Zeiss TIRF 3, Руководство по эксплуатации

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INSTRUMENT DESCRIPTION
Carl Zeiss Operating Principle Laser TIRF 3
24 423683-7144-001 02/2014
2.2 Operating Principle
The analysis of images obtained with conventional wide-field fluorescence excitation is often difficult
because of background fluorescence emitted in out-of-focus planes. This is due to the comparatively low
Z resolution achievable with this illumination technique.
By contrast, fluorescence excited by total internal reflection (TIRF:
T
otal
I
nternal
R
eflection
F
luorescence)
yields excellent Z resolution. The evanescent field is typically 200 nm thick.
The TIRF method is based on the behavior of light when making the transition between media with
different refractive indices, for example from glass to water. When traveling from the optically denser to
the optically less dense medium, the light beam is refracted away from the normal to the surface at the
point of incidence.
Here, Snell’s law of refraction applies (Fig. 2-1/
A ):
2 2 1 1
sin * sin *
φ φ
n n
=
Φ
1
Angle of incident beam
1 Objective
Φ
2
Angle of refracted beam
2 Immersion oil
n
1
Refractive index of optically denser medium 3 Cover slip
n
2
Refractive index of optically less dense medium 4 Evanescent field
Fig. 2-1 Schematic drawing of the beam path (A: beam at critical angle; B: total reflection)