Eppendorf AF2200 Plate Reader - Manual (Page 40)

Operation
Eppendorf
® PlateReader AF2200
English (EN)
40
In this equation, U is the voltage, Gain is the selected amplification, 255 is the maximum amplification and
1250 V is the highest PMT voltage supply possible. Example: A gain setting of 100 corresponds to a supply
voltage of 490 V:
6.3.3 Flash settings
Quick measurements with 1 flash (measurement) per well can be completed with all plate types. However,
the measuring precision at a low light intensity depends on the measuring time during which the
fluorescence signal can be received.
For prompt fluorescence, increasing the standard integration time does not help because the detector can
no longer receive a signal after the flash has disappeared.
6.3.4 Settle time
A settle time can be set before measuring a well. Because of the stop-and-go movement of the plate
compartment, the meniscus of the dispensed liquid may continue to vibrate while the signal is being
integrated. This can lead to fluctuations in the measured values. This effect has been observed in the wells
of 96-well plates and larger wells.
6.3.5 Recommended filter
To ensure an acceptable, low background, the upper cut-off value for excitation wavelengths and lower
cut-off value for emission wavelengths must be separated, i.e. the filter bandpasses should not overlap. This
compromise depends on the filter properties. For many fluorescent molecules, the emission and the quality
can be improved by moving the filter bandpass width away from the other bandpass filter.
6.3.6 Recommended microplate types
Generally, black microplates are recommended for high fluorescence sensitivity. For measuring in the
Lower fluorescence mode, we recommend black plates with a transparent bottom. You should not use
volumes that are less than one-third of the maximum volume. When using low volumes, check to see if a
suitable plate type is available.
Increase the number of flashes (measurements) per well until the S/N ratio of the empty wells
stops improving or until the measuring time per well becomes unacceptable.
If the maximum values for fluorophore excitation and emission are nearly the same, they
should not be directly translated into central wavelengths for fluorescence filters.
V = 490V 1250 *
255
100
U =