Lec6_TIRF_Microscopy_ML

Lec6_TIRF_Microscopy_ML - Total Internal Reflection...

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1 Total Internal Total Internal Reflection Fluorescence Reflection Fluorescence (TIRF) (TIRF) Microscopy Microscopy Interface between media with different optical density The behavior of light as it passes from one medium to another is described by Snell’s Law: n 1 sin " 1 = n 2 sin " 2 Glass n 1 =1.5 Air n 2 =1.0 E I E R E T n is the refractive index of the medium Θ 2 Θ 1
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2 At shallow angles all light is reflected Critical angle: sin Θ c = n 2 /n 1 , n 1 >n 2 TIR light is present in only a thin layer above the interface 65º Θ > Θ c Water, n 2 =1.37 Glass, n 1 =1.518 Evanescent light ~100 nm TIRF Microscopy Intensity vs. Angle TIR p-pol metal film (20nm Al) p-pol bare glass s-pol bare glass Intensity in the low index medium at z=0 INCIDENCE ANGLE (degrees) Fused Silica/ Water interface From D. Axelrod, figure 4.
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3 TIRF Microscopy Biophysical Methods Intermediate Films Water n 2 =1.33 Glass n 1 =1.518 Intermediate Layer, n INT n 1 sin " 1 = n INT sin " INT TIRF Microscopy Biophysical Methods TIR Illumination Depth Light intensity in the second medium decays exponentially: I(z) = I(0)exp( " z d p )
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4 What is the penetration depth for TIR illumination? Calculating the TIR illumination penetration depth
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5 Calculating the TIR illumination penetration depth E T r r ( ) = E 0 T " exp # i r k " r r ( ) = E 0 T " exp # kz n 2 " n 1 2 sin 2 $ 1 # n 2 2 % & ( ) * " exp # ikx " n 1 n 2 sin $ 1 % & ( ) * exponential decay wave in z-direction propagating in x direction We are usually interested in the intensity of the excitation light, rather than the amplitude of the electric field. The Intensity is proportional to E 2 . We can thus write the z-dependence of the intensity as: I(z) ~ exp # 2kz n 2 n 1 2 sin 2 + 1 # n 2 2 % & ( ) * = exp # z d P % & ( ) * With the penetration depth d p : d P = n 2 2 k 1 n 1 2 sin 2 + 1 # n 2 2 using the conversions for the wave vector, k = 2 , - , and the wavelength in the second medium relative to vacuum (or air), - = - 0 n 2 we can write this as d P = - 0 4 , n 1 2 sin 2 + 1 # n 2 2 TIRF Microscopy Biophysical Methods TIR Fluorescence excitation is confined to the surface The penetration depth of the evanescent wave depends on incident angle and wavelength of light TIR - Generated Fluorescence Glass I(z) = I(0)exp( " z d p ) d p = " 0 4 # n 1 2 sin 2 $ 1 % n 2 2
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6 TIRF Microscopy Biophysical Methods Practical TIRF Implementation using Prisms From D. Axelrod, figure 6. TIRF Microscopy Biophysical Methods A more convenient TIRF Implementation: Excitation through the objective http://www.microscopyu.com/articles/ fluorescence/tirf/tirfintro.html
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7 TIRF Microscopy Biophysical Methods A more convenient TIRF Implementation: Excitation through the objective http://www.microscopyu.com/articles/
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  • Fall '08
  • LINDAU
  • Total internal reflection, Total internal reflection fluorescence microscope, TIRF microscopy, evanescent wave, Biophysical Methods, ) Microscopy

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