Online Fingerprinting
The functionality of
Online Fingerprinting
can be
used to separate overlapping emissions even while
a time series is being recorded. This may be of
decisive importance where dynamic processes are
investigated.
Here, a reference spectrum is assigned to each
image channel before image acquisition starts.
During the experiment proper, lambda stacks are
acquired and immediately unmixed in a back-
ground operation. The user sees the unmixed mul-
tichannel image during the acquisition of the time
series.
Online Fingerprinting
is of advantage espe-
cially in spectral FRET experiments and in studies
of dynamic processes with fluorescent proteins.
Visualization of FRET by means of acceptor photobleaching
Expression of a FRET-positive protein construct (CFP linker citrin)
in HEK 293 cells.
Recording conditions: Simultaneous excitation with 458 and 514 nm.
Spectral detection from 462 to 655 nm in Lambda Mode.
Online Fingerprinting and simultaneous display of the two-channel image
(CFP blue, citrin green).
During the combined time-&-bleaching series (bleaching region marked),
citrin (green channel) as a FRET partner is destroyed by intensive
irradiation with 514 nm. The decrease in FRET is visible as an increase
in CFP fluorescence (blue channel).
Specimen: PD Dr. M. Schäfer,
Charité University Hospital,
Berlin, Germany
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