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
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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
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TIRF Microscopy
Biophysical Methods
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What is the penetration depth for TIR illumination?
TIRF Microscopy
Biophysical Methods
Calculating the TIR illumination penetration depth
TIRF Microscopy
Biophysical Methods
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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
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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
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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
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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
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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
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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$1 % n 2 2
!
Using a 1.45 NA objective and 488 laser illumination the penetration depth can be varied between 224 nm and 87 nm. In practice the laser spot in the back focal plane has a finite diameter such that the range will be slightly less.
TIRF Microscopy
Biophysical Methods
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TIRF Excitation through the Objective without Laser:
TIRF Microscopy
Biophysical Methods
TIRF Illumination with a Range of Incident Angles:
dP (" max ) =
#0
2 4$ n12 sin 2 " max % n 2
= 87nm
dP (" min ) =
#0
2 4$ n12 sin 2 " min % n 2
= 224nm
!
TIRF Microscopy
Biophysical Methods !
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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.
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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
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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
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Single Molecule Studies
TIRF Microscopy
Biophysical et al A. Yildiz, Methods
Single Molecule Studies
~ MOVIE ~
TIRF Microscopy
Biophysical et al A. Yildiz, Methods
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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
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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
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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
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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'
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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)
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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
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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
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