Molecular Devices FLIPR Tetra, User Manual

Page: 33 / 302

FLIPR
®
Tetra High Throughput Cellular Screening System User Guide
0112-0109 H 33
CCD Camera Options
Two camera options are available for purchase with the FLIPR
®
Tetra
System. A standard, EMCCD (Electron Multiplying CCD) camera is
recommended for fluorescence only experiments, while the more
sensitive Aequorin ICCD (Intensified CCD) camera is designed for both
fluorescence as well as luminescence assays.
Standard EMCCD Camera
The CCD camera is located directly beneath the read plate on the five-
position stage. The camera is thermoelectrically cooled and requires
about five minutes to reach its operating temperature of -60 ± 2 °C.
The camera uses frame-transfer technology that shifts pixel values
from the exposed portion of the chip to the data processing portion.
This method allows a high acquisition rate and eliminates the possibility
of camera shutter failure.
The camera is an integrating-type detector using temporal integration
to build up the signal-to-noise ratio. Depending on the intensity of the
emitted light (reliant on dye efficiency and LED power), it may be
necessary to use longer camera exposures. This prevents the measured
fluorescence signal from being dominated by detector noise.
Images are taken of the bottom of the entire plate for a time specified
in the ScreenWorks Software protocol; exposure time can be set from
0.05 to 30 seconds. In ratiometric experiments for example , where two
or more wavelengths are measured, the number of images captured
increases so that an image is taken for each wavelength at the specified
rate. From each image a relative light value is calculated for each well.
The FLIPR
®
Tetra System reports relative light units (RLUs) in a range
from zero to approximately 12,000.
The light intensity detected by each pixel on the CCD chip can be
amplified using the Gain setting in ScreenWorks Software ( Settings
Process ). This parameter has a range from zero to 240. Amplification
is exponential, with increments increasing as you go higher up the
range. Fluorescence assays typically use a gain of 130 whereas
WARNING! Do not use the camera before it has reached its
operating temperature—this will result in noisy data. Check the
camera temperature on the Instrument Status panel before
starting an experiment.
Note: Relative light units in the FLIPR
®
Tetra System do not have the
same value as those of previous FLIPR
®
Tetra Systems.