Evanescent wave induced Time-resolved Fluorescence Myoung Hee Lee

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

Microspheres in TIR and far-field Fluorescence http://www.nikon.com

Time lapse Sequence of Protein Dynamics http://www.nikon.com

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 http://www.phys.virginia.edu/education/outreach/8thgradesol/InternalReflectionFrm.htm

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 http://motility.york.ac.uk/poster/tirfm.shtml

Induced Evanescent Wave What happens ? n 1 evanescent wave

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

Decay length of Evanescent Wave 1 d for example, air/glass, 400nm excitation, d : about 57 nm

Induced Evanescent Wave http://www.nikon.com

Common Setups of TIRFM Setup with prism or with objective lens http://www.nikon.com

Two types of TIRFM Prism type objective lens type http://www.nikon.com

High numerical Aperture Objective TIRFM http://www.nikon.com

Time-Resolved TIRF study on polarity at a liquid/liquid interface Anal. Chem. 2001, 73, 2421-2428

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, 2421-2428

Coordinate system of anisotropy experiment X-Y plane is the plane of the interface Anal. Chem. 2001, 73, 2421-2428

Molecular structures of SRB, SR101 and AB1 Anal. Chem. 2001, 73, 2421-2428

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, 2421-2428

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, 2421-2428

Photophysical parameters of SRB and Polarities at the water/oil interfaces Anal. Chem. 2001, 73, 2421-2428

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, 2421-2428

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

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

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

Appendix evanescent wave light source prism air