Fluorescent Lifetime Imaging Microscopy.
This is a fluorescence microscopy technique that can be used
to measure the time individual flurophores and fluorescent proteins remain in
the excited state.
Every flurophore has a unique natural lifetime and this
lifetime can be changed by environmental factors such as ion concentration,
oxygen concentration, pH and protein-protein interactions.
There are different methodes of measuring FLIM. Our Lambert
systems are based on the Frequency domain method.
Frequency Domain Method
This determination requires a modulated light source and a modulated image
intensifier as detector. The excitation light is modulated in intensity at the
frequency of 10 - 100 MHz. Therefore, the induced fluorescence emission will be
intensity-modulated as well. Due to the decay of the emission, the emitted light
will show a phase-shift (delay in time) and a decrease in modulation-depth
(lower maximum intensity and higher minimum intensity than the excitation light,
while the average intensity remains the same) with respect to the excitation
light. This phase-shift and decrease in modulation-depth depend on the decay
constants of the fluorescent material and the modulation frequency. The lifetime
can be calculated from each of these two parameters.
To extract the phase shift and the decrease in modulation
depth from the emission signal relative to the excitation signal, the
sensitivity of the image intensifier (coupled to the CCD camera) is modulated
with the same radio frequency. Several phase steps are introduced in the
sensitivity of the image intensifier. The resulting signal is detected at each
phase step during a small integration period of the CCD camera. In the phase
step that the emission signal is in phase with the sensitivity of the
intensifier, a high detector signal level is measured. Similarly, when the
emission signal is out phase with the sensitivity of the intensifier, a low
detector signal level is measured.
These measurements are also done with a reference of known
lifetime, so the phase shift and the decrease in modulation depth can be
calculated by comparing the measurements of the sample with those of the
reference. The lifetime is calculated out of both of these parameters ,so that
two values of the lifetime are given.
We mainly use two different reference solutions.
The choice will depend on
your donor fluorophores lifetime and excitation wavelegnth:
- 1 mg/ml Erythrosin (in
water) to use for GFP and the Red FPs (lifetime = 0.086 ns)
- 10 µM Fluorescein (in
Tris, pH>11) to use for CFP (lifetime = 4.0 ns)
Other FluoO Two concentrations of
Fluorescein - 0.1 µM and 1.0 µM - are available to use for GFP for historical
reasons, but we strongly recommend using Erythrosin as it shows no lifetime
Above diagram showing shift in modulation depth and phase between reference and
Above image showing lifetime images for GFP and GFP+RFP
The above information and more information on the
LIFA system can be found on the Lambert website: