Measuring Förster Resonance Energy Transfer
FD FLIM measurements from cells producing the FP-labeled proteins, the cells are fi rst identifi ed by epi-fl uores- cence microscopy. Frequency domain FLIM measurements are then acquired from the selected cells. T e fi eld of view used here is 256×256 pixels, and frame averaging was used to accumulate about 200 peak counts per pixel, which typically requires about 45 seconds. T e FLIM images were analyzed with the VistaVision soſt ware by selecting regions of interest (ROI, typically 10 μ m 2 ) with average intensity >100 counts.
Figure 4 : A composite phasor plot showing the distributions of fl uorescence lifetime for Coumarin 6 and HPTS. For Coumarin 6, the centroid of the distribution falls on the universal semicircle, indicating a single exponential lifetime of 2.5 ns. For HPTS, the distribution for the longer lifetime probe is shifted to the left along the semicircle. The centroid of the distribution falls on the semicircle, indicating a single exponential lifetime of 5.3 ns.
T e microscope is equipped with a 60 × 1.2 numerical aperture water-immersion objective lens, an objective warmer, and a stage-top environmental control system (Pathology Devices, Inc.) to maintain the temperature and CO 2 levels of the sample. For FRET studies using the cyan and yellow FPs, the 5 mW 440 nm diode laser was modulated by the Alba FastFLIM system at a fundamental frequency of 10 MHz, with additional measurements at 13 harmonics (10–140 MHz). T e modulated laser is coupled to the confocal scanning system that is controlled by the VistaVision soſt ware (Build 218, ISS Inc., Champaign, IL). T e fl uorescence signals emitted from the specimen are routed by a 495 nm long-pass beam splitter through the 530/43 nm (acceptor emission) and 480/40 (donor emission) band-pass emission fi lters, and the signals are detected using two identical avalanche photodiodes (APD).
Calibration and data acquisition . T e system is calibrated
with 50 μM Coumarin 6 dissolved in ethanol, which has a lifetime of 2.5 ns and is used as a reference for the soſt ware. T e calibration is verifi ed by measurement of the lifetime of 2 mM HPTS (8-hydroxypyrene-1,3,6-trisulfonic acid) dissolved in phosphate buff er at pH 7.8 (reference lifetime of 5.3 ns). For live-cell
Table 1 : FD FLIM analysis of the variants of Cerulean.
Fluorescent Protein a
Cerulean
Turquoise Cerulean3
2-component lifetime (Fraction)
2.2 ns (0.66) 4.6 ns (0.34) 3.9 ns (0.99) 3.9 ns (0.99)
Tau(f) (±SD) b
3.0±0.05
3.9±0.06 3.9±0.06
X2
(±SD) c 1.1±0.4
3.6±2.0 5.7±2.5
a Expressed in cells at 37º, n = 10 or more. b Tau(f) is the average lifetime. c Chi-square for the fi t of measurements at 12 frequencies from 10–120 MHz.
2015 May •
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Results Verifi cation of the calibration . Following calibration, FLIM measure- ments of Coumarin 6 were obtained from a 256 × 256 pixel fi eld of view (65,536 pixels). T e Φ and M of the emission signal at each image pixel was acquired simultaneously at the fundamental frequency (10 MHz) and
13 harmonics (20–140 MHz). T en lifetime measurements were acquired from a second reference standard, 10 mM HPTS. T e multi-frequency response curves in Figure 3 show both the Φ and M of the emission signal for Coumarin 6 and HPTS at each frequency, and the chi-square values determine the quality of the data fi t. T e fl uorescence lifetime of both Coumarin 6 and HPTS were then analyzed using the phasor plot method [ 5 – 7 ]. T e phasor plot is a simple geometric representation of the Φ and M of the emission signal. T e time delays from each image pixel are transformed into vectors with the length determined by M and the angle determined by the Φ ( Figure 4 ). T e lifetime distribution for all image pixels at a particular frequency is then plotted relative to a universal semicircle, with the incidence of lifetime values indicated from blue (highest) to red to yellow (lowest; see Figure 4 ). T e semicircle is universal in the sense that the lifetime distribution for any species with a single- exponential decay will fall directly on the semicircle irrespective of the lifetime or modulation frequency [ 6 ]. Here, the lifetime distribution for Coumarin 6 falls directly on the universal semicircle at a position corresponding to an average lifetime of 2.5 ns ( Figure 4 ). In contrast, the lifetime distribution for HPTS is shiſt ed to the leſt along the semicircle relative to Coumarin 6, refl ecting its longer lifetime. Again, the distribution falls directly on the semicircle, indicating a single lifetime component of 5.3 ns ( Figure 4 ).
Optimized donor FPs for FRET-FLIM . Here, the FLIM imaging method is demonstrated in living cells expressing the "FRET standard" proteins. T e use of the FRET standards provides a straightforward approach to verify both the biological model and the imaging system used for the measurement of FRET. T e original FRET standard fusion proteins consist of monomeric (m)Cerulean directly coupled to mVenus through linkers of diff erent lengths [ 8 ]. However, because mCerulean has more than
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