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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

Joel Hagman

Supervisors

Anne-Marie Hermansson

Niklas Lorén


Outline
Outline photobleaching (FRAP) in different materials

  • 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) photobleaching (FRAP) in different materials


Principles of frap
Principles of FRAP photobleaching (FRAP) in different materials

Braeckmans PhD thesis


Brief history of frap

1974 - Peters et al. photobleaching (FRAP) in different materials

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 photobleaching (FRAP) in different materials

Stationary beam

Fringe pattern

Huyck

Huyck

Bleached disk

Bleached line


Stationary beam
Stationary beam photobleaching (FRAP) in different materials

  • Axelrod, 1976

  • 2D diffusion

  • Easy

  • Does not account for homo/heterogenety

  • “Far from correct”


Stationary beam1
Stationary beam photobleaching (FRAP) in different materials

Soumpasis 1983


Fringe pattern
Fringe pattern photobleaching (FRAP) in different materials

  • 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 photobleaching (FRAP) in different materials

Tinland 2000


Bleached disk
Bleached disk photobleaching (FRAP) in different materials

  • Braeckmans, 2003

  • 2D/3D diffusion

  • Eliminates beam edge effects

  • Requires a large bleach area

  • Sensitive for heterogeneity

Braeckmans 2006


Bleached disk1
Bleached disk photobleaching (FRAP) in different materials

Huyck

Braeckmans 2003


Bleached line
Bleached line photobleaching (FRAP) in different materials

  • Braeckmans, 2007

  • 1D diffusion

  • Less accurate then disk model

  • Can be performed in more heterogene areas

  • Faster then disk model


Bleached line1
Bleached line photobleaching (FRAP) in different materials

Huyck

Braeckmans 2007


Important parameters for frap
Important parameters for FRAP photobleaching (FRAP) in different materials

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 photobleaching (FRAP) in different materials

Effect of Numerical Aperture (NA)

Effect of bleach intensity distribution

Braeckmans PhD thesis

Blonk 1993


Frap in different materials
FRAP in different materials photobleaching (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)


Gels photobleaching (FRAP) in different materials

Probe molecular weight (Stokes radius) combined with

gelconcentration and dynamic viscosity

Mw decreases

Mw decreases

Dynamic viscosity

de Smedt 1997


Gels photobleaching (FRAP) in different materials

Enzymatic treatment

Burke 2000

Enzyme modification time (min)

β-mannanase creates backbone cleaving

α-galactosidase creates side chain cleaving


Solutions
Solutions photobleaching (FRAP) in different materials

Enzymatic treatment

Mw and gel concentration

Increasing gel concentration

Mw

Bu 1994

Burke 2000


Cells
Cells photobleaching (FRAP) in different materials

“FRAP revealed rapid dynamics of actin in spines”

Star 2002


Cells1
Cells photobleaching (FRAP) in different materials

Nuclear dynamics of AML1 fusion proteins in HeLa cells

Qiu 2006


During electrophoresis
During electrophoresis photobleaching (FRAP) in different materials

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


Fatty acid diffusion in heterogeneous chocolate determined using FRAP

3 samples

Diffusion rate evaluated using Braeckmans uniform disk model


Conclusions
Conclusions using FRAP

  • 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 using FRAP

Questions?


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