1.  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

2. 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

3. TIRF illuminates fluorescent molecules localized at or close to the cell surface
GFP-clathrin light chain
Caveolin-1-GFP
Mattheyses et al 2010 J Cell Sci 123: 3621

4. n2 ~ 1.38
n2 = 1.515
Mattheyses et al 2010 J Cell Sci 123: 3621

5. 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 nm

6. Refraction and reflection
Millis (2012) Methods in Molecular Biology 823:295

7. Critical angle
Millis (2012) Methods in Molecular Biology 823:295

8. 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= ) 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

9. Total internal reflection generates an evanescent wave
Millis (2012) Methods in Molecular Biology 823:295

10. 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
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
d increases with increasing 
d decreases with increased 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

11. Achieving TIRF using through-the-objective 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
Higher NA lenses allow for larger incidence angles

12. TIRF test sample- fluorescent beads
Focusing at coverslip
Focusing deeper into sample
epifluorescence
TIRF
Mattheyses et al 2010 J Cell Sci 123: 3621

13. Kinase-regulated quantal assemblies and kiss-and-run recycling of caveolae
Lucas Pelkmans and Marino Zerial
Nature 436, (7 July 2005)
doi: /nature03866

14. Kinase-regulated quantal assemblies and kiss-and-run recycling of caveolae
Lucas Pelkmans and Marino Zerial
Nature 436, (7 July 2005)
doi: /nature03866

15. Fluorescence recovery after photobleaching (FRAP) and photoactivation

17. 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

18. Fluorescence recovery after photobleaching (FRAP)

19. 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)

20. 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)

21. A. Highlight molecules for tracking
B. Diffusion
C. Pulse-chase
G. Patterson
Methods in Cell Biology Volume 85, 2008, Pages 45-61
Fluorescent Proteins

22. Figure 21. 6. 3. Selective photoactivation of PA-GFP
Figure 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 413-nm light. The images were acquired at sec 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

23. 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

24. An example of FRAP in action

25. Autophagy is a major lysosomal degradation pathway
1. Cytosolic materials are captured in double membrane vesicle
Lysosome
2. Trafficked to the lysosome for degradation
Autophagosome
Autophagy or self-eating is a major lysosomal degradation pathway. The process involves capturing cytosolic materials including protein aggregates, invading pathogens, and even entire organelles in a double membrane bound vesicle called the autophagosome. This vesicle is subsequently trafficked to the lysosome for degradation and recycling.

26. LC3 functions in autophagosomal membrane expansion/fusion and cargo selection
Lc3 has been proposed to perform two important functions. The first, is in membrane expansion and fusion where lipid modified LC3 is thought to contribute membrane and energy to the process. The second, is in selective targeting of autophagy substrates. Make this shorther, just say it recognizes ubiquitinated protein aggregates with the help of other proteins in the pathway and targets them for degradation.

27. EGFP-LC3 is widely utilized as a reporter of autophagy
Drake et al (2010)

28. EGFP-LC3, a marker for autophagosomes… is enriched in the nucleus ???
Drake et al (2010)

29. Is active nuclear transport involved in the nucleo-cytoplasmic transport of LC3?

30. LC3 contains a putative NES, but is unaffected by blockade of nuclear export or mutation of the NES
Drake et al (2010)

31. Does EGFP-LC3 passively equilibrate across the nuclear envelope?

32. LC3 enters the nucleus slowly compared to EGFP
Drake et al (2010)

33. LC3 exits the nucleus slowly compared to EGFP
Drake et al (2010)

34. LC3 undergoes little active or passive nuclear transport
Is it trapped in the nucleus?

35. 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

36. If EGFP-LC3 exists as a freely diffusing monomer, its diffusion should be similar to that of EGFP
kBT
= viscosity
R = hydrodynamic radius
D =
(6πR)
Stokes-Einstein equation

37. Setup for FRAP analysis of diffusion
Kraft and Kenworthy, 2012

38. prebleach
t=0
0.14s 1s 5s
EGFP
EGFP-LC3
tfLC3
EGFP-p53
2 m

39. LC3 diffuses more slowly than predicted by its molecular weight
Drake et al (2010)

40. 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

41. Considerations for experimental design, data analysis, and interpretation

42. Getting started Define beaching conditions Define imaging 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

43. What to look for: a “good” experiment
Prebleach
0 s
10 s
70 s 3 1 2 B C

44. A B Fluorescence Intensity Time (s)
Photobleaching during the recovery and/or focal plane changes A
Fluorescence Intensity B
Bumping the microscope
Time (s)
Goodwin and Kenworthy 2005

45. 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

46. Lippincott-Schwartz et al 2001