Previously, we reported on the spatial resolution and quantitative accuracy of temperature-modulated fluorescence tomography (TM-FT) using simulation studies. TM-FT is a novel fully integrated multimodality imaging technique that combines fluorescence diffuse optical tomography (FT) with focused ultrasound. Utilizing unique thermo-reversible fluorescent nanocapsules (ThermoDots), TM-FT provides high-resolution cross-sectional fluorescence images in thick tissue (up to 6 cm). Focused ultrasound and temperature-sensitive ThermoDots are combined to provide accurate localization of these fluorescent probes and functional a priori information to constrain the conventional FT reconstruction algorithm. Our previous simulation studies evaluated the performance of TM-FT using synthetic phantoms with multiple fluorescence targets of various sizes located at different depths. In this follow-up work, we perform experimental studies to evaluate the performance of this hybrid imaging system, in particular, the effect of size, depth, and concentration of the fluorescence target. While FT alone is unable to accurately locate and resolve the fluorophore target in many cases, TM-FT is able to resolve the size and concentration of the ThermoDots within a thick turbid medium with high accuracy for all cases. The maximum error in the recovered ThermoDots concentration and target sizes with TM-FT are 12% and 25%, respectively.

Previously, we demonstrated that temperature-modulated fluorescence tomography (TM-FT) could provide fluorescence images with high quantitative accuracy and the spatial resolution of focused ultrasound. TM-FT is based on scanning the focused ultrasound across the medium to activate temperature-reversible fluorescent nanoprobes (ThermoDots). This technique can resolve small fluorescent targets located several centimeters deep in turbid media with millimeter resolution. Our past studies with this multimodality technique used agar phantoms, which could not represent the true heterogeneous nature of the acoustic and optical properties of biological tissue. In this work, we report the results of the first TM-FT study performed on ex vivo chicken breast tissue. In order to improve the spatial resolution of this technique, diffuse optical tomography is also used to better estimate the optical property maps of the tissue, which is utilized as functional a priori for the TM-FT reconstruction algorithm. These ex vivo results show that TM-FT can accurately recover the concentration and position of a 1.5  mm×5  mm inclusion filled with ThermoDots. Since the inclusion is embedded 2 cm deep in the chicken breast sample, these results demonstrate the great potential of TM-FT for future in vivo small animal imaging.

Conventional fluorescence tomography provides images of the distribution of fluorescent agents within highly scattering media, but suffers from poor spatial resolution. Previously, we introduced a new method termed "temperature-modulated fluorescence tomography" (TM-FT) that generates fluorescence images with high spatial resolution. TM-FT first uses focused ultrasound to locate the distribution of temperature-sensitive fluorescence probes. Afterward, this a priori information is utilized to improve the performance of the inverse solver for conventional fluorescence tomography and reveal quantitatively accurate fluorophore concentration maps. However, the disadvantage of this novel method is the long data acquisition time as the ultrasound beam was scanned in a step-and-shoot mode. In this Letter, we present a new, fast scanning method that reduces the imaging time 40 fold. By continuously scanning the ultrasound beam over a 50 mm by 25 mm field-of-view, high-resolution fluorescence images are obtained in less than 29 min, which is critical for in vivo small animal imaging.

Fluorescence tomography (FT) has become a powerful preclinical imaging modality with a great potential for several clinical applications. Although it has superior sensitivity and utilizes low-cost instrumentation, the highly scattering nature of bio-tissue makes FT in thick samples challenging, resulting in poor resolution and low quantitative accuracy. To overcome the limitations of FT, we previously introduced a novel method, termed temperature modulated fluorescence tomography (TMFT), which is based on two key elements: (1) temperature-sensitive fluorescent agents (ThermoDots) and (2) high-intensity focused ultrasound (HIFU). The fluorescence emission of ThermoDots increases up to hundredfold with only several degree temperature elevation. The exceptional and reversible response of these ThermoDots enables their modulation, which effectively allows their localization using the HIFU. Their localization is then used as functional a priori during the FT image reconstruction process to resolve their distribution with higher spatial resolution. The last version of the TMFT system was based on a cooled CCD camera utilizing a step-and-shoot mode, which necessitated long total imaging time only for a small selected region of interest (ROI). In this paper, we present the latest version of our TMFT technology, which uses a much faster continuous HIFU scanning mode based on an intensified CCD (ICCD) camera. This new, to the best of our knowledge, version can capture the whole field-of-view (FOV) of 50×30m m ² at once and reduces the total imaging time down to 30 min, while preserving the same high resolution (∼1.3m m) and superior quantitative accuracy (<7% error) as the previous versions. Therefore, this new method is an important step toward utilization of TMFT for preclinical imaging.

Fluorescence molecular tomography (FMT) is widely used in preclinical oncology research. FMT is the only imaging technique able to provide 3D distribution of fluorescent probes within thick highly scattering media. However, its integration into clinical medicine has been hampered by its low spatial resolution caused by the undetermined and ill-posed nature of its reconstruction algorithm. Another major factor degrading the quality of FMT images is the large backscattered excitation light component leaking through the rejection filters and coinciding with the weak fluorescent signal arising from a low tissue fluorescence concentration. In this paper, we present a new method based on the use of a novel thermo-sensitive fluorescence probe. In fact, the excitation light leakage is accurately estimated from a set of measurements performed at different temperatures and then is corrected for in the tomographic data. The obtained results show a considerable improvement in both spatial resolution and quantitative accuracy of FMT images due to the proper correction of fluorescent signals.