Biophotonics_Concepts_to_Applications

offluorescence from excited molecules, FLIM can detectfluorophore-fluorophore
interactions and environmental effects onfluorophores [ 13 – 17 ].
As is noted in Fig.9.4, following a transition to an excited energy state a
molecule will transition to a lower ground-state level in the order of 10−^9 s. As
Fig.9.6shows, in an ensemble of excited molecules, thefluorescence intensity
from spontaneously emitted photons will decay with time t according to the
exponential function

I(t)¼I 0 expðt=s 0 Þð 9 : 2 Þ

Here τ 0 is the normally observed fluorescence lifetime and I 0 is the initial
fluorescence intensity at time t = 0. The time interval needed for thefluorescence
intensity to decay to 1/e of its initial intensity is called thefluorescence lifetimeτ 0.
By examining Eq. (9.2) it can be seen that thefluorescence lifetime can be found
from the slope of a plot of the natural logarithm ln [I(t)/I 0 ] versus time t.

Example 9.3Consider thefluorescence decay for the particularfluorophore

shown in Fig.9.6. Using the illustrated intensity decay curve, what is the

fluorescence lifetimeτ 0?

Solution: Thefluorescence lifetime is the time interval needed for the

fluorescence intensity to decay to 1/e of its initial value I 0. In the curve in

Fig.9.6thefluorescence lifetime occurs at the time where I(t) = I 0 /e = 0.368

I 0. This occurs at t =τ 0 = 1.45 ns.

A primary application of FLIM is to identify different fractional amounts of the
samefluorophore when it is in different states of interaction with its environment.
This is done by using the basic property thatfluorescence lifetimes are independent
offluorophore concentration and laser excitation intensity. Because thefluores-
cence lifetime of afluorophore is sensitive to the local environment (for example,
pH, molecular charge, the presence offluorescence quenchers, refractive index, and

Time t (ns)

Intensity (arbitrary units)

13245

Fluorescence decay: exp[-t/τ 0 ]

0.2

0.4

0.6

0.8

1.0

Excitation event: 10-15s

1/e point

τ 0

Fig. 9.6 Example of
fluorescence decay for a
fluorophore with lifetimeτ 0

9.2 FRET/FLIM 267