Florescent proteins have opened the possibility to observe protein distribution and localization by fluorescence microscopy. Directly observing these nanometer-size molecules is not possible, however. As an alternative, indirect methods such as Förster resonance energy transfer (FRET) have become very popular. FRET is a nonradiative process in which energy from an excited molecule (donor) is transferred to an acceptor molecule, which leads to changes in the fluorescence intensity and the fluorescence lifetimes of the two chromophores. The rate of energy transfer is sensitive to the distance between these two molecules. Hence, this technique can measure intermolecular distances on a nanometer scale. In intensity-based microscopy, FRET can be measured by ratiometric techniques or by acceptor photobleaching. Ratiometric methods, however, require careful calibration, while acceptor photobleaching can be performed only once for a given sample. These limitations can be overcome by measuring the fluorescence lifetime of the FRET-donor using fluorescence lifetime imaging microscopy (FLIM). A FLIM-FRET measurement images the FRET efficiency and therefore visualizes directly the proximity of the donor and the acceptor molecules. Only the fluorescence lifetime of the donor molecule, which is concentration-independent, is used as a probe. The FRET process can be identified by a decrease of the fluorescence lifetime (quenching) of the donor in comparison with the lifetime of the individual molecule as a result of the energy transfer to the acceptor molecule. FLIM-FRET measurements of live mouse adipocyte cells transfected with CFPF46L, A206K-C/EBPa DBD and YFPF46L, A206K-C/EBPa DBD were imaged before (A) and after photobleaching (B) of the FRET-acceptor YFP in the cell indicated by the white circle. The donor fluorescence originating from CFP is shown, and the fluorescence lifetime is indicated by the false-color representation. As an example, we used FLIM to visualize the dimerization process of the CCAAT/enhancer binding protein alpha (C/EBPα), with samples provided by Richard N. Day and Amnasi Periasami of the University of Virginia in Charlottesville. The C/EBP family plays a key role in developmental gene expression. C/EBPα is known to form obligate dimers and to bind to certain regions of pericentric heterochromatin in mouse adipocyte cells. To visualize the distribution and dimerization of C/EBFα, mouse adipote cells were transfected with plasmids encoding the DNA binding domain of C/EBFα tagged with CFP and YFP. The dimerization of the proteins can be identified by the FRET process between them, which in turn leads to a decreased lifetime of CFP in comparison with a sample without YFP staining. FLIM images of the live cells were taken using an Olympus FluoView 1000 upgraded to FLIM and fluorescence correlation spectroscopy capabilities with a dedicated kit from PicoQuant GmbH. The Upgrade Kit uses a pulsed picosecond diode laser emitting at 440 nm, a single-photon avalanche photodiode and a time-correlated single-photon counting board. The measurement principle is based on the precise measurement of the time difference between the moment of excitation and the arrival of the first fluorescence photon at the detector. This measurement is repeated several million times to account for the statistical nature of fluorescence emission, and all measured time differences are sorted into a histogram. This histogram can be analyzed to extract the fluorescence lifetime and signal amplitude for every pixel. The figure shows a FLIM image of a heterogeneous cell stained with CFP-C/EBPα DNA binding domain; the areas with high signal intensity are pericentric heterochromatin regions (A), and the FRET process can be identified in the blue areas. The lifetime of CFP is shorter than that of a sample without YFP staining, which was found to be 2.4 ns on average. To prove this, YFP was photobleached in the marked area in A by a 514-nm CW laser. After the photobleaching (B), the pixels corresponding to a fluorescence lifetime of less than 2.2 ns are greatly reduced, while the fluorescence lifetime in other cellular regions is unaffected. The average lifetime distribution of the acceptor bleached cell becomes much more uniform, as shorter components mainly disappear, indicating that shorter measured average CFP-lifetimes in pericentric heterochromatin have been caused by FRET. A further analysis of these images yields the FRET efficiency as well as the distance between the donor and the acceptor molecule. Meet the authors Volker Buschmann is senior scientist for microscopy, Felix Koberling is head of the microscopy group and Andreas Bülter, in sales and marketing at PicoQuant GmbH, Berlin; e-mail: photonics@picoquant.com.