PerkinElmer EnVision, Instrument Manual

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Functional description
25
AlphaScreen / HTS AlphaScreen mode
Unlike the other modes, AlphaScreen uses a laser to excite the sample. A
special dichroic mirror module is used to direct the excitation laser light into
the sample well. In the case of standard AlphaScreen a light guide is used to
direct the light to this mirror module. In the case of HTS AlphaScreen the
light is directed straight to the sample from the laser. The excitation
wavelength is 680 nm. AlphaScreen donor beads are excited by the laser
beam. A photosensitizer in the donor bead converts ambient oxygen to a
more excited singlet state. These oxygen molecules diffuse to the bound
acceptor bead where they react with a thioxene derivative generating
chemiluminescence at 370 nm. This activates fluorophores in the bead
which emit fluorescence light in the range 520 to 620 nm. The long half-life
of the signal permits the measurement to be time-resolved to reduce the
contribution of background fluorescence. The fact that the excitation
wavelength is longer than the emission, further reduces the background, as
does the fact that wavelength itself is long.
In the case of AlphaScreen the emission light pathway involves the
fluorescence light passing back through the mirror module and then through
the filter into the photomultiplier tube. There is also a shutter in this
pathway which is open during the emission phase but closed during
excitation to protect the detector from the laser light.
Figure 29. Laser excitation from above/reading from above. Mirror module
must be the special AlphaScreen dichroic
In the case of HTS AlphaScreen, the detector is directly above the plate and
reads the well adjacent to the one excited by the laser. Light passes through
an aperture and into the detector.