2.

Materials and Methods

The method of photoacoustic imaging of FRET was reported in our previous article.²¹ FRET imaging involves nonradiative dipole–dipole interaction that transfers the excited state energy from a donor fluorophore to an acceptor chromophore.²² The excited state fluorophore generally decays through radiative $k_f$ and nonradiative $k_{\mathrm{nr}}$ pathways, generating fluorescence and thermal/photoacoustic emission, respectively. FRET adds another excited state decay mechanism with decay rate $k_t$. The fluorescence quantum yield $Q$ of the donor is determined by the ratio of the rate of fluorescence emission to the sum of all decay rates of the excited state:

where $k_f$ is the fluorescence emission rate and $\sum k$ is the sum of all decay rates. Therefore, FRET reduces the donor fluorophore quantum yield $Q$ and leads to a decrease in the donor fluorescence intensity $F$. In fluorescence imaging, we have where the subscripts $D$ and $A$ denote the presence of the donor and the acceptor, respectively.

With a nonfluorescence acceptor (quencher), the transferred energy eventually decays through the acceptor’s nonradiative pathway, yielding a 100% conversion efficiency of the transferred energy to thermal/photoacoustic emission. Therefore, in the case of a quenching acceptor, FRET manifests itself through a reduction of donor fluorescence and a concomitant increase of the total photoacoustic signal. The FRET efficiency $E$ can be computed from photoacoustic measurement as follows²¹:

The ratio of the fluorescence emission wavelength to the excitation wavelength ${\lambda }_f/{\lambda }_{\mathrm{ex}}$ accounts for the Stokes shift of fluorescence emission. For rhodamine 6G, ${\lambda }_f/({\lambda }_{\mathrm{ex}}{Q}_D)$ is measured to be 1.20 at an excitation wavelength of 523 nm.²¹

The photoacoustic emissions $P_D$ and $P_{\mathrm{DA}}$ in Eq. (4) are attributed to the excited state energy from light absorbed by the donor fluorophore. However, the spectral overlap of the donor and acceptor required for FRET imaging leads to the acceptor bleed-through (ABT) contamination, i.e., the direct excitation of the acceptor at the donor’s excitation wavelength.²³ Therefore, for quantitative treatment of FRET, the ABT background signal needs to be subtracted from the raw photoacoustic amplitude measured from the FRET dye system. The corrected photoacoustic signal $P_{\mathrm{DA}}$ at ${\lambda }_{\mathrm{ex}}$ is obtained as follows:

where $P_{\mathrm{DA}}^{\mathrm{raw}}({\lambda }_{\mathrm{ex}})$ is the raw photoacoustic amplitude measured from the FRET dye system at ${\lambda }_{\mathrm{ex}}$, and $P_{\mathrm{ABT}}({\lambda }_{\mathrm{ex}})$ is the photoacoustic amplitude from the acceptor chromophore alone.

Figure 1 is a schematic of the photoacoustic tomography system whose design and performance have been detailed previously.^24,25 The photoacoustic tomography system employs a 512-element full-ring transducer array. The transducer array has a 5-MHz central frequency (80% bandwidth) and a 50-mm ring diameter. A tunable OPO laser (Newport, basiScan 280) with a 12-ns pulse duration and a 10-Hz pulse repetition rate was the illumination source. The laser beam was homogenized using an optical diffuser, and the maximum light intensity at the surface of the sample was approximately $10\mathrm{mJ}/{\mathrm{cm}}^2$. Within the imaging plane, the system provided 0.10 to 0.25 mm tangential resolution and relatively uniform 0.10-mm radial resolution.²⁶ The raw data from each element was first Wiener deconvolved to account for the ultrasonic transducer’s impulse response. A back-projection algorithm was then implemented using matlab to reconstruct photoacoustic images.

We used solutions containing a controlled amount of donor rhodamine 6G and acceptor DQOCI to analyze FRET efficiencies. Seven stock ethanol solutions were prepared with concentrations of donor rhodamine 6G and acceptor DQOCI as tabulated in Fig. 2(a). Fluorescence imaging of the sample solutions sealed in glass tubes was performed.²⁷ Figure 2(b) shows the fluorescence emission from solution containing only donor rhodamine 6G and from mixtures containing both donor rhodamine 6G and acceptor DQOCI. It can be seen that more acceptor DQOCI quenched rhodamine 6G fluorescence more effectively. The fluorescence intensity acquired at 523 nm from the pure donor rhodamine 6G (tube no. 1) and the mixture solutions with different acceptor concentrations of 0.125, 0.25, and 0.5 mM (tube nos. 3, 5, and 7) are plotted in Fig. 2(c). From the ratios of the diminished and unperturbed fluorescence intensities, the absolute FRET efficiencies were quantified using Eq. (3) and are shown in Fig. 2(d).