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Why lifetime imaging?
The fluorescence lifetime is the signature of
a fluorescent material; it is the exponential
decay in emission after the excitation of a
fluorescent material has been stopped. FLIM
(Fluorescence Lifetime Imaging Microscopy)
is a technique to map the spatial distribution
of lifetimes within microscopic images and it
allows measurements in living cells as well
as in fixed materials.
Because of the fact that some phenomena
do affect fluorescence lifetimes, the lifetime
is used to detect these phenomena leading
to various applications such as: ion imaging
(pH measurements), oxygen imaging,
probing microenvironment, and medical
diagnosis. Moreover, the most powerful
FLIM-application in biology is Fluorescence
Resonance Energy Transfer (FRET). When
two fluorescent molecules (or two fluorescent
labeled epitopes within a protein) are in very
close proximity, i.e. less than 9 nm, the energy
of the one fluorescent (donor) molecule (e.g.
GFP) is transferred in a nonradiative process
to the other fluorescent (acceptor) molecule
(e.g. mCherry). In this way, the lifetime of the
donor molecule decreases and this change
can be measured quantitatively by FLIM.
Frequency domain method
The homodyne frequency domain FLIM
method requires a modulated light source
and a modulated detector. In the LIFA system
these are the LED and the intensified CCD
camera. Both are modulated at exactly the
same frequency, but with an adjustable
difference in phase. The emission intensity
shows a phase-shift (or delay) with respect
to the excitation as well as a decrease of
modulation-depth. These two parameters
depend on the fluorescence lifetime of the
sample and the modulation frequency and
are measured to calculate the fluorescence
lifetime in each pixel of the image.
Fluorescence Lifetime Imaging
Microscopy (FLIM)
Pseudo colored lifetime image of
mammalian cells stably expressing
GFP in the nuclei. Additionally,
two cells are transiently
transfected with GFP-RFP at the
plasmamembrane. The lifetime
of the GFP in the membrane
is shorter (blue) compared to
the lifetime of GFP in the nuclei
(green). This is due to FRET
between GFP and RFP. (Courtesy of
BCF/ IRI Lille, France)
