tecan infinite 200 Instructions For Use Manual page 97

Flash Settings
Timing Parameters for Time Resolved Fluorescence
Settle Time
2008-07
Where U is the voltage, Gain is the selected gain setting, 255 is the maximum
possible gain and 1250 V is the maximum voltage supply of the PMT.
Example:
A gain of 100 corresponds to a voltage supply of 490 V:
100
= =
U * 1250 490
255
On the fly measurements with 1 flash (read) per well are possible for all plate
types; however, measurement precision at low light levels depends on the
reading time while fluorescence signal can be received.
Increase the number of flashes (reads) per well until noise of BLANK
wells does not further improve, or until measurement time per well
For prompt fluorescence it does not help to increase the default integration time,
because the detector will not receive more signal once the flash has vanished.
For TRF, signal integration parameters need to be adjusted according to the
label. The start of the signal Integration Time is delayed against the preceding
flash by a Lag Time. TRF timing parameters may be established with the
following procedure:
1. As a starting point you may take the Fluorescence Lifetime of the label for
both Integration Time and Lag Time.
2. Coarse tuning: With Integration Time being fixed reduces the Lag Time to
maximize Signal to Background (S/B).
3. Fine tuning: With Lag Time being fixed extends the Integration Time and
check, if S/B further improves.
4. Optional Fine-tuning: With either timing parameter being fixed you may vary
the other one and check, if S/B further improves.
Before measuring a well, a settle time may be set. Due to the stop and go motion
of the plate carrier the meniscus of the dispensed liquid may still vibrate while
signal is integrated. This can give rise to fluctuations of the measured values. The
effect has been observed in wells of 96-well plates and larger wells. In particular,
it is critical with absorbance measurements.
Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4
6. Operating the infinite® 200
V
Note
becomes unacceptable.
97