Biology Reference
In-Depth Information
Time
Intensity decay
Instrumental
response function
Exponential decay
Figure 5.10 Illustration of the convolution product. When the instrumental response
function (IRF) of the FLIM system is temporally short, the measured intensity decay is
almost identical to the sample decay. If the IRF is large, the collected decay becomes
very much different of the sample decay, and it becomes thus necessary to take into
account this IRF to obtain correct lifetime estimations.
Quantitative measurements require multiexponential decay analysis. 6 The
proportion of donor molecules involved in energy transfer is given by the ratio
of the two exponential decay amplitudes, a 2 / a 1 , while the average coupling
efficiency of the FRET pairs is given by the lifetime ratio t 2 / t 1 .
Amajor complication of many in vivo FRET experiments is the large and
differential contribution of cellular autofluorescence to the measured donor
and acceptor fluorescence. Autofluorescence contributions can be some-
times corrected by acquiring an additional image, 25 or they can be mini-
mized by the use of spectral lifetime image microscopy. 26
When autofluorescence is substantial, determination of FRET efficiency
by fluorescence lifetime measurement might be advantageous because
autofluorescence lifetime is usually very short and can be included in the
fit of lifetime data. Finally, one has to consider the limitations imposed by
the available instrumentation. For example, lifetime measurements require
relatively sophisticated instrumentation, which is not yet widely available.
3.8. Few considerations on fluorescent proteins
Green fluorescence proteins from the jellyfish Aequorea victoria are well char-
acterized and have provided myriad applications in cellular biology. 27,28
GFP engineering has generated a large range of fluorescent proteins of
Search WWH ::




Custom Search