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### Lec6_TIRF_Microscopy_ML

Course: A&EP 470, Fall 2008
School: Cornell
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Internal Total Reflection Fluorescence (TIRF) Microscopy Interface between media with different optical density The behavior of light as it passes from one medium to another is described by Snells Law: n1sin&quot;1 = n 2sin&quot; 2 EI Glass n1=1.5 1 ER 2 Air n2=1.0 ET n is the refractive index of the medium ! TIRF Microscopy Biophysical Methods 1 At shallow angles all light is reflected...

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Internal Total Reflection Fluorescence (TIRF) Microscopy Interface between media with different optical density The behavior of light as it passes from one medium to another is described by Snells Law: n1sin"1 = n 2sin" 2 EI Glass n1=1.5 1 ER 2 Air n2=1.0 ET n is the refractive index of the medium ! TIRF Microscopy Biophysical Methods 1 At shallow angles all light is reflected Evanescent light Water, Water, Air, n2=1.0 n2=1.37 2 n2=1.37 Glass, n1=1.5 Glass, Glass, n1=1.518 1 n1=1.518 <c <c >c = c = 65 ~100 nm Critical angle: sinc= n2/n1, n1>n2 TIR light is present in only a thin layer above the interface Intensity vs. Angle Intensity in the low index medium at z=0 Fused Silica/ Water interface p-pol metal film (20nm Al) TIR p-pol bare glass s-pol bare glass TIRF Microscopy INCIDENCE ANGLE (degrees) Biophysical Methods From D. Axelrod, figure 4. 2 Intermediate Films Water n2=1.33 Intermediate Layer, nINT Glass n1=1.518 >c n1sin"1 = n INT sin" INT = n 2sin" 2 TIRF Microscopy Biophysical Methods ! TIR Illumination Depth Light intensity in the second medium decays exponentially: I(z) = I(0)exp(" z d ) p ! TIRF Microscopy Biophysical Methods 3 What is the penetration depth for TIR illumination? TIRF Microscopy Biophysical Methods Calculating the TIR illumination penetration depth TIRF Microscopy Biophysical Methods 4 Calculating the TIR illumination penetration depth rr % kz ( % ( r n 2 E T ( r ) = E 0T " exp #ik " r = E 0T " exp'# " n12 sin 2 \$1 # n 2 * " exp'#ikx " 1 sin\$1 * n2 & n2 ) & ) ( ) e xponential decay in z-direction wave p r o p a gating in x direction ! We are usually interested in the intensity of the excitation light, rather than the a mplitude of the electric field. The Intensity is proportional to E2. We can thus write the z-dependence of the intensity as: % 2kz 2 2 ( % z( I(z) ~ exp'# n1 sin +1 # n 2 * = exp' # * 2 & n2 ) & dP ) ! With the penetration depth dp: dP = n2 2k 1 2 n12 sin 2 +1 # n 2 ! using the conversions for t he wave vector, k = 2, - , and the wavelength in the second medium relative to vacuum (or air), - = 0 n we can write this as 2 4, n12 2 ! 2 TIRF Microscopysin +1 # n 2 dP = -0 ! Biophysical Methods ! TIR Fluorescence excitation is confined to the surface I(z) = I(0)exp(" z d ) p dp = "0 2 4# n1 sin 2\$1 % n 2 2 ! ! The penetration depth of the evanescent wave depends on incident angle and wavelength of light Glass TIR - Generated Fluorescence TIRF Microscopy Biophysical Methods 5 Practical TIRF Implementation using Prisms TIRF Microscopy Biophysical Methods From D. Axelrod, figure 6. A more convenient TIRF Implementation: Excitation through the objective TIRF Microscopy Biophysical Methods http://www.microscopyu.com/articles/ fluorescence/tirf/tirfintro.html 6 A more convenient TIRF Implementation: Excitation through the objective TIRF Microscopy Biophysical Methods http://www.microscopyu.com/articles/ fluorescence/tirf/tirfintro.html Through the Lens TIRF TIRF Microscopy From D. Axelrod, figure 5. Biophysical Methods 7 Excitation through the objective: What is the penetration depth? I(z) = I(0)exp(" z d ) p Example: ! dp = Cell, n2=1.38 Water, n2=1.37 Glass, n1=1.518 >c "0 2 4# n1 sin 2\$1 % n 2 2 ! TIRF microscopy using Green Fluorescent Protein as label. Use 488 nm Argon laser as excitation light source: 0=488 nm. Refractive index of microscope cover glass is n1=1.518 Refractive index of cell is typically n2~1.38. What is the minimum angle C for total internal reflection? " > " C , with sin" C = n2 n1 "C = arcsin n2 = 65.38 n1 ! TIRF Microscopy ! Biophysical Methods Excitation through the objective: What is the penetration depth? I(z) = I(0)exp(" z d ) p Example: ! dp = Cell, n2=1.38 Water, n2=1.37 Glass, n1=1.518 >c "0 2 4# n1 sin 2\$1 % n 2 2 ! The minimum angle C for total internal reflection is 65.38 For practical use the minimum angle needs to be ~1 larger min=C + 1= 66.38, which gives a penetration depth dP (" min) = #0 2 4\$ n12 sin 2 " min % n2 = 224nm ! TIRF Microscopy Biophysical Methods 8 Excitation through the objective: What is the penetration depth? I(z) = I(0)exp(" z d ) p Example: ! dp = Cell, n2=1.38 Water, n2=1.37 Glass, n1=1.518 >c "0 2 4# n1 sin 2\$1 % n 2 2 ! The maximum angle of the incident light is determined by the numerical aperture of the objective: NA = n1 " sin# max Giving a maximum angle " max = arcsin NA = 72.79 n1 ! ! and a corresponding penetration depth dP (" max ) = TIRF Microscopy #0 2 4\$ n12 sin 2 " max % n 2 = 87nm Biophysical Methods ! Excitation through the objective: What is the penetration depth? I(z) = I(0)exp(" z d ) p Example: ! dp = Cell, n2=1.38 Water, n2=1.37 Glass, n1=1.518 >c "0 2 4# n1 sin 2\$1 % n 2 2 ! Using a 1.45 NA objective and 488 laser illumination the penetration depth can be varied between 224 nm and 87 nm. In practice the laser spot in the back focal plane has a finite diameter such that the range will be slightly less. TIRF Microscopy Biophysical Methods 9 TIRF Excitation through the Objective without Laser: TIRF Microscopy Biophysical Methods TIRF Illumination with a Range of Incident Angles: dP (" max ) = #0 2 4\$ n12 sin 2 " max % n 2 = 87nm dP (" min ) = #0 2 4\$ n12 sin 2 " min % n 2 = 224nm ! TIRF Microscopy Biophysical Methods ! 10 TIRF Illumination with a Range of Incident Angles: 1.0 0.8 0.6 Intensity curves spaced in 10 nm All the individual of d intervalsexponentials P 0.4 0.2 dP (" max ) = #0 2 4\$ n12 sin 2 " max % n 2 #0 = 87nm 0.0 0 4 2 0 -2 1.0 100 200 300 400 500 dP (" min ) = ! 2 4\$ n12 sin 2 " min % n 2 = 224nm x10 -3 Normalized fit Sum to Normalized Sum Res_Normal_sum 0.8 ! TIRF microscopy is currently the most confocal technique 0.6 The overall intensity decay is well fitted by a single exponential with dP=156 nm 0.4 0.2 0 100 200 300 400 500 TIRF Microscopy Biophysical Methods Advantages of TIRF Microscopy Background light is nearly nonexistent. Less photobleaching. Full field of view all at once. Thinner section then confocal Ability to see single fluorescent molecules. Can observe cellular kinetics Can be combined with other methods. GFP fluorescence in Chromaffin cells excited by 488nm light. EPI TIRF Microscopy Biophysical Methods TIRF From Axelrod, figure 1. 11 Macrophage grown on a coverslip Wide field illumination TIR illumination TIRF Microscopy Biophysical Methods Examples of TIRF in Use Exocytosis and Endocytosis Single Molecule Detection Folding/Interactions of Molecules Molecular Motors Biosensors List expands TIRF Microscopy Biophysical Methods 12 Exocytosis and Trafficking From cover of Cell, March 20, 1998. TIRF Microscopy Biophysical Methods From the cover of Science, Nov. 6, 1992 Exocytosis Mechanism TIRF Microscopy Biophysical Methods An and Almers 13 Single Molecule Studies TIRF Microscopy Biophysical et al A. Yildiz, Methods Single Molecule Studies ~ MOVIE ~ TIRF Microscopy Biophysical et al A. Yildiz, Methods 14 A single-fluorophore is characterized by a high fluorescence intensity and step-wise photobleaching 12000 Fluorescence (D.U) 10000 8000 6000 4000 2000 0 Signal Photobleaching rate depends strongly on: -Illumination intensity -dye environment -solution conditions 0 1 2 3 4 5 6 7 Time (Sec) Signal-to-noise (S/N= <(If)>/std(If)) can be tuned by illumination intensity, data acquisition rate and solution conditions Photobleaching rate and S/N have been optimized Cy3 lifetime 140 120 Data: Count1_pop Model: ExpDec1 Equation: y = A1*exp(-x/t1) + y0 Weighting: y No weighting Chi^2/DoF = 5.77022 R^2 = 0.99477 A1 146 t1= ~4 sec 0 t1 3.99724 y0 0 0 0.05884 Cy3 S/N 25 20 Count Data: SNR_Count Model: Gauss Equation: y=y0 + (A/(w*sqrt(PI/2)))*exp(-2*((x-xc)/w)^2) Weighting: y No weighting Chi^2/DoF = 5.6732 R^2 = 0.9328 y0 xc w A 0 0 8.00535 4.75858 146.08526 0.15951 0.32113 8.50044 Population Population 100 80 60 40 20 0 15 10 5 0 S/N= ~8:1 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Time (sec) 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 SNR Time (Sec) Time (Sec) At a frame rate of 40ms, typical experiments have ~8:1 S/N and ~4 sec Cy3 lifetime 15 9-base pair DNA oligo Single-molecule Fluorescence 20000 15000 10000 5000 0 0 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0 8 16 24 32 40 48 56 8 16 24 32 40 48 56 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 -0.1 Cy5 Ensemble Cy3 Biotin Single-frequency h FRET 0.5 1.0 1.5 2.0 Time (seconds) Time (seconds) 18-base pair DNA oligo 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0.5 1.0 1.5 2.0 15-base pair DNA oligo 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0.5 1.0 1.5 2.0 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 12-base pair DNA oligo 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 -0.1 9-base pair DNA oligo 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 -0.1 6-base pair DNA oligo 0.5 1.0 1.5 2.0 0.5 1.0 1.5 2.0 0.5 1.0 1.5 2.0 FRET ~0.15 = ~10 16 h Single-molecule Total Internal Reection (TIR) Fluorescence Microscopy (principle) Quartz Slide F Objective Long-pass Filter Image analysis Lens CCD Dichroic Filter Mirror Biological activities of molecular assemblies Cy3 (50S) Cy5 (aminoacid) h Quartz surface 3 100 m 17 Single-molecule observations of tRNA selection E h P A + EF-Tu(GTP)Phe-tRNAPhe 3' (Cy3 linked to modied residue at position 8) (Cy5 linked to modied residue at position 47) Single-molecule observations of tRNA selection E h P A Expected result: FRET corresponding to ~40 3' 18 Fluorescence TIR image volume (~0.5 fL) 1100 1000 900 800 700 600 500 400 300 200 100 0 -100 -200 -300 0 Arrival 2 4 6 8 10 12 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 -0.1 0 2 4 6 8 10 12 Inject ------50M------ FRET At least three states have been identified Time (0.1 seconds) FRET data can be quantified in a manner in the same manner as ion channel recordings using Hidden-Markov modeling Qin et al. (1997) McKinney et al. (2006) 19 TIRFwith Other Methods Fluorescence Correlation Spectroscopy AFM Two-Photon Flash Photolysis Epifluorescence Forrester Resonance Energy Transfer (FRET) Electrochemical measurements TIRF Microscopy Biophysical Methods TIRF Limitations Only able to view the footprint of a cell. Light can be scattered/ redirected inside cells Laser illumination may not be uniform Can be tricky and/or expensive to set up. Too many acronyms!! TIRF Microscopy TIR-FM Evanescent Wave/Field Microscopy TIRF Microscopy Biophysical Methods 20 References Toomre, D., Manstein, DJ. 2001. Lighting Up the Cell Surface with Evanescent Wave Microscopy. TRENDS in Cell Biology; 11: 298-303. Axelrod, D. 2001. Total Internal Reflection Fluorescence Microscopy in Cell Biology. Traffic; 2: 764-774. An,S.J., Almers, W. Tracking SNARE Complex Formation in Live Endocrine Cells. Science 306:1042 (2004) Yildiz, A., et al. 2003. Myosin V Walks Hand-Over-Hand: Single Fluorophore Imaging with 1.5-nm Localization. Science; 300: 2061 (2003) Mathur, A.B., et al. Atomic Force and Total Internal Reflection Fluorescence Microscopy for the Study of Force Transmission in Endothelial Cells. Biophysical Journal; 78: 1725 (2000) Schapper, F. et al. Fluorescence imaging with two-photon evanescent wave excitation. Eur Biophys J; 32: 635643 (2003) http://www.microscopyu.com/articles/fluorescence/tirf/tirfintro.html http://www.olympusmicro.com/primer/techniques/fluorescence/tirf/tirfhome. html Biophysical Methods TIRF Microscopy 21
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