Fluorescence Molecular Tomography (FMT) is a novel optical imaging approach which has been investigated for about two decades. Motivations of FMT are low cost, non-ionization, high sensitivity and wide availability of the contrast agents. In vivo FMT imaging allows 3D visualization of molecular activities in the tissues of live small animals. Typical applications of FMT include protease activity detection, cancer detection, bone regeneration imaging, drug delivery study and so on.
Our lab has developed a prototype FMT imaging system with a conical mirror for whole surface measurement. With this prototype imaging system, we studied systematically the performance of the conical mirror-based FMT imaging system. In the FMT imaging system, the object is placed inside a conical mirror and scanned with a line pattern laser that is mounted on the rotary stage. The rotary laser scanning approach was introduced into the imaging system for casting the excitation laser pattern conveniently. After being reflected by the conical mirror, the emitted fluorescence photons pass through central hole of the rotation stage and then the band pass filters in a motorized filter wheel, and finally are collected by a CCD camera. To improve the measurement dynamic range, we applied different neutral density filters. We also tested different measuring modes to compare their effects on the FMT reconstruction accuracy. Experimental results indicate that the conical mirror based FMT system can reconstruct targets with high accuracy after its optimization.
Another optimization of the FMT imaging system is the application of 3D optical profilometry for obtaining the object geometry. We utilized a phase shifting method to extract the mouse surface geometry. Nine fringe patterns with a phase shifting of 2π/9 are projected onto the mouse surface by a pico-projector. The fringe patterns are captured using a webcam to calculate a phase map that is converted to the geometry of the mouse surface with the algorithms. We used a DigiWarp approach to warp a finite element mesh of a standard digital mouse to the measured mouse surface so that the tedious and time-consuming procedure from a point cloud to a finite element mesh is removed. Experimental results indicated that the proposed method is accurate with errors less than 0.5 mm. Phantom experimental results have demonstrated that the proposed new FMT imaging system can reconstruct the target accurately.
Moreover, we applied Monte Carlo raytracing to study the multiple reflection effect of the conical mirror. Conical mirror is a preferred choice for FMT imaging systems because of its ability to collect fluorescent emission photons from the whole surface of the imaged object. However, the conical mirror might have a fraction of photons to be reflected back to the mice surface, including excitation photons and emission photons, which result in inaccurate source positions and measurements errors in the forward modeling and the reconstruction of FMT. Based on Monte Carlo simulations, we have investigated different conical mirror designs to select one design with the minimum multiple reflection. We first generated a multiple reflected photon map for each design of the conical mirror, and then we applied Monte Carlo simulations to model photon propagation inside tissues. Finally, we evaluated the ratio of the multiple reflected photons to the total photons and figured out the optimized size of the conical mirror. Our simulations demonstrated that a single conical mirror configuration could minimize the multiple reflection issues while keep the imaging system setup simple when its small aperture radius is larger than 5 centimeters. We then fabricated a conical mirror with the optimized size and performed phantom experiments with both the optimized conical mirror and the non-optimized one. Phantom experiment results show that noises in the reconstructed images are reduced with the optimized conical mirror and the reconstruction accuracy is improved as well. Other mirror setups, such as pyramid mirror and two-side flat mirror setups for bioluminescence optical tomography and Cerenkov luminescence imaging were studies by simulations as well.
Finally, we performed euthanized mice imaging to validate the optimized FMT imaging system. To reduce the effect of autofluorescence from mice skin, we compared a point laser with the line laser to scan the mouse surface. Soft prior obtained from MicroCT images was utilized to guide the FMT reconstruction. The reconstructed FMT images with both the point laser and the line laser were compared. We found that the line laser performed better than the point laser. Moreover, we applied a demixing method with measurements at four different emission wavelengths and used the demixed measurements at 720 nm as the input for the FMT reconstruction. The soft prior method was adopted as well. Reconstruction results show that the demixing method improves the accuracy of the reconstructed FMT images.
In the future, we will perform mice imaging using a laser at longer wavelengths (such as 780 nm) because the autofluorecence from longer wavelength lasers is weaker. We will incorporate a MicroCT imaging system into the FMT imaging system as well so that the anatomical guidance extracted from CT images can be used to guide the FMT reconstruction precisely and conveniently. We will also perform in vivo mice experiments with the optimized FMT imaging system and evaluate the quality of the reconstruction results.