OPERATION
Carl Zeiss
Calibration of the Angle Using the Motorized Slider Laser TIRF
Laser TIRF 3
82
423683-7144-001
02/2014
4.8
Calibration of the Angle Using the Motorized Slider Laser TIRF
4.8.1
Measurement of Penetration Depth
The penetration depth of the evanescent field under TIRF conditions is determined using the following
equation.
( )
( )
[
]
1
sin
/
sin
4
2
1
2
0
−
=
g
n
d
α
α
π
λ
Where
0
λ
= 488 nm,
1
n
= 1.518,
2
n
= 1.33
→
g
α
= 61.2°
The calculated average refractive index of "normal" cover slips is used below: n
1
=1.5255.
4.8.2
Snell's Law
( )
( )
2
2
1
1
sin
*
sin
*
α
α
n
n
=
The critical angle is reached at
2
α
= 90°. Therefore:
( )
1
*
sin
*
2
1
1
n
n
=
α
This corresponds to the point at which the intensity of the grey-scale value has fallen to half the
maximum in the pupil.
1
2
1
)
sin(
n
n
=
α
Summary of Contents for TIRF 3
Page 1: ...Laser TIRF 3 Imaging System Operating Manual...
Page 110: ......
