1.

Method

A picture of the combined FT-DOT-MRI interface is shown in Fig. 1(a). Optical fibers transmitted light to and from the optic interface located in the MRI bore. The optical interface had eight source and eight detector positions with radial adjustable holders. A 16-leg birdcage coil was built into the interface for transmitting and receiving RF signals. MRI and optical measurements were obtained simultaneously with this hybrid system.

Fig. 1

(a) Optic fiber interface integrated with a birdcage MRI coil; (b) the schematic diagram of the frequency-domain FT system.

An FD photomultiplier tube-based FT system was used for the experiments [Fig. 1(b)]. The details of the instrumentation were previously given by Lin et al.³² A network analyzer was used to provide the RF signal for the laser diode amplitude modulation as well as measure the amplitude and phase of the detected fluorescence-emission signals. The fluorophore ICG (IC-Green, Akorn, Inc.) was excited by a 785-nm laser diode. Two cascaded bandpass filters (830 nm, MK Photonics, Albuquerque, New Mexico) were used to eliminate the excitation light. The optical properties of the animal were obtained through DOT measurements at the excitation wavelength. Since the emission and excitation wavelength were close to each other and on the opposite sides of the isobetic point (800 nm) for oxy- and deoxy-hemoglobin, the properties were assumed to be the same at the emission wavelength. The DOT measurements were obtained using our fully automatic FD system reported earlier by Gulsen et al.³⁶ In this study, however, the detector fibers were sequentially connected to the FT and DOT systems that operated at a modulation frequency of 100 MHz, using a computer controlled translation stage.

For optical data analysis, the FD diffusion equation was used to model light propagation in tissue. A Levenberg-Marquardt nonlinear optimization algorithm was used for reconstructing both fluorescence concentration and lifetime. When structural a priori information from the MRI was available, Laplacian-type soft a priori was utilized to guide and constrain the FT reconstruction algorithm. More details on the mathematical framework are described in our previous studies.³⁷

For the MRI data acquisition, a 4T system was used to acquire high-resolution spin-echo T1-weighted MR images. The MR acquisition parameters were 300-ms repetition time, 14-ms echo time, 90-deg flip-angle, 120-mm field of view, 4-mm slice-thickness, and $256\times 256$ matrix size. All animal procedures were approved by the Institutional Animal Care and Use Committee at the University of California, Irvine. Transparent thin-wall NMR tubes were implanted in a six-week-old Fischer rat. The tube had an inner diameter of 4.2 mm and a wall thickness of 0.3 mm. 1% Intralipid and 669 nM ICG were added as the scatterer and fluorophore, respectively. The rat was anesthetized using combination of ketamine and xylazine, and positioned on a custom-made holder after the surgical placement of the tube. The tube was placed deep inside the abdominal cavity. The MRI image showed that the tube was 15 mm under the skin (Fig. 2). This was a very difficult case due to the position of the inclusion, which was located deep in heterogeneous tissue.

Fig. 2

The results for different combinations of functional a priori information. The MRI image of the animal is shown at the top of the figure. The ICG inclusion is indicated by the arrow and can be identified as the dark circle in the MR image. The reconstructed images for each case, with and without MR a priori information is provided.

The image co-registration procedure is critical to utilize MRI for anatomical guidance during the FT reconstruction. Fiducial markers made from agar powder were used for accurate co-registration of the MRI and optical images. The exterior boundary of the animal was obtained from MRI images and used to generate the finite element mesh for optical analysis as described in our earlier studies.³² Following the generation of the FEM mesh, FT reconstructions were performed with three different combinations of functional a priori. Two different methods of optical background heterogeneity correction were compared: Born normalization method and reconstructed DOT optical maps. The details of the three cases are summarized in Table 1.

Table 1

Three different combinations of functional a priori information.

For each case, the fluorescence parameters were reconstructed with and without MRI structural a priori information. When MRI a priori information was used, the fluorophore location obtained from the MR image was utilized to guide and constrain the FT reconstruction. The results obtained from these three cases were compared to demonstrate the benefit of using DOT functional and/or MRI anatomical a priori information for obtaining quantitative FT reconstruction results.

2.

Results and Discussion

The MRI image of the animal is shown in Fig. 2 and the location of the tube filled with the fluorophore is indicated by the red arrow. The results of the reconstructed concentration and lifetime maps for all cases are shown in Fig. 2, and the recovered ICG values are listed in Table 2. As shown in the first row of Fig. 2, the ICG inclusion can be clearly located in the reconstructed concentration and lifetime maps using FT data. However, the recovered ICG concentration and lifetime has 98% and 52% error, respectively. Even when structural a priori information from MRI is utilized, the recovered ICG concentration and lifetime still has 87% and 45% error, respectively. When the Born normalization is used to correct for background optical heterogeneity, the recovered ICG concentration error is reduced to 94%, while the recovered ICG lifetime maintains 55% error. On the other hand, when structural a priori information from the MRI is used, the error is dramatically reduced to 16% and 30% for recovered concentration and lifetime, respectively. When background optical heterogeneity is corrected using the absorption and reduced scattering coefficient maps obtained from the DOT measurements, error in the recovered parameters further reduced to 94% and 27%. Finally, when both MRI structural and DOT functional a priori information are used together, the error in the recovered ICG concentration and lifetime are both reduced to 5%.

Table 2

The recovered ICG concentration and lifetime for the animal study.

In conclusion, a hybrid frequency domain FT-DOT-MRI small animal system was evaluated in vivo. A 4.2-mm fluorescence inclusion embedded deep inside a rat was accurately recovered using this system. Without MRI structural a priori information, the recovered ICG concentration and lifetime maps are only qualitative. Both ICG concentration and lifetime can be accurately recovered in a heterogeneous medium using both DOT functional a priori information and MRI structural a priori information.

It has been demonstrated that a stand-alone FT system cannot recover fluorescence parameters accurately. Several studies have shown the recovered fluorophore concentration is highly dependent on the sizes and depth of the inclusion.^18,19 To overcome this limitation, much effort has been spent to integrate FT with XCT or MRI to incorporate structural a priori information for FT reconstruction guidance. To the best of our knowledge, all current FT-MRI systems operate in the continuous wave mode. These systems are not capable of resolving fluorophore lifetime parameters. In order to obtain both fluorescence parameters accurately, we built a first-of-its-kind FD hybrid FT-MRI system that can resolve both fluorophore concentration and lifetime accurately.

Furthermore, two background-optical-heterogeneity correction methods are compared in this study. The Born normalization method is more straightforward and simpler to implement, and already used in many FT studies. However, background optical heterogeneity correction using recovered optical property map from DOT is demonstrated to provide much better accuracy than the Born normalization method.³⁸ Our study also confirms that better quantitative accuracy is achieved when the absorption and scattering maps of the medium are reconstructed from DOT and used in FT reconstruction. Perhaps in most small animal imaging applications, the Born normalization method is sufficient to correct background optical heterogeneity in FT reconstruction. However, this is not the case for breast imaging due to large tissue volume. In breast imaging, multi-wavelength DOT measurements are taken for tissue chromophore reconstruction to extract physiologically relevant parameters such as the total hemoglobin concentration, oxygen saturation, water concentration, and fat content. The FT measurements can be taken in addition to DOT measurements to provide complementary information about the fluorescence contrast agents or targeted molecular probes. In such a case, functional a priori information from DOT reconstruction should be used for quantitative imaging.

In our current setting, the DOT measurements need to be acquired using a separate system. This is not only inconvenient but also greatly increases the data acquisition time. The next step is to integrate the fluorescence detection unit to the DOT system so that the high-speed DOT and FT measurements can be taken with the same instrument.