Measuring Förster Resonance Energy Transfer
decreasing the average lifetime. T is change in the fl uorescence lifetime can be accurately quantifi ed by FLIM. T is approach is particularly useful for biological applications because measure- ments made in the time-domain are not aff ected by variations in probe concentration, by changes in excitation intensity, nor by light scatter in the sample, all of which can impact steady- state intensity measurements. T e FLIM techniques are broadly subdivided into time domain and frequency domain (FD) methods. T e physics that underlies these two methods is identical, and they diff er only by the analysis of the measure- ments [ 3 ]. T e frequency domain (FD) FLIM approach described here uses a laser modulated at high frequencies (10–140 MHz) to excite the fl uoro- phores. Because of the lifetime of the excited state, there is a phase delay (Φ ) and a change in the modulation (M) of the emission signal relative to the excitation waveform ( Figure 2 ). Fluorescence lifetimes are directly determined from both the Φ and the M of the emission signal simultane- ously measured at many diff erent modulation frequencies (ω ). T e data are then represented using phasor plots that do not require fi tting algo- rithms or any a priori knowledge of the system.
Materials and Methods DNA preparation, cell culture, and
transfection . T e cDNAs encoding the monomeric (m) Turquoise, mCerulean3, and mVenus FPs, were obtained from Michael Davidson (Florida State University, Tallahassee, FL). Standard recombinant DNA methods were used to generate the plasmids encoding the FRET standard proteins and fusion proteins described below. All plasmid inserts were confi rmed by direct seq- uencing. T e plasmid DNAs were used in transfection of mouse pituitary GHFT1 cells by electroporation as described previously [ 4 ]. Immediately aſt er elec- troporation, the cells are recovered and diluted in phenol red-free tissue culture medium containing serum. T e cells are transferred into sterile two- well-chambered coverglasses (Lab-Tek II, T ermo Scientifi c), which are maintained at 37ºC and 5% CO 2 prior to the FLIM analysis.
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Figure 2 : The FD FLIM system. The excitation source for the FD FLIM system in this study is a 440 nm diode laser that is modulated by the ISS FastFLIM module at the fundamental frequency of 10 MHz and 13 harmonic frequencies (ω ). The modulated laser is coupled to the ISS scanning system that is attached to an Olympus IX71 microscope with an environmentally controlled stage. The emission signals from the specimen travel through the scanning system (de-scanned detection), and are routed by a beam splitter through the donor and acceptor emission channel fi lters to two identical APDs.
Figure 3 : Multi-frequency response curves for Coumarin 6 and HPTS. The phase delays (Φ ) and modulation ratios (M) of the emission signals for Coumarin 6 and HPTS were measured at the fundamental frequency (10 MHz) and 13 harmonics (ω =10–140 MHz). The average lifetime (τ ) and chi-square values for fi tting the data are shown.
FD FLIM measurements . Lifetime measurements are made using the ISS Alba FastFLIM system (ISS Inc., Champaign, IL) coupled to an Olympus IX71 microscope ( Figure 2 ).
www.microscopy-today.com • 2015 May
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