Fluorescence Depolarisation Effects

Consequently, a signal proportional to I = Ip + 24 must be recorded to reject the rotational relaxation from a lifetime measurement. However, for detection along X, Z or any other direction in the X-Z plane 4 + 4 deteeted. The problem can be solved by placing a polariser in the detection path. The polariser is rotated by 54.7° from the polarisation of the excitation. The detection efficiency of 4 is then twice the effieieney of 4, resulting in detecting a signal proportional to 7 = 4 + 24. 

Except for a few special cases, there are similar polarisation effects for other angles between the optical axis of the excitation and the X-Z plane, different polarisation of the excitation, and even for unpolarised excitation. Methods to reject depolarisation effects from the measured decay curves in different geometric configurations are discussed in detail in [355, 389, 476]. 

The considerations above apply strictly only for negligible aperture angles of the excitation and detection light cones. However, optical systems designed for 

For NA= 1.4 the field components. Ex and Ez, are expected to be 7.5% and 40.6% of Ey. Bahlmann and Hell [18] measured an X component of the intensity, Ix, of 1.51%. The X component causes a small crosstalk offs into the Ip channel. The Z component results in excitation of molecules with dipoles oriented along the Z axis, and, consequently, in an increase of Is. 

A similar effect occurs in the detection light path. A part of the Z component of the fluorescence is detected both in the Is and in the Ip channel. As a result, a substantial amount of fluorescence polarised in the Z direction is recorded, i.e. additional Is. The total intensity is therefore not / = 7, 4- 2 /j as in the case of narrow beam angles but 

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