GenePix 4100A Microarray Scanner User Guide
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PMT
The 4100A Microarray Scanner uses a high-sensitivity, low-noise PMT to
detect the emitted fluorescent light. A PMT converts incident photons
into electrons through the photoelectric effect. When an incident
photon impinges on the active surface of the PMT (the photocathode),
an electron is generated. The electron flows through a series of electron
multipliers (dynodes) to the anode. The amount of current that flows
from the anode is directly proportional to the number of photons at the
photocathode.
The amount of amplification a PMT can produce depends on the number
of dynodes in the PMT, and the voltage that is applied to the PMT. It is
possible to achieve a signal amplification of 10
7
. Increasing the PMT
voltage setting in the GenePix Pro Software, increases the signal
amplification of the PMT.
The output of a PMT is typically linear over a wide range of incident light
intensities. However, there is a very non-linear relationship between
the voltage of the PMT and the PMT output. Therefore, a slide scanned
at a PMT voltage of 800 V will not be twice as bright as the same slide
scanned at 400 V.
Signal amplification by the PMT is controlled by adjusting the PMT gain
in the GenePix Pro Software. The PMT gain is related to the PMT voltage
by a calibration constant such that:
PMT gain = PMT voltage / calibration constant
The calibration constant is derived by performing the system calibration
in Hardware Diagnostics and will ensure that the scanner performance
is self-consistent over time. For an uncalibrated scanner the constant is
1.000.
Note:
When the PMT gain is increased, the sensitivity to non-specific
fluorescence is also increased, as is the electronic noise in the system.
The increase in noise can often be overcome by line averaging;
however, the result is decreased acquisition speed and increased
photobleaching. In general, the signal-to-noise ratio is not improved
by increasing the PMT voltage.
GenePix_4100A.book Page 27 Friday, October 22, 2010 3:21 PM
