1. Evanescent wave induced Time-resolved Fluorescence
Myoung Hee Lee

2. Can we see single fluorophores ?
far field
near field
TIRF or TIRFM
(Total Internal Reflection Fluorescence Microscopy)

3. Microspheres in TIR and far-field Fluorescence

4. Time lapse Sequence of Protein Dynamics

5. total internal reflection
What is TIRF ?
total internal reflection
evanescent wave (EW)
~ a few hundreds nm
this optical section thickness
~ 1/10 for confocal microscopy technique

6. What is TIRF ? Key advantage of TIRF Surface selectivity
fluorophores
Surface selectivity
(Shallow penetration depth of EW)
Not excited beyond the EW
High Signal-to-Noise ratio

7. Induced Evanescent Wave
What happens ? n 1
evanescent wave

8. Induced Evanescent Wave
What happens ? n 1 d
for 1D evanescent wave, plane wave

9. Decay length of Evanescent Wave1 d
for example, air/glass, 400nm excitation, d : about 57 nm

10. Induced Evanescent Wave

11. Common Setups of TIRFM Setup with prism or with objective lens

12. Two types of TIRFM
Prism type
objective lens type

13. High numerical Aperture Objective TIRFM

14. Time-Resolved TIRF study on polarity at a liquid/liquid interface
Anal. Chem. 2001, 73,

15. Schematic diagram of a system for TIRF
TCSPC
Ti:sapphire laser
100 kHz, 200 fs
Glan-laser prism
IA : 80O
R3809U-50
Anal. Chem. 2001, 73,

16. Coordinate system of anisotropy experiment
X-Y plane is the plane of the interface
Anal. Chem. 2001, 73,

17. Molecular structures of SRB, SR101 and AB1
Anal. Chem. 2001, 73,

18. Schematic diagram of water / oil interface- +
- Oil -
cyclohexane
carbon tetrachloride
tolene
Chlorobenzene (CB)
o-dichlorobenzene (DCB)
1,2-dichloroethane (DCE)
oil
Anal. Chem. 2001, 73,

19. Fluorescence decay curves of SRB
nonradiative decay rate const. increases
as solvent polarity increases
means fast decay
At a water/DCE interface
(DCE : dichlorobenzene)
(b) In water
Single exponential function
Anal. Chem. 2001, 73,

20. Photophysical parameters of SRB and
Polarities at the water/oil interfaces
Anal. Chem. 2001, 73,

21. Schematic illustrations of flat and rough interfaces
At water/oil interface
For low polarity of oil
(CCl4 and toluene)
: thin and flat interface (a)
For high polarity of oil
(DCB and DCE)
: thin but rough interface (b)
(for SRB, single
exponential decay)
Anal. Chem. 2001, 73,

22. Propagation of a ray in an optical fiber
Appendix
Propagation of a ray in an optical fiber
dcot  z
take
time  d
z : an additional distance for the time ,
The penetration of a ray undergoing total internal reflection (or Effective lateral shift of the ray) is known as the

23. What happens ? Appendix Propagation factor for the refracted wave xz
Propagation factor for the refracted wave
not propagated to the z axis
propagated to the surface

24. D : transverse displacement
Appendix
Time-averaged normal component of the Poynting vector inside the surface d x z D
purely imaginary
D : transverse displacement

25. Appendix
evanescent wave
light source
prism
air