Biomedical Engineering Reference
In-Depth Information
Tabl e 3. 2
The advantages and limitations of FLIM over steady-state (intensity-based) imaging
microscopy
Aspects
Advantages
Limitations
General
applications
The fluorescence lifetime of a
fluorophore is sensitive to its
environmental changes; FLIM
can be a good choice for
visualizing those changes that
cannot be revealed by
steady-state imaging methods,
such as calcium binding,
change in pH, etc.
FLIM is preferably applied to live
specimens in life-science
applications, since it can be
difficult to distinguish the real
fluorescence lifetime changes
from those caused by the
fixative in fixed specimens
FRET measure-
ments
Since only donor signals are
measured for determining the
energy transfer efficiency in
FLIM-FRET, the method does
not usually require the
corrections for spectral
bleedthrough that are necessary
for intensity-based
measurements of sensitized
emission from the acceptor (see
Sect.
3.4
)
In FLIM-FRET, it is important to
make sure that the donor
fluorophores reside in the same
microenvironment in both
donor-alone control specimens
and specimens containing both
donor and acceptor
fluorophores, since the
fluorescence lifetime of a
fluorophore can be affected by
its microenvironment
The FLIM-FRET approach has the
capability to estimate the
percentage of the interacting
and noninteracting donor
populations, which cannot be
distinguished by most of
intensity-based FRET methods
The donor molecule whose
intrinsic lifetime has multiple
components is not suitable for
FLIM-FRET, since it will
complicate the data analysis
Data acquisition
and analysis
FLIM can provide both lifetime
and intensity information
As technology improves, the FLIM
data acquisition time is
expected to decrease. Currently
this time varies from seconds to
minutes depending on the setup
of a FLIM system - particularly
the detector, as well as the
specimens to be imaged
Fluorescence lifetime
measurements are insensitive to
the change in fluorophore
concentration, excitation
intensity, or light scattering and
to some extent of
photobleaching - all these
factors commonly induce false
information in intensity-based
imaging
Some understanding of the physics
of fluorescence lifetime is
required for processing the
FLIM data and interpretation of
the results, particularly for a
biological system
FLIM can help to discriminate the
intrinsic fluorescence of a
specimen, i.e., autofluorescence
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