Chapter 5: Read Modes and Read Types
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You do not designate luminescence read times based on multiple reads per well, but rather
by choosing the total integration time that is between 1 ms and 1,500 ms. Typical
luminescence assays require between 500 ms and 1,000 ms for integration time.
If wells incubate for a long period of time, then you should use the Automix function to mix
the wells before the read.
If the signal is always higher in the first wells read (for example, column A), you might need to
dark adapt the plate to reduce the auto-luminescence of the white plastic. Because the auto-
luminescence decreases quickly, use the control panel Drawer button to load the plate, and
then wait 1–2 minutes before you initiate the read and determine if the read-out is more
consistent across the plate.
Time-Resolved Fluorescence Read Mode
The Time-Resolved Fluorescence read mode is a secondary read mode.
Time-Resolved Fluorescence 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 cutoff of each light pulse and the
start of signal collection. During the delay, the unspecific fluorescence (caused by test
compounds, assay reagents, and the plate) 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 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 plate 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.
