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0:00
Today, we'll talk about
time-resolved
fluorescence microscopy
in high-resolution
brain imaging.
0:10
This lecture will
consist of two parts;
the first one will be about
fluorescence lifetime
imaging, or FLIM,
exemplified by the monitoring
of intracellular nanomolar
calcium concentrations,
and the second
part will be about
time-resolved fluorescence
anisotropy imaging which
you'll try to explain by
measuring molecular
mobility on the nanoscale.
0:41
First, we have to
understand why and when we
actually need
time-resolved imaging
inflorescence microscopy
of the brain.
Well, you probably know that
in the majority of cases,
fluorescent indicators increase
their emission intensity
upon binding to the signal
molecule of interest.
However, the emission
intensity signal
might depend directly on
the local concentration of
the indicator as well as
on the optical properties
of the tissue.
This actually makes it pretty
difficult to assess
quantitatively how
much of the signal and molecule
is reported by the
fluorescence indicator,
or historically,
concentration measurements
of signaling molecules
were, therefore, achieved using
so-called ratiometric
indicators.
The ratiometric
indicators changed
either the absorption or
emission spectrum
upon binding to
the signaling molecule so that
the ratio between
different parts
of the emission spectrum
reports the signaling
molecule concentration which,
therefore, does not depend on
the indicator concentration.
