Why does the phase lifetime differ from the modulation lifetime?

The lifetime from phase and modulation are the same only if all the fluorophores behave according to a single exponential decay. In this case we speak of a mono-exponential fluorescence lifetime distribution and the lifetime calculated from the phase shift and the lifetime calculated from the modulation depth are in this case identical. In case of multi-exponential lifetime distributions, the lifetimes calculated from the phase shift and from modulation depth will be different because the equations on which the lifetimes are based assume mono-exponentiality. The more exponents the more both lifetimes differ. For FRET or for tracking lifetime changes the lifetime from phase can be taken to determine the changes in lifetimes.

For multi-exponential distributions, a multi-frequency recording can be used and the phase and modulation behaviour can be fitted with a multi-exponential model, in a similar way as in time domain FLIM (TCSPC). This allows one to retrieve multiple lifetime components and their relative fractions.

How can I export data?

LI-FLIM stores its data in its own native .fli format. If you choose to save a Sample or Reference file, the phase stack is being stored in this file format including all the acquisition settings and other metadata. The *.fli files can be opened outside LI-FLIM by either the Lambert Instruments off-line version of LI-FLIM or with ImageJ Fiji (Fiji an ImageJ distribution). Optionally, you can choose to store the calculated lifetime images:

  1. Select the Lifetime tab in the image window (it is automatically highlighted once a Reference and Sample have been recorded or loaded)
  2. Select "File" in the upper menubar and choose "Save Active Tab as..."
  3. Here you can select a destination, give it a name and choose a data type. Currently it is possible to export for example *.raw (pure binary data, without the header), *.tiff and to the ICS data format.

Is LI-FLIM compatible with MATLAB?

For automated acquisition it is possible to script the LI-FLIM functions through Windows via an Active-X connection. It is possible to access and control LI-FLIM from many different programming languages (e.g. C, Java, Visual Basic) or scripting languages (e.g. MATLAB, Perl, Python). Please look here for further information on automated FLIM. Lambert Instruments also provides Script support for those who would like to try these.

What does modulation lifetime mean?

From the modulation depth decrease between the excitation and emission light, an estimate of the fluorescence lifetime can be calculated. This estimate assumes that the fluorescence lifetime distribution is mono-exponential. For definitions and more information please refer to 'Principles of Fluorescence Spectroscopy' by J.R. Lackowicz, Chapter 5.

What does phase lifetime mean?

From the phase shift between the excitation and emission light, an estimate of the fluorescence lifetime can be calculated. This estimate assumes that the fluorescence lifetime distribution is mono-exponential. For definitions and more information please refer to 'Principles of Fluorescence Spectroscopy' by J.R. Lackowicz, Chapter 5.

What reference should I use?

The perfect reference is a uniform fluorescing sample (e.g. solution of fluorophores), containing fluorophores that have a mono-exponential lifetime, are excited at the same wavelength and emit with the same wavelength as the sample, and do not bleach. We recommend 1 micromolar fluorescein solution pH 10 (4.00 ns) or erythrocine solution (86 ps) for GFP samples and 10 micromolar fluorescein solution pH10 (4.00 ns) for CFP samples.

Please make sure that your reference can be excited at the same excitation wavelength as your sample. Most ideal is to get similar intensity values for the reference stack as for the sample stack. It is highly recommended that you keep the MCP gain of the image intensifier and the LED DC current the same during the reference and sample acquisitions. To get similar intensity values, you can change the exposure time, use ND filters, or change the concentration of your reference solution.