Additional correction for energy transfer efficiency calculation in filter-based Forster resonance energy transfer microscopy for more accurate results

Abstract

Forster resonance energy transfer (FRET) microscopy is commonly used to monitor protein interactions with filter-based imaging systems, which require spectral bleedthrough (or cross talk) correction to accurately measure energy transfer efficiency (E). The double-label (donor+acceptor) specimen is excited with the donor wavelength, the acceptor emission provided the uncorrected FRET signal and the donor emission (the donor channel) represents the quenched donor (qD), the basis for the E calculation. Our results indicate this is not the most accurate determination of the quenched donor signal as it fails to consider the donor spectral bleedthrough (DSBT) signals in the qD for the E calculation, which our new model addresses, leading to a more accurate E result. This refinement improves E comparisons made with lifetime and spectral FRET imaging microscopy as shown here using several genetic (FRET standard) constructs, where cerulean and venus fluorescent proteins are tethered by different amino acid linkers.

Figure 1

FLIM-FRET data representation. Both TCSPC [Becker and Hickl (Bh, Berlin, Germany) SPC 150, Biorad Radiance 2100 Ex820 nm] and FD [ISS (Champaign, Illinois) ALBA Ex440 nm] FLIM-FRET measurements were carried out to verify the intensity-based FRET results. The lifetime results were obtained through TCSPC—fitting the decay data given an estimated instrument response function (IRF) in Bh SPCImage software; and FD—fitting the phase shifts and amplitude attenuations in ISS Vista Vision software. For all donor-alone controls and the CTV construct, a single exponential fitting was sufficient to yield good approximation of the raw data, while double exponential fitting had to be applied to the C32V, C17V, and C5V constructs. (a) TCSPC shows the estimated IRF and the representative decay profiles of the cerulean-alone (C) and FRET-standard constructs, clearly demonstrating a faster decay (a shorter lifetime) from C to CTV to C32V to C17V to C5V. (b) FD displays the representative phasor plots drawn from raw data measured at 20 MHz [semicircle as the single lifetime curve; (1,0) zero lifetime; and (0,0) infinite lifetime],, clearly demonstrating a longer lifetime from C5V to C17V to C32V to CTV to C, and also showing the corresponding CTV, C17V, C32V, and C5V lifetime images. The distribution of C or CTV almost centers on the semicircle, indicating they both follow a monoexponential decay. The center of the distribution of C32V, C17V, or C5V falls inside the semicircle, conforming why they require double exponential fittings. (For both: objective-Nikon 60×∕1.2 NA W; and emission filter 480∕30 nm).

Figure 2

FRET efficiency (E) comparison. The average Es of CTV, C32V, C17V, and C5V constructs measured in the four FRET microscopy methods are compared as columns and actual numbers in the inset (n⩾12 for each construct measured in each method; the bar on the top of each column indicates the standard deviation). Es in filter-based FRET were calculated using both Eqs. 6, 7 (see Sec. 2). For the C5V, C17V, or C32V construct, ANOVA analyses indicate conventional Eq. 6 results in a statistically different E of the same construct estimated by other methods (spectral, TCSPC, and FD) (p<0.05). In contrast, Eq. 7 statistically matches the other methods (p>0.05). For the CTV construct, the intensity-based Es are found to be different than the FLIM-FRET Es based on ANOVA analysis, because the very low FRET signal level of the CTV construct results in a poor signal-to-noise ratio (SNR) in the intensity-based methods, and in turn affects the accuracies of their E estimations. However, it is still clearly shown that the average E obtained with Eq. 7 is closer to those obtained by other methods than the average E obtained with Eq. 6 within their small variations. [For filter based and spectral FRET: Zeiss 510 Meta; 63X∕1.4NA Oil, Ex. 458 nm (donor), Ex. 415 nm (acceptor); filter-based: Em. 470~500 nm (donor) and Em. 535~590 nm (acceptor); spectral: Em. 458~561 nm.]