Time-Resolved Fluorescence Read Mode - Molecular Devices SpectraMax Paradigm User Manual

SpectraMax Paradigm Multi-Mode Detection Platform User Guide

Time-Resolved Fluorescence Read Mode

Time-resolved fluorescence (TRF) is a measurement technique that depends on three
characteristics that lead to better discrimination between the specific signal, proportional to
the quantity of label, and the unspecific fluorescence resulting from background and
compound interference:
Pulsed excitation light sources
Time-gated electronics faster than the fluorescence lifetime
Labels with prolonged fluorescence lifetime
The time-gating electronics introduce a delay between the cut off of each light pulse and the
start of signal collection. During the delay, the unspecific fluorescence (caused by test
compounds, assay reagents, and the microplate) vanishes while only a small portion of the
specific fluorescence from the label is sacrificed. Enough of the specific signal remains during
the decay period with the added benefit of reduced background.
In Time-Resolved Fluorescence read mode, the instrument detects the extremely long
emission half-lives of rare earth elements called lanthanides such as europium (lifetime of
about 700 µs), samarium (lifetime of about 70 µs), or terbium (lifetime of about 1000 µs).
Applications of Time-Resolved Fluorescence
Time-resolved fluorescence (TRF) is widely used in high throughput screening applications
such as kinase assays, and is useful in some fluorescence immunoassays, such as DELFIA
(dissociation-enhanced enzyme linked fluorescence immunoassay). TRF is also useful in
some assay variants of TR-FRET (time-resolved fluorescence resonance energy transfer) in
which the FRET acceptor label acts as a quencher only and does not emit fluorescence. The
proximity between donor label and acceptor (quencher) is then quantified by the intensity
decrease of the donor label.
DELFIA requires washing steps as in an ELISA, but the TR-FRET assay involving quenching is a
homogeneous microplate assay technique and requires only mixing and measuring—no
wash steps are required. It can also be miniaturized, which makes it useful for high-
throughput screening applications.
The Cisbio Bioassays HTRF (Homogeneous Time-Resolved Fluorescence) technology is a
proprietary time-resolved fluorescence technology that overcomes many of the drawbacks
of standard Fluorescence Resonance Energy Transfer (FRET) techniques, such as the
requirements to correct for autofluorescence and the fluorescent contributions of unbound
fluorophores. See
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HTRF Read Mode on page 45.
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