From FRET Imaging to Practical Methodology for Kinase Activity Sensing in Living Cells - Progress in Molecular Biology and Translational Science - page 171

A

Laser pulses Measured photons

D U ~D T

D U ~D T D U ~D T 1

B

Titanium:Sapphire
Pulsed laser source

Synchronization with each laser pulse

Synchronization
frame/line

Lifetime image

Confocal
Scanner Analysis software

PMT signal

LP

Dichroic
mirror

PMT

BP

Iris

100x,
1.4
NA
oil

Figure 5.14 ( A) Principle of the time amplitude conversion. On the left, a fast photon is
measured and starts the linear tension ramp resulting on a large D U corresponding to
the difference in time between a photon emission and the following laser pulse. While
the excitation is at constant frequency, the measured D U allows the retrieval of the pho-
ton emission time. The same explanation is also valid for a slow photon (middle
scheme). However, if two photons are emitted between two laser pulses, only the first
one is measured. This effect, called “pulse pile-up,” induces an artifactual decrease in the
measured fluorescence lifetime. (B) Scheme of a typical TCSPC acquisition setup with a
laser source allowing two-photon excitation. Pictures on the left show the injection of
the infrared laser in a confocal scan head (upper panel) and the detection module
adapted on the descanned position of the confocal microscope.

4.1.5 Frequency domain: Phase and modulation
In many experimental FD FLIM systems described in the literature, the
modulated excitation light source is composed of a laser (diode, solid-state,
gas, or dye lasers) combined with an external modulator (either an acousto-
optic or an electro-optic modulator). 54-56 The advent of commercially
available LEDs (light-emitting diodes), which can be directly modulated,