Imaging of glioma tumor with endogenous fluorescence tomography.

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.

Early-photon fluorescence tomography: spatial resolution improvements and noise stability considerations.

In vivo tissue imaging using near-infrared light suffers from low spatial resolution and poor contrast recovery because of highly scattered photon transport. For diffuse optical tomography (DOT) and fluorescence molecular tomography (FMT), the resolution is limited to about 5-10% of the diameter of the tissue being imaged, which puts it in the range of performance seen in nuclear medicine. This paper introduces the mathematical formalism explaining why the resolution of FMT can be significantly improved when using instruments acquiring fast time-domain optical signals. This is achieved through singular-value analysis of the time-gated inverse problem based on weakly diffused photons. Simulations relevant to mouse imaging are presented showing that, in stark contrast to steady-state imaging, early time-gated intensities (within 200 ps or 400 ps) can in principle be used to resolve small fluorescent targets (radii from 1.5 to 2.5 mm) separated by less than 1.5 mm.

A microcomputed tomography guided fluorescence tomography system for small animal molecular imaging.

A prototype small animal imaging system was created for coupling fluorescence tomography (FT) with x-ray microcomputed tomography (microCT). The FT system has the potential to provide synergistic information content resultant from using microCT images as prior spatial information and then allows overlay of the FT image onto the original microCT image. The FT system was designed to use single photon counting to provide maximal sensitivity measurements in a noncontact geometry. Five parallel detector locations are used, each allowing simultaneous sampling of the fluorescence and transmitted excitation signals through the tissue. The calibration and linearity range performance of the system are outlined in a series of basic performance tests and phantom studies. The ability to image protoporphyrin IX in mouse phantoms was assessed and the system is ready for in vivo use to study biological production of this endogenous marker of tumors. This multimodality imaging system will have a wide range of applications in preclinical cancer research ranging from studies of the tumor microenvironment and treatment efficacy for emerging cancer therapeutics.

Automatic exposure control and estimation of effective system noise in diffuse fluorescence tomography.

A diffuse fluorescence tomography system, based upon time-correlated single photon counting, is presented with an automated algorithm to allow dynamic range variation through exposure control. This automated exposure control allows the upper and lower detection levels of fluorophore to be extended by an order of magnitude beyond the previously published performance and benefits in a slight decrease in system effective noise. The effective noise level is used as a metric to characterize the system performance, integrating both model-mismatch and calibration bias errors into a single parameter. This effective error is near 7% of the reconstructed fluorescent yield value, when imaging in just few minutes. Quantifying protoporphyrin IX concentrations down to 50 ng/ml is possible, for tumor-sized regions. This fluorophore has very low fluorescence yield, but high biological relevance for tumor imaging, given that it is produced in the mitochondria, and upregulated in many tumor types.

Fluorescence tomography characterization for sub-surface imaging with protoporphyrin IX.

Optical imaging of fluorescent objects embedded in a tissue simulating medium was characterized using non-contact based approaches to fluorescence remittance imaging (FRI) and sub-surface fluorescence diffuse optical tomography (FDOT). Using Protoporphyrin IX as a fluorescent agent, experiments were performed on tissue phantoms comprised of typical in-vivo tumor to normal tissue contrast ratios, ranging from 3.5:1 up to 10:1. It was found that tomographic imaging was able to recover interior inclusions with high contrast relative to the background; however, simple planar fluorescence imaging provided a superior contrast to noise ratio. Overall, FRI performed optimally when the object was located on or close to the surface and, perhaps most importantly, FDOT was able to recover specific depth information about the location of embedded regions. The results indicate that an optimal system for localizing embedded fluorescent regions should combine fluorescence reflectance imaging for high sensitivity and sub-surface tomography for depth detection, thereby allowing more accurate localization in all three directions within the tissue.

Subsurface diffuse optical tomography can localize absorber and fluorescent objects but recovered image sensitivity is nonlinear with depth.

Subsurface tomography with diffuse light has been investigated with a noncontact approach to characterize the performance of absorption and fluorescence imaging. Using both simulations and experiments, the reconstruction of local subsurface heterogeneity is demonstrated, but the recovery of target size and fluorophore concentration is not linear when changes in depth occur, whereas the mean position of the object for experimental fluorescent and absorber targets is accurate to within 0.5 and 1.45 mm when located within the first 10 mm below the surface. Improvements in the linearity of the response with depth appear to remain challenging and may ultimately limit the approach to detection rather than characterization applications. However, increases in tissue curvature and/or the addition of prior information are expected to improve the linearity of the response. The potential for this type of imaging technique to serve as a surgical guide is highlighted.