Measurement Techniques; Fluorescence - tecan Infinite 200 PRO Instructions For Use Manual

2. General Description
2.4

Measurement Techniques

2.4.1

Fluorescence

A) Fluorescence Intensity (FI)
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The following sections provide an introduction to the Infinite 200 PRO
measurement techniques when fully equipped. To keep this compact, a few
simplifications have been made. For details see the references.
The Infinite 200 PRO offers the basic fluorescence measurement technique and
some even more sophisticated variants:
•
Fluorescence Intensity (FI) (or simply Fluorescence)
•
Fluorescence Resonance Energy Transfer (FRET)
•
Fluorescence Time Resolved (TRF)
•
Fluorescence Polarization (FP)
FI may also be used to measure Fluorescence Resonance Energy Transfer
(FRET). For some microplate applications, FRET offers advantages over FI and
TRF, because they simplify assay preparation. These preferably apply for mix
and measure binding studies. Compared to FP, FRET requires both binding
partners to be labeled in a suitable way. On the other hand, FRET may utilize
TRF labels for increased sensitivity, then being referenced as HTRF
(Homogeneous TRF).
TRF should not be confused with Fluorescence Lifetime measurements.
Fluorescent molecules emit light of specific wavelength when struck by light of
shorter wavelength (Stokes Shift). In particular, a single fluorescent molecule can
contribute one fluorescence photon (quantum of light). This is a part of the
energy, which has been absorbed before (electronic excitation), but could not be
released quickly enough into thermal energy.
The average time it takes between excitation and emission is called the
fluorescence lifetime. For many fluorescent molecular species, fluorescence
lifetime is on the order of nanoseconds (prompt fluorescence). After excitation,
fluorescence emission occurs with a certain probability (quantum yield), which
depends on the fluorescent species and its environmental conditions.
For a detailed treatise on fluorescence techniques and applications see:
Principles of Fluorescence Spectroscopy by Joseph R. Lakowicz, Plenum
Press.
In many microplate applications, the intensity of fluorescence emission is
measured to determine the abundance of fluorescent labeled compounds. In
these assays, other factors having an influence on fluorescence emission need to
be controlled experimentally. Temperature, pH-value, dissolved oxygen, kind of
solvent etc. may significantly affect the fluorescence quantum yield and therefore
the measurement results.
IFU for Infinite 200 PRO No. 30052730 Rev. No. 1.6 2016-04