrical field & gel strength

Ekani-Nkodo 2002


Fatty acid diffusion in heterogeneous chocolate determined using frap

Fatty acid diffusion in heterogeneous chocolate determined using FRAP

Bleached area

Before

bleaching

Directly after

bleaching

10 s after

bleaching

100 s after

bleaching

Fatty acid analogue with Mw = 320 Da


Literature review on the use of fluorescence recovery after photobleaching frap in different materials

Fatty acid diffusion in heterogeneous chocolate determined using FRAP

3 samples

Diffusion rate evaluated using Braeckmans uniform disk model


Conclusions

Conclusions

  • Possible to measure slow diffusion

  • Versatile tool

  • The technique have still a lot of growth potential

  • The technique is “again” under development


Thank you for your attention

Thank you for your attention

Questions?


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