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 M Fluorescein (in Tris, pH>11) to use for CFP (lifetime = 4.0 ns)


Other FluoO    Two concentrations of Fluorescein - 0.1 M and 1.0 M - are available to use for GFP for historical reasons, but we strongly recommend using Erythrosin as it shows no lifetime variability.

Above diagram showing shift in modulation depth and phase between reference and sample.


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: Lambertinstruments.com






Send mail to m.oprey@beatson.gla.ac.uk with questions or comments about this web site.
Copyright 2009 Beatson Advanced Imaging Reaource
Last modified: 02/25/16