2. General Description
2011-09
Instructions for Use for INFINITE M1000 PRO No. 30064852 Rev. No. 1.0
15
2.2 Measurement
Techniques
The following sections provide an introduction to the
INFINITE M1000 PRO
measurement techniques. To keep this chapter compact, a few simplifications
have been made. For details, see the references.
2.2.1 Fluorescence
The
INFINITE M1000 PRO
offers the basic fluorescence measurement technique
and some even more sophisticated variants:
A.
Fluorescence Intensity (FI, or simply Fluorescence)
B.
Fluorescence Time Resolved (TRF)
C.
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 fluorescence polarization (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 and then be referenced as
HTRF (TR-FRET). Fluorescence Time Resolved (TRF) measurements should not
be confused with Fluorescence Lifetime measurements.
Fluorescence Intensity
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 fast 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
A) Fluorescence Intensity (FI)
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, type of
solvent, etc. may significantly affect the fluorescence quantum yield and therefore
the measurement results.
Flash Fluorescence and FI Kinetic
For high sensitivity Flash Fluorescence assays, the measurement is done just
after dispensing the activating reagent or after a short delay time.
The measurement position is not identical to the injector position. The movement
between measurement position and inject position takes
≤
500 ms.
