Fluorescence Recovery After Photobleaching (FRAP 荧光漂白后恢复(FRAP
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2000 Nature Cell Biology)
Experimental Setup
• Laser beam focused or through small field diaphragm
• Rapid shutter to switch from high powered beam for bleach to attenuated beam for recovery same power won’t work-Keep bleaching)
Real FRAP data
More Diffusion types
Fx,t/F()12texpxt2/w2t Fullyrecovers
with
w2tw021t/D at/1t/
Important for Large macromolecules: Collisions, obstacles, binding
• Detector: PMT, camera ?
• Original apparatus used stationary laser spot (still sometimes used)
• Later improvement included scanning mirror to scan spot over sample.
A
intra
extracellular
B
Both A and B will
have similar D in
Membrane
although
Very different
sizes
Binding to immobilized matrix will reduce fraction of molecules diffusing
Cartoon of FRAP
Bleach creates “hole” of fluorophores, Diffusion is measured by “hole filling in”
Bleach high power Monitor low power
Analysis of membrane
Problems with FRAP of cytoplasmic components (2 orders of magnitude faster
than membranes)
1. Diffusion is fast compared to bleaching and monitoring rate D=ms : cannot truly scan
Spot Photobleaching
•Bleach and monitor single diffraction limited spot •Assumes infinite reservoir of fluorescent molecules (hole can fill back in) •Use D = w2/4D to obtain D •Determine w = nominal width of Gaussian spot by other optical method 1/e2 point •Fit fluorescence recovery curve to obtain D
Scanning over bleach spot improves ability to characterize recovery curves
• Allows accurate characterization of the bleach geometry and size for each individual experiment
Different bleaching geometries yield different types of information
Pre-bleach Photo源自文库leach image
Post-bleach image
No recovery Recovery
FRAP of GFP in Mitochondria
• Simplifies fits of recovery curves to:
t
2
D 2 ax where is a constant reflecting
extent of bleaching.
Koppel, 1979 Biophys. J. 281
• Allows compensation for photobleaching during monitoring and sample drift.
• As shown by Saffman and Delbruck, the translational diffusion coefficient for membrane components depends only on the size of the membrane spanning domain
x
Verkman, J. Cell Biology 2019
Heavy dextrans very slow Mobile fraction low: binding More polarizable
FRAP in Cytoplasm
J D
x
• Problem is much more complicated because of three dimensional freely diffusing geometry.
Diffusion of membrane components can be seen as a two dimensional diffusion problem
• Membrane is modeled as infinite plane
• Viscosity of the lipid bilayer is ~ 2 orders of magnitude higher than water
compartments
Cells expressing VSVG–GFP were incubated at 40 °C to retain VSVG–GFP in the endoplasmic reticulum (ER) under control conditions (top panel) or in the presence of tunicamycin (bottom panel). Fluorescence recovery after photobleaching (FRAP) revealed that VSVG– GFP was highly mobile in ER membranes at 40 °C but was immobilized in the presence of tunicamycin(Nehls et al,
A way of understanding diffusion: Random Walk
Spread of molecules from one spot is proportional to square root of time for random walk. Therefore, to go 2X as far takes 4X as long.
Axelrod et al., 1976
PSF and Beam Waist
Imaging sub-resolution 100 nm fluorescent beads
Use 1/e2 pointstDo =gewt 2ω/4bDeam waist (87%)
Different bleaching geometries yield different types of information
• Now can be done with laser scanning confocal instruments. (for some cases-e.g. membranes)
Idealized photobleaching data
Y = mobile fraction X
D =w2/4D
• Random thermal motions: D = kTv/f
• f depends on size of particle and viscosity of solution.
Spheres: scale as m1/3 (radius scaling)
FRAP - Fluorescence Recovery After Photobleaching
Fast limit: cytoplasm Slow limit: membrane bound
Suggests barriers (cristae) need to be large
Occlude 90% space
Verkman, TIBS, 2019
Size dependence of dextrans (polysaccharides) diffusion in solution
NNNooA way of understanding diffusion: Fick’s Law J D x
age • Jisflux • D is diffusion constant • is concentration
Diffusion Constant: What controls it?
J D
x
Not simple spheres: Random coils No simple m1/3 scaling
Verkman, J. Cell Biology 2019
Diffusion of FITC Dextrans, Ficolls in MDCK Cell Cytoplasm
J D
e.g. Luby-Phelps et al., 1994. SekSek et al. 2019.
D = kT/f
D =w2/4D
Not reliable, cytoplasm complicated collection of fluid, cytoskeletal components, endosome, etc: simple viscosity not sufficient
Recovery is convolved With depth of field
2 If use small bleach regions, redistribution may occur during bleaching. In fact, often cannot observe bleach of small region at all.
3. By enlarging the size of the bleach region, can overcome this problem: but lose localization
Photobleaching of cytoplasmic components
One solution is to measure cytoplasmic diffusion by comparing to characteristic times of known samples in solutions of known viscosity.
1. Line photobleaching generates a one-dimensional diffusion problem
Note that beam is still Gaussian Line scan of single points
F
x
Allows collection of more fluorescence, averaging
Photobleaching of cytoplasmic components
Another solution is to use geometry such depth of field is comparable to thickness of cell
High NA lens
Low NA lens
Experimental Setup
• Laser beam focused or through small field diaphragm
• Rapid shutter to switch from high powered beam for bleach to attenuated beam for recovery same power won’t work-Keep bleaching)
Real FRAP data
More Diffusion types
Fx,t/F()12texpxt2/w2t Fullyrecovers
with
w2tw021t/D at/1t/
Important for Large macromolecules: Collisions, obstacles, binding
• Detector: PMT, camera ?
• Original apparatus used stationary laser spot (still sometimes used)
• Later improvement included scanning mirror to scan spot over sample.
A
intra
extracellular
B
Both A and B will
have similar D in
Membrane
although
Very different
sizes
Binding to immobilized matrix will reduce fraction of molecules diffusing
Cartoon of FRAP
Bleach creates “hole” of fluorophores, Diffusion is measured by “hole filling in”
Bleach high power Monitor low power
Analysis of membrane
Problems with FRAP of cytoplasmic components (2 orders of magnitude faster
than membranes)
1. Diffusion is fast compared to bleaching and monitoring rate D=ms : cannot truly scan
Spot Photobleaching
•Bleach and monitor single diffraction limited spot •Assumes infinite reservoir of fluorescent molecules (hole can fill back in) •Use D = w2/4D to obtain D •Determine w = nominal width of Gaussian spot by other optical method 1/e2 point •Fit fluorescence recovery curve to obtain D
Scanning over bleach spot improves ability to characterize recovery curves
• Allows accurate characterization of the bleach geometry and size for each individual experiment
Different bleaching geometries yield different types of information
Pre-bleach Photo源自文库leach image
Post-bleach image
No recovery Recovery
FRAP of GFP in Mitochondria
• Simplifies fits of recovery curves to:
t
2
D 2 ax where is a constant reflecting
extent of bleaching.
Koppel, 1979 Biophys. J. 281
• Allows compensation for photobleaching during monitoring and sample drift.
• As shown by Saffman and Delbruck, the translational diffusion coefficient for membrane components depends only on the size of the membrane spanning domain
x
Verkman, J. Cell Biology 2019
Heavy dextrans very slow Mobile fraction low: binding More polarizable
FRAP in Cytoplasm
J D
x
• Problem is much more complicated because of three dimensional freely diffusing geometry.
Diffusion of membrane components can be seen as a two dimensional diffusion problem
• Membrane is modeled as infinite plane
• Viscosity of the lipid bilayer is ~ 2 orders of magnitude higher than water
compartments
Cells expressing VSVG–GFP were incubated at 40 °C to retain VSVG–GFP in the endoplasmic reticulum (ER) under control conditions (top panel) or in the presence of tunicamycin (bottom panel). Fluorescence recovery after photobleaching (FRAP) revealed that VSVG– GFP was highly mobile in ER membranes at 40 °C but was immobilized in the presence of tunicamycin(Nehls et al,
A way of understanding diffusion: Random Walk
Spread of molecules from one spot is proportional to square root of time for random walk. Therefore, to go 2X as far takes 4X as long.
Axelrod et al., 1976
PSF and Beam Waist
Imaging sub-resolution 100 nm fluorescent beads
Use 1/e2 pointstDo =gewt 2ω/4bDeam waist (87%)
Different bleaching geometries yield different types of information
• Now can be done with laser scanning confocal instruments. (for some cases-e.g. membranes)
Idealized photobleaching data
Y = mobile fraction X
D =w2/4D
• Random thermal motions: D = kTv/f
• f depends on size of particle and viscosity of solution.
Spheres: scale as m1/3 (radius scaling)
FRAP - Fluorescence Recovery After Photobleaching
Fast limit: cytoplasm Slow limit: membrane bound
Suggests barriers (cristae) need to be large
Occlude 90% space
Verkman, TIBS, 2019
Size dependence of dextrans (polysaccharides) diffusion in solution
NNNooA way of understanding diffusion: Fick’s Law J D x
age • Jisflux • D is diffusion constant • is concentration
Diffusion Constant: What controls it?
J D
x
Not simple spheres: Random coils No simple m1/3 scaling
Verkman, J. Cell Biology 2019
Diffusion of FITC Dextrans, Ficolls in MDCK Cell Cytoplasm
J D
e.g. Luby-Phelps et al., 1994. SekSek et al. 2019.
D = kT/f
D =w2/4D
Not reliable, cytoplasm complicated collection of fluid, cytoskeletal components, endosome, etc: simple viscosity not sufficient
Recovery is convolved With depth of field
2 If use small bleach regions, redistribution may occur during bleaching. In fact, often cannot observe bleach of small region at all.
3. By enlarging the size of the bleach region, can overcome this problem: but lose localization
Photobleaching of cytoplasmic components
One solution is to measure cytoplasmic diffusion by comparing to characteristic times of known samples in solutions of known viscosity.
1. Line photobleaching generates a one-dimensional diffusion problem
Note that beam is still Gaussian Line scan of single points
F
x
Allows collection of more fluorescence, averaging
Photobleaching of cytoplasmic components
Another solution is to use geometry such depth of field is comparable to thickness of cell
High NA lens
Low NA lens