TIRF, FRAP, photoactivation
Anne Kenworthy
•Total internal reflection fluorescence
(TIRF) microscopy
•FRAP and photoactivation
-General principles
-An example of FRAP in action
-Considerations for experimental
design
QFM 2014
Total Internal Reflection
Fluorescence (TIRF) Microscopy
• The problem: how to visualize events occurring
at the plasma membrane in the presence of
“stuff” going on in the rest of the cell?
• What TIRF does: illuminates only fluorescent
structures very near the coverslip (≤100 nm)
• How it does it: excites molecules using an
evanescent field
• Advantages: Low background fluorescence, no
out-of-focus fluorescence, minimal exposure of
the sample to light except at the surface
TIRF illuminates fluorescent molecules
localized at or close to the cell surface
GFPclathrin light
chain
Caveolin-1GFP
Mattheyses et al 2010 J Cell Sci 123: 3621
n2 ~ 1.38
n2 = 1.515
Mattheyses et al 2010 J Cell Sci 123: 3621
TIRF essentials
• Total internal reflection occurs at angles greater
than the critical angle and generates an
evanescent wave in the sample
• The intensity of the evanescent wave decays
exponentially
• Only fluorophores within the evanescent wave are
excited
• The depth of penetration d depends on the
wavelength of excitation light used, the angle, and
the refractive indices of the media and coverslip
– Typical values of d range from 60 -100 nm
Refraction and reflection
Millis (2012) Methods in Molecular Biology 823:295
Critical angle
Millis (2012) Methods in Molecular Biology 823:295
Critical angle
Total internal reflection occurs when light propagating through
a transparent medium of high refractive index (ex. glass, n =
1.518) encounters a planar interface of a medium of a lower
refractive index (ex. water/cell, n= 1.33-1.37) for angles of
incidence greater than the critical angle 
c = sin-1 (n1/n2)
where n1 and n2 are the refractive indices of the sample and
coverslip, respectively
For  < c most light is refracted (and enters the
sample)
For  > c all of the light reflects back into the
solid; evanescent field is formed
Total internal reflection generates an
evanescent wave
Millis (2012) Methods in Molecular Biology 823:295
Properties of the evanescent field
The intensity of the evanescent wave decays
exponentially at a distance z perpendicular to the interface
as
I(z) = I(0)-z/d
d increases with increasing 
d decreases with increased incidence angle
where
d = (o/4π)(n22 sin2 - n12)-1/2
and o is the wavelength of the incident light, n1 and n2 are
the refractive indices of the sample and coverslip,
respectively, and  is the incidence angle
The depth of penetration depends on the wavelength
of excitation light used, the angle, and the refractive
indices of the media and coverslip
Achieving TIRF using through-theobjective TIRF
• To achieve TIRF, the NA of
the objective must be
greater than the refractive
index of the sample
– Refractive index of cell ~ 1.38
– Common TIRF objectives:
1.45 NA and 1.49 NA
• To achieve an angle of
illumination greater than
the critical angle, the laser
beam is focused off axis on
the back focal plane
http://www.microscopyu.com/articles/fluorescence/tirf/tir
fintro.html
– Higher NA lenses allow for
larger incidence angles
TIRF test sample- fluorescent beads
Focusing at
coverslip
Focusing
deeper into
sample
epifluorescence
TIRF
Mattheyses et al 2010 J Cell Sci 123: 3621
Kinase-regulated quantal
assemblies and kiss-and-run
recycling of caveolae
Lucas Pelkmans and Marino Zerial
Nature 436, 128-133(7 July 2005)
doi:10.1038/nature03866
Kinase-regulated quantal
assemblies and kiss-and-run
recycling of caveolae
Lucas Pelkmans and Marino Zerial
Nature 436, 128-133(7 July 2005)
doi:10.1038/nature03866
Fluorescence recovery after
photobleaching (FRAP) and
photoactivation
Fluorescence recovery after photobleaching
(FRAP)
• In FRAP, a population of fluorescent molecules in a region
of interest is irreversibly photobleached
• Recovery of fluorescent molecules back into that same
region is monitored over time
• This allows you to measure the characteristic kinetics of
underlying motion of the fluorescently tagged molecules
• This approach can be readily used in measurements of
diffusion and other forms of transport, including vesicular
trafficking
• FRAP can be carried out on most confocal microscopes
and for many commonly used fluorophores
Fluorescence recovery after
photobleaching (FRAP)
Large-scale confocal FRAP
measurements of protein diffusion
Prebleach
t=0
10s
100s
10 mm
GFP-KRas, COS-7, 37º C
Kenworthy et al (2004)
Photoactivation/photoconversion
• Certain fluorophores and fluorescent proteins
exhibit little of no fluorescence at certain
wavelengths until they are photoactivated
• This allows one to control the time and place at
which fluorescence is “turned on”
• The redistribution of the photoactivated
molecules can then be monitored over time
• Applications of photoactivatable FPs include:
–
–
–
–
Protein dynamics
Fluorescence pulse labeling
Photoquenching FRET
Photoactivated Localization Microscopy (PALM)
A. Highlight
molecules for
tracking
B. Diffusion
C. Pulse-chase
G. Patterson
Methods in Cell Biology Volume 85,
2008, Pages 45-61
Fluorescent Proteins
Figure 21.6.3.
Selective
photoactivation of
PA-GFP. A COS-7 cell
expressing PA-GFP was
imaged using low levels
of 488-nm light before
and after photoactivation
of the nuclear region
(indicated by the white
circle) with ~1 sec of 413nm light. The images
were acquired at 15.75sec intervals.
Current Protocols in Cell Biology
UNIT 21.6
Photoactivation and Imaging of Photoactivatable Fluorescent Proteins
George H. Patterson1
1Cell
Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health
Why use FRAP or photoactivation?
• FRAP and photoactivation can measure diffusion
(Brownian motion), which depends on
– Molecular size
– Local environment
– Binding interactions
• FRAP and photoactivation can also provide
information about
–
–
–
–
Reversible membrane binding
Intracellular transport
Continuity of compartments
Stability of molecular complexes
An example of FRAP in action
Autophagy is a major lysosomal degradation
pathway
Autophagosome
1. Cytosolic
materials are
captured in
double
membrane
vesicle
Lysosome
2. Trafficked to
the lysosome for
degradation
LC3 functions in autophagosomal membrane
expansion/fusion and cargo selection
EGFP-LC3 is widely utilized as a reporter of
autophagy
Drake et al (2010)
EGFP-LC3, a marker for autophagosomes…
is enriched in the nucleus ???
Drake et al (2010)
Is active nuclear transport involved in
the nucleo-cytoplasmic transport of
LC3?
LC3 contains a putative NES, but is
unaffected by blockade of nuclear export
or mutation of the NES
Drake et al (2010)
Does EGFP-LC3 passively equilibrate
across the nuclear envelope?
LC3 enters the nucleus slowly compared
to EGFP
Drake et al (2010)
LC3 exits the nucleus slowly compared to
EGFP
Drake et al (2010)
LC3 undergoes little active or
passive nuclear transport
Is it trapped in the nucleus?
Models
• LC3 binds to nuclear components such as
chromatin
• LC3 binds to other proteins and forms a
complex that is too large to pass through
nuclear pores
Both of these models predict that LC3
should not diffuse like a monomeric
protein
If EGFP-LC3 exists as a freely diffusing
monomer, its diffusion should be similar
to that of EGFP
D =
k BT
(6πmR)
m = viscosity
R = hydrodynamic radius
Stokes-Einstein equation
Setup for FRAP analysis of diffusion
Kraft and Kenworthy, 2012
prebleach
t=0
0.14s
1s
5s
EGFP
EGFPLC3
tfLC3
EGFPp53
2 mm
LC3 diffuses more slowly than
predicted by its molecular weight
Drake et al (2010)
Implications
• Our findings suggest that the nuclear localization of
LC3 is not an artifact, rather that the protein has a
currently unrecognized, novel nuclear function
• Our diffusion measurements indicate that LC3 either
transiently binds immobile structures in the cell in
both the nucleoplasm and cytoplasm, or diffuses as
part of a high molecular weight (~1 MDa!)
macromolecular complex
• How can we distinguish between these possibilities?
– FRET
– FCS
Considerations for experimental
design, data analysis, and
interpretation
Getting started
• Define beaching conditions
– Bleach region geometry and size
• A smaller region will recover faster
– Region of interest to monitor recovery
• Bleach ROI only vs surrounding area of cell
– Bleach depth/bleach iterations
• More iterations is not always better
• Confirm the minimal number needed using a fixed
sample
• In general bleach time should be less than 0.1 t1/2
• Define imaging conditions
– Minimize photobleaching during the recovery phase
– Recovery time is sufficiently long to reach plateau
What to look for: a “good” experiment
A
Prebleach
0s
10 s
70 s
3
1
2
B
C
Photobleaching during
the recovery and/or
focal plane changes
B
Fluorescence Intensity
A
Bumping the
microscope
Goodwin and Kenworthy 2005
Time (s)
Hallmarks of fast vs slow diffusion
• Fast diffusion is
characterized by
– Low apparent bleach
depth in live vs fixed
sample
– Short halftime of
recovery
– Depletion of
fluorescence outside
the bleach region
immediately
following the bleach
Lippincott-Schwartz et al 2001