Biology Reference
In-Depth Information
5.2.3 Nonfitting-based approaches
In the previous section, it was mentioned that the correct analysis of the ac-
quired fluorescent signal with fitting methods is time consuming and neces-
sitates a high level of expertise. In order to simplify the analysis of FLIM
images and to make it accessible to the nonexpert user, novel methods based
on nonfitting approaches have been developed recently.
89-92
This section
limits the review to two approaches that are applicable to a large range of
lifetime acquisition techniques: the minimal fraction of interacting donor
(
mf
D
)
90
and the polar approach or phasor.
89,91
The rapid lifetime
determination, which is restricted to time-gated FLIM images with two
temporal channels, is voluntarily omitted.
93,94
The minimal fraction of interacting donor
mf
D
, which was introduced by
Padilla-Parra
et al.
,
90
can be applied with all TD FLIM techniques (TCSPC,
time-gated, etc.). It is defined by
1
hi=
t
2
hi=
mf
D
¼
½
5
:
37
2
ð
2t
ðÞ
1
Þ
where t
2
is the lifetime of the donor alone and
is the mean lifetime de-
fined by Eq.
(5.12)
(see previous section). The computation of
h
t
i
is straight-
forward and can be performed pixel by pixel and recovered online during
FLIM acquisition. When the lifetime of the donor alone t
2
is mono-
exponential and known (implying that it has been measured in a previous
experiment which is typically not performed in a biosensor experiment),
it can be deduced from Eq.
(5.37)
that the computation of the
mf
D
is a simple
calculation that can be performed online on a standard computer. This is a
major advantage when following the biosensor activity over time in live
cells, since the user has immediate access to quantitative information, which
is the minimal fraction of the interacting donor. This indicator varies be-
tween 0 when there is no interaction and 1 when all the donors interact.
It is a robust indicator, but the reader is reminded that it gives information
on the minimum of the interacting proportion alone and not on the exact
quantity a
1
.
The polar approach or phasor is another nonfitting method that has
been successfully applied in FLIM experiments. This method was initially
described by Jameson
et al.
95
and then successively improved by differ-
ent groups.
96-98
It consists in converting the lifetime image into a new
two-dimensional histogram called “polar” or “phasor.” In this polar
representation, each point
h
t
i
that
is defined with [
u; v
] coordinates
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