Excitation and emission spectra of mTFP and mKO2. The spectra of mTFP and mKO2 are available from Allele Biotechnology and MBL International, respectively (see Sec. 2A1). The spectral overlap between the mTFP emission and the mKO2 excitation spectra (cross-hatched) is more than 50%; the 458-nm argon laser used for exciting the donor (mTFP) is almost at its peak excitation wavelength (462 nm); the 540-nm excitation wavelength was selected from the WLL to excite the acceptor (mKO2). Excitation of mKO2 at the 458-nm donor laser excitation determines the acceptor spectral bleedthrough (ASBT) and is low at 4.3%. The donor spectral bleedthrough (DSBT) is relative to the spectral overlap between the mTFP and the mKO2 emission spectra (shaded). The Förster distance (Ro=5.29 nm) for mTFP and mKO2 was calculated using Eqs.2, 3 (see Sec. 3A).

Comparison of two measured instrument response functions (IRFs). Both IRFs were measured through collecting the second-harmonic generation (SHG) signals emitted from urea crystals. At the 880-nm excitation wavelength, the 440-nm SHG emission signals were collected using a 460∕50-nm emission filter. Under the 940-nm excitation wavelength, the 470-nm SHG signals were collected using a 480∕40-nm emission filter. The two IRFs (solid— the IRF measured at excitation 880 nm, emission 440 nm versus dashed—the IRF measured at excitation 940 nm, emission 470 nm) are almost overlaid with each other, confirming that the IRF did not change for the two different emission signals.

Data processing in sensitized spectral FRET (sFRET) microscopy. All mTFP-5aa-mKO2 and mKO2-alone λ-stacks acquired by spectral imaging were linearly unmixed, and signals in each λ-stack were decomposed into the mTFP and the mKO2 emission channels. The amount of the quenched donor [qDonor; (a)] signals in the mTFP emission channel was unmixed from the λ-stack acquired by exciting the mTFP-5aa-mKO2 cell with the donor excitation. The image at the mKO2 emission channel unmixed from the same λ-stack included both the FRET and the acceptor spectral bleedthrough (ASBT) signals, called the uncorrected spectral FRET [usFRET; (b)] image. ASBT was determined from the image at the mKO2 emission channel unmixed from the λ-stack acquired from the same mTFP-5aa-mKO2 cell excited by the acceptor excitation (c), and also the images at the mKO2 emission channel unmixed from the λ-stacks obtained through exciting the mKO2-alone cell with both the donor (d) and the acceptor (e) excitation wavelengths. The unmixed images were then processed using the established PFRET software (see Sec. 2B2) to obtain the processed spectral FRET [psFRET; (f)] image. Based on Eq. 3 and given the qDonor and psFRET values at each pixel, the apparent FRET efficiency [E%; (g)] image was calculated.

Data acquisition in sensitized spectral FRET (sFRET)microscopy. λ-stacks in sFRET microscopy for evaluating E% of mTFP-5aa-mKO2 were acquired with both the donor (458 nm) and the acceptor (540 nm) excitation wavelengths. (a) The emission spectrum was plotted from the λ-stacks acquired from an mTFP-5aa-mKO2 cell excited by the 458-nm argon laser. The emission signals at 568 nm clearly indicate sensitized FRET signals, although the spectrum still contains both donor and acceptor spectral bleedthroughs. (b) The same cell was also excited by the 540-nm wavelength selected from the WLL, and the emission spectrum was plotted. (c) As discussed in Sec. 3B, to remove the acceptor spectral bleedthrough (ASBT) from FRET signals, the acceptor-alone expressed cells were excited by both the donor and the acceptor excitation wavelengths, and the emission spectra of one selected cell were plotted. The two emission spectra were normalized by the same factor. The comparison between the two emission spectra produced by the acceptor (solid line) and the donor (dashed line) excitations indicates that the ASBT was trivial. Reference spectra of (c) mKO2 (solid) and (d) mTFP for linear unmixing were extracted from the λ-stacks collected from singly expressed cells.

Data analysis in FLIM-FRET microscopy. Change in donor (mTFP) lifetime in the absence and the presence of the acceptor (mKO2) was measured from cells expressing mTFP alone or mTFP-5aa-mKO2, respectively. The fluorescent lifetime decay kinetics for mTFP (donor) alone and in the presence of mKO2 (acceptor) were determined by fitting the data into a single or double exponential decay, respectively. These lifetimes were estimated using our measured instrument response function (IRF) (see Sec. 2B5). The measured IRF (300 picoseconds at FWHM) of the FLIM system (dotted line) is shown in (a), where the fitting curve and the raw data points of Cresyl Violet in ethanol (dotted-dashed line and +) used for verifying the utility of the measured IRF (see Sec. 2B5) is also shown. The comparison between the decay curves and the raw data points (a) clearly shows mTFP-5aa-mKO2 (solid line and ×) decayed faster than mTFP (coarse dashed line and °), since mTFP was quenched by mKO2 due to FRET (see Secs. 2B4, 3C). The representative single-exponential decay curve together with the corresponding raw data points of mKO2 (fine dashed line and −) expressed in mKO2 single-label cells is also shown in (a) (see Secs. 2B4, 3D). The corresponding residuals of the mTFP-5aa-mKO2, mTFP, mKO2, and Cresyl Violet fitting curves are shown in (b), (c), (d), and (e) with the same nanosecond (ns) scale used in (a), respectively. The lifetime distributions for quenched (left: mTFP-5aa-mKO2) and unquenched (right: mTFP) mTFP are shown in (f) with a picosecond (ps) time scale. A wider distribution of the quenched mTFP lifetime was observed, compared to the unquenched mTFP, since the distance variations existed between the mTFP and mKO2 folded proteins in cells even though they were tethered by a 5 amino acid linker (see Sec. 3C).