Which factors can influence my FLIM measurements?

Lifetime imaging microscopy often involves performing delicate experiments, such as the detection of protein-protein interactions inside cells, or dynamics of certain proteins. Fluorescence decay can be influenced by many factors in the micro-environment, and each factor should be considered carefully.

CONCENTRATION
For diluted, non-interacting chromophores, fluorescence lifetimes are independent of the chromophore concentration, and this is actually one of the major advantages of the FLIM techniques in quantification of e.g. FRET. [1] and oxygen imaging.

However, significant concentration-dependent quenching is possible. For example, a shortening of the CFP fluorescence lifetime was observed in cells expressing simultaneously the YFP acceptor. This probably reflects primarily intermolecular energy transfer between unlinked CFPs and YFPs. But also in the absence of YFP acceptors this quenching was observed, which might be attributed to pseudo-homo-FRET. [1]

Homo-FRET may lead to significant perturbations of the measured fluorescence decay, if different sub-populations of the chromophore are associated to slightly shifted emission spectra, which we know is actually the case for purified CFP. Therefore, it is called pseudo-homo-FRET. In this situation, energy transfer will preferably take place from the blue emitting sub-populations, and will lead to a specific decrease in the apparent fluorescent lifetimes determined on the blue edge of the emission spectrum (<495nm emission). This decrease in fluorescence lifetime will be maximum if the collected fraction of the emission spectrum does not include any contribution from the “red” acceptors emission. [1]

PHOTOBLEACHING
Photobleaching of the fluorophores used in an experiment may affect the fluorescence lifetime results. However, as long as the acquisition time scale for measuring the fluorescence lifetime is small compared to the characteristic timescale for photobleaching, the effect of photobleaching on the lifetime determination can be neglected.

Acceptor photo-bleaching is a method widely used for quantifying energy transfer in intensity-based FRET imaging, although it cannot be used for time lapses. In FLIM-based FRET imaging it is very hard to obtain a full recovery of the donor lifetime only after photobleaching the acceptor. In next example [1], the CFP-YFP FRET pair was used; the lifetime of CFP only was 2,5ns and the lifetime of CFP in presence of YFP was1,68ns. After 98% photodestruction of YFP, the lifetime of CFP only increased up to 1,83ns. However, irradiating YFP in the absence of CFP leads to a significant increase in the fluorescence detected in the “CFP” detection channel (up to 28% above background after an 80% photobleaching of YFP). Therefore, it seemed that some photoproducts were created during YFP irradiation, which contributed to fluorescence in the same wavelength ranges than CFP, leading to a contamination of the CFP signal. [1]

AUTOFLUORESCENCE
Media and cells often contain substances that are autofluorescent. Examples are NADH, riboflavins, collagen and lipofuscins [5]. This means that those substances can be excited at the same wavelength as used to excite the fluorophore of interest. Every substance that is excited and emits photons (thus fluorescence) has its own characteristic fluorescence lifetime. The emission of the autofluorescence material mixes with the emission of the fluorophore of interest. If it is not filtered out, it will influence the measured lifetime. To suppress autofluorescence FLIM users generally use phosphate buffered saline (PBS) as medium for the lifetime measurements. One can also use phenol red-free medium.

Frequency domain FLIM can be used to discriminate against autofluorescence, using either the polar (or phasor) plot [6] or a multi-frequency or multi-harmonic FLIM acquisition.

FIXATION
Fixation can also affect fluorescence lifetimes. The important thing is that you need to use the appropriate controls for your experiments. If you are looking for FRET in fixed cells then you need to have donor only and donor + acceptor cells treated under the same conditions. [3]

Various labs use paraformaldehyde (PFA) fixation protocols, methanol fixation and combined methanol/PFA fixation protocols; which protocol to use is mostly determined upon the protein of interest. You should check that the protein localisation is not perturbed by the fixation method and also that the fluorescence is not completely diminished. [3]

MORE FACTORS
There are more factors that should be taken into account. For example, the work of Klaus Suhling [4] shows that the refractive index of the medium can influence the fluorescence lifetime of GFP. Also GFP expressed alone can have a different lifetime to when it is part of a protein construct.

Even temperature can have severe influence on lifetime measurements. Therefore, the temperature of the sample should be kept as stable as possible, e.g. by use of a climate control chamber.

REFERENCES
1. Regis Grailhe, Fabienne Merola, Jacqueline Ridard, Stephen Couvignou, Chantal Le Poupon, Jean-Pierre Changeux, Helene Laguitton-Pasquier. “Monitoring protein interactions in the living cell through the fluorescence decays of the Cyan Fluorescent Protein”, Chemphyschem 7:1442-1454 (2006).
2. D. A. Zacharias, J. D. Violin, A. C. Newton, R. Y. Tsien, Science 296:913-916 (2002).
3. Roland Brock, Irene H.L. Hamelers, and Thomas M. Jovin. “Comparison of Fixation Protocols for Adherent Cultured Cells Applied to a GFP Fusion Protein of the Epidermal Growth Factor Receptor”. Cytometry 35:353-362 (1999).
4. Klaus Suhling, Jan Siegel, David Phillips, Paul M. W. French, Sandrine Leveque-Fort, Stephen E. D. Webb, and Daniel M. Davis. “Imaging the environment of green fluorescent protein”. Biophysical Journal 83:3589-3595 (2002).
5. Billinton N, Knight AW. “Seeing the wood through the trees: a review of techniques for distinguishing green fluorescent protein from endogenous autofluorescence”. Anal Biochem 291(2):175-97 (2001).
6. Stringari, C. et al., PNAS, 108, 33, 13582“Phasor approach to fluorescence lifetime microscopy distinguishes different metabolic states of germ cells in a live tissue” (2011).