5 Maintenance and Repair
DC3100
34
6
Application Note
6.1
Fluorescence Lifetime Imaging (FLIM)
FLIM (Fluorescence Lifetime Imaging) is an imaging technology that is primarily
used with confocal microscopy, but the FLIM method has also been applied in other
microscopy methods such as wide field and multiphoton imaging. Especially in the
field of cell and tissue research FLIM is a powerful tool to analyze the distribution of
biological materials.
If a dye molecule is exposed to energy of a specific wavelength, it emits another also
specific wavelength. The color of the emitted light as well as the lifetime of a
fluorescent dye are particular properties of the fluorophore. Due to different
fluorescent dyes the lifetime can vary between a few nanoseconds and several
milliseconds. The lifetime is depended on the ion concentration as well as further
factors like molecular binding, oxygen concentration or hydrophobic properties,
which will provide information about molecules of living cells. The concentration of
the dye, the dimension of the cell or the excitation light intensity will not affect the
lifetime. Therefore, the fluorescence lifetime imaging is a precise measure for ion
concentration.
6.1.1
Time Domain FLIM
The sample will be excited with a light pulse in the ps range. After the pulse the
electrons of the dye will stay in the excited state for a certain time (lifetime). They
return into their ground state and emit a fluorescence photon. The lifetime of the
excited electrons are quite short (ns range). The fluorescence intensity is highest
right after the excitation pulse and decays quickly since there are only a few
electrons with a longer lifetime. The decay can be described with a simple
exponential function: I(t)=I(t
0
)*e
-t/
t
.
Mostly lasers are used to generate pulses with several ps pulse width.
Figure 35
Time Domain FLIM
