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The Measure step contains the
following measurement types with their own parameters (only “Single” is
described further):
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Single
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Dual
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Kinetic
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Dual Kinetic
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Scanning -
Monitor
Incubate Step:
The incubate step sets the time and temperature for the incubation in
the instrument.
Shake Step:
The shake step shakes the plate using the given parameters. It is also
possible to use shaking as a background task. Shaking is then on when
the reader does not have any other task to perform.
Dispense Step:
The Dispense Step dispenses the defined volume against the wall of the
well.
Dispense and Measure Step:
Dispensing and Measuring can be started at the same time. The step has a
check box “Overwrite general step settings”, which breaks up the
dispensing and measuring to work in loops (i.e, if the “Execute by 1…n
wells” value is 1, dispensing and measuring is carried out well-by-well
through the selected area.)
Pause Step:
The “Pause” step pauses the run, drives the plate out and gives the
alarm, if desired.
Save/Load Step:
The Save/Load step can be used to save a Results Desktop sheet in a
file or to load a sheet from a file to the Results Desktop.
In this type of step, each of the selected wells is
measured once. A single measurement result is taken from each
measurement point.
The general parameters for all the measurement types are:
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Step name:
The name is generated automatically or given by the user and must be
unique.
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Unit:
The measurement unit for the reading
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Step time:
The time spent before advancing to the next step. Expressed as hh::mm::ss.s.
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Measurement method:
Selection of the measurement method, either fluorometric or luminometric.
This field can be changed if
Overwrite general step settings
is selected.
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Integration time:
Defines the length of time used to obtain one measurement result from a
measurement point.
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Lag time:
The time before the measurement is started.
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Filter:
It is the wavelength of the filter in nanometers.
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Overwrite general step
settings: By checking this checkbox, you can
define the area and plate layout for this step to be different from
those defined in the general step. When this step is selected, the area
definition, layout and the settings of the General step are used as
default for the current step.
Parameters:
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Fluorometric
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Luminometric
The fluorometric parameters are :
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Excitation: Wavelength for the excitation filter selected from the
drop-down list (expressed in nanometers).
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Emission: Wavelength for the emission filter expressed in nanometers.
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Pairs: The users can select a validated filter pair.
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Beam: Normal or Small beam selection. If the beam of the instrument is
changed, the beam state is updated.
The
Luminometric parameters
are (FL only):
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Filter: The appropriate wavelength is selected from the drop-down
list.
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Use default PMT voltage: By selecting this check-box, the standard
factory calibration PMT (photomultiplier tube) voltage is used in the
measurements. The software obtains the actual voltage from the
instrument and it can be viewed in the Instrument Status sheet.
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PMT voltage: If the Use default PMT voltage check box is deselected,
you can change the PMT voltage value from 300 to 1000.The initial value
is factory-calibrated. Changing this value may become necessary if very
high signals are measured.
| LUMINOMETRIC MEASUREMENT: |
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This has several phases:
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The anode voltage of the PMT is set according to the selected value of
the software.
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The excitation filter wheel is driven in between the two filter
positions and the excitation light is blocked even when the light is
off. The emission filter slot 8 is held in place until the plate door is
closed. The selected filter is then driven to the measurement positions.
The emission filter slot 8 is also used for the blanking procedure to
compensate for the PMT drift.
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In the blanking procedure the instrument reads the PMT dark signal if
the dark signal is drifting. If the bottom value is drifting, the
results are compensated. To obtain the most accurate results, the
measurement time of the dark must be as long as the measurement time of
the sample. The first half is measured before the sample measurement and
the second half after the sample measurement.
Note: The selection in the settings of a session, ‘Execute by 1 – n
wells’, also controls blanking so that the whole selected group of wells
is measured using common blanking.
Luminometric Scaling:
The measured results are obtained in Relative light Units(RLU) as no
standard luminescence units exist. Scaling is performed similarly as in
fluorometric scaling.
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