Tomographic imaging of a glioma tumor with endogenous fluorescence is demonstrated using a noncontact single-photon counting fan-beam acquisition system interfaced with microCT imaging. The fluorescence from protoporphyrin IX (PpIX) was found to be detectable, and allowed imaging of the tumor from within the cranium, even though the tumor presence was not visible in the microCT image. The combination of single-photon counting detection and normalized fluorescence to transmission detection at each channel allowed robust imaging of the signal. This demonstrated use of endogenous fluorescence stimulation from aminolevulinic acid (ALA) and provides the first in vivo demonstration of deep tissue tomographic imaging with protoporphyrin IX.
Fluorescence molecular tomography (FMT) has the potential to become a powerful quantitative research tool for pre-clinical
applications such as evaluating the efficacy of experimental drugs. In this paper, we show how a time-domain
FMT/microCT instrument can in principle be used to monitor volumetric fluorescence intensity over time for low
fluorophore concentration levels. The experimental results we present relate to Protoporphyrin IX which has a quantum
efficiency as much as two orders of magnitude lower compared to more conventional extrinsic dyes used for molecular
imaging (e.g., Alexa Fluor dyes, Cyanine dyes). Our results highlight the high sensitivity of the single photon counting
technology on which the optical system we have built is based. In conjunction with this system we have developed a
diffuse optical fluorescence reconstruction technique that is robust and shown here to perform adequately even in cases
when the contribution of noise to the data is important. Related to this, we show that the regularization scheme we have
developed is reliable even for low fluorophore concentration values and that no adjustment of the regularization
parameter needs to be made for different levels of noise. This generic reconstruction approach insures that images
reconstructed from data sets acquired at different times and for different fluorescence levels can be compared on an
A fully non-contact CCD-based approach to sub-surface fluorescence diffuse optical imaging is presented. An
overview of CCD-noise sources are described and a possible solution for obtaining an adequate SNR in CCD-based
diffuse optical imaging is implemented. To examine the impact of excitation and remission light attenuation in this
geometry, the linearity of response in recovering object position was examined in simulations, with respect to changes
in target size, target-to-background contrast, and depth. To provide insight regarding the technological complications of
sub-surface imaging, liquid phantom experiments were performed for targets of size 4mm, 8mm and 14mm having 10:1
target-to-background contrast. Overall, the results indicate that steps must be taken to eliminate blooming artifacts,
perhaps by physically blocking the active source as it is projected onto the CCD chip. In general, response linearity in
the recovered target centroid position, size, and fluorophore concentration as well as complications arising due to partial
volume sampling effects are expected to improve if prior structural images obtained from another modality are
incorporated into the DOT reconstruction algorithm.
A non-contact fluorescence diffuse optical tomography (DOT) system capable of producing B-scan-type images of localized fluorescence regions up to depths of 15mm is presented. The B-Scan mode is analogous to ultrasound where the excitation and remission signals are delivered from the same surface of the tissue. This optical fluorescence system utilizes a 635 nm diode laser and two orthogonal galvanometers to raster scan the position of the source along the tissue surface. Using Protoporphyrin IX (PpIX) as a fluorescent agent, the amplitude of the remitted signal is separated by a 650 nm long pass filter and the fluorescence is then detected by a cooled CCD camera. Images are acquired for all source positions along the surface of the tissue, providing remission intensity images for each. This volume data set is then used in image reconstruction of the sub-surface volume, via a finite element method of modeling the fluorescence diffusion. The optimal remission imaging geometry, in terms of depth sensitivity, computation time, and image contrast-to-noise, was determined by performing sensitivity and singular-value decomposition analysis of the Jacobian for various source/detector combinations. The simulated results indicate that the fluorophore concentration and the inclusion size can not be recovered accurately in this mode; however, it is shown that the inclusion depth can accurately be predicted. Finally, by performing simulations on a mesh created from an MR image, we show that our system may prove useful in predicting the regions of local tissue fluorescence in the application of resection of residual brain tumor tissue under fluorescence guidance. This non-contact diagnostic system is being calibrated and finalized for potential use in this application of sub-surface imaging in the brain.
This paper summarizes ten approaches to quantifying fluorescence in tissues, and contrasts their strengths and weaknesses, relative to what their common applications are, and should be. The major issues involved in this analysis are to compare the accuracy of the method and the ability to quantify the active (i.e. non-aggregated) fraction of fluorophore in the tissue. In addition, issues of the depth of penetration and the availability of the method come into play when clinical applications are required. In general, tissue extraction and liquification methods are the 'gold standard' in this field, yet these are plagued by large variance in the values, raising questions about their ability to report on the true active fraction of drug in the tissue. Confocal and fiber optic microsampling methods allow direct quantification of the active fluorescence in vivo and are able to quantify the heterogeneity in the tissue. Yet both of these methods sample the most superficial layers of a tissue, unless invasive injection of the probe is done. Macroscopic sampling of the tissue is therefore the preferred choice for clinical use, yet there is truly no optimum method which can sample the drug concentration to arbitrary accuracy. Empirical bulk tissue sampling methods are the most commonly used, yet without model-based interpretation of the values it is generally not possible to be quantitative. Even relative uptake values can be distorted by the shape of the tissue, and so raster scanning or model-based assessment of the fluorescent yield is preferable, if available. Extending this concept further, tomographic methods can be implemented to quantify fluorescence, and can even be coupled into existing clinical imaging systems, but development and optimization of these methods will be required in the coming years. These are outlined, and case examples illustrated in this paper.