Quantitative performance characterization of three-dimensional noncontact fluorescence molecular tomography

© 2016 The Authors.Fluorescent proteins and dyes are routine tools for biological research to describe the behavior of genes, proteins, and cells, as well as more complex physiological dynamics such as vessel permeability and pharmacokinetics. The use of these probes in whole body in vivo imaging would allow extending the range and scope of current biomedical applications and would be of great interest. In order to comply with a wide variety of application demands, in vivo imaging platform requirements span from wide spectral coverage to precise quantification capabilities. Fluorescence molecular tomography (FMT) detects and reconstructs in three dimensions the distribution of a fluorophore in vivo. Noncontact FMT allows fast scanning of an excitation source and noninvasive measurement of emitted fluorescent light using a virtual array detector operating in free space. Here, a rigorous process is defined that fully characterizes the performance of a custom-built horizontal noncontact FMT setup. Dynamic range, sensitivity, and quantitative accuracy across the visible spectrum were evaluated using fluorophores with emissions between 520 and 660 nm. These results demonstrate that high-performance quantitative three-dimensional visible light FMT allowed the detection of challenging mesenteric lymph nodes in vivo and the comparison of spectrally distinct fluorescent reporters in cell culture

MULTIMODAL NONCONTACT DIFFUSE OPTICAL REFLECTANCE IMAGING OF BLOOD FLOW AND FLUORESCENCE CONTRASTS

In this study we design a succession of three increasingly adept diffuse optical devices towards the simultaneous 3D imaging of blood flow and fluorescence contrasts in relatively deep tissues. These metrics together can provide future insights into the relationship between blood flow distributions and fluorescent or fluorescently tagged agents. A noncontact diffuse correlation tomography (ncDCT) device was firstly developed to recover flow by mechanically scanning a lens-based apparatus across the sample. The novel flow reconstruction technique and measuring boundary curvature were advanced in tandem. The establishment of CCD camera detection with a high sampling density and flow recovery by speckle contrast followed with the next instrument, termed speckle contrast diffuse correlation tomography (scDCT). In scDCT, an optical switch sequenced coherent near-infrared light into contact-based source fibers around the sample surface. A fully noncontact reflectance mode device finalized improvements by combining noncontact scDCT (nc_scDCT) and diffuse fluorescence tomography (DFT) techniques. In the combined device, a galvo-mirror directed polarized light to the sample surface. Filters and a cross polarizer in stackable tubes promoted extracting flow indices, absorption coefficients, and fluorescence concentrations (indocyanine green, ICG). The scDCT instrumentation was validated through detection of a cubical solid tissue-like phantom heterogeneity beneath a liquid phantom (background) surface where recovery of its center and dimensions agreed with the known values. The combined nc_scDCT/DFT identified both a cubical solid phantom and a tube of stepwise varying ICG concentration (absorption and fluorescence contrast). The tube imaged by nc_scDCT/DFT exhibited expected trends in absorption and fluorescence. The tube shape, orientation, and localization were recovered in general agreement with actuality. The flow heterogeneity localization was successfully extracted and its average relative flow values in agreement with previous studies. Increasing ICG concentrations induced notable disturbances in the tube region (≥ 0.25 μM/1 μM for 785 nm/830 nm) suggesting the graduating absorption (320% increase at 785 nm) introduced errors. We observe that 830 nm is lower in the ICG absorption spectrum and the correspondingly measured flow encountered less influence than 785 nm. From these results we anticipate the best practice in future studies to be utilization of a laser source with wavelength in a low region of the ICG absorption spectrum (e.g., 830 nm) or to only monitor flow prior to ICG injection or post-clearance. In addition, ncDCT was initially tested in a mouse tumor model to examine tumor size and averaged flow changes over a four-day interval. The next steps in forwarding the combined device development include the straightforward automation of data acquisition and filter rotation and applying it to in vivo tumor studies. These animal/clinical models may seek information such as simultaneous detection of tumor flow, fluorescence, and absorption contrasts or analyzing the relationship between variably sized fluorescently tagged nanoparticles and their tumor deposition relationship to flow distributions

Noncontact Speckle Contrast Diffuse Correlation Tomography of Blood Flow Distributions in Tissues with Arbitrary Geometries

A noncontact electron multiplying charge-coupled-device (EMCCD)-based speckle contrast diffuse correlation tomography (scDCT) technology has been recently developed in our laboratory, allowing for noninvasive three-dimensional measurement of tissue blood flow distributions. One major remaining constraint in the scDCT is the assumption of a semi-infinite tissue volume with a flat surface, which affects the image reconstruction accuracy for tissues with irregular geometries. An advanced photometric stereo technique (PST) was integrated into the scDCT system to obtain the surface geometry in real time for image reconstruction. Computer simulations demonstrated that a priori knowledge of tissue surface geometry is crucial for precisely reconstructing the anomaly with blood flow contrast. Importantly, the innovative integration design with one single-EMCCD camera for both PST and scDCT data collection obviates the need for offline alignment of sources and detectors on the tissue boundary. The in vivo imaging capability of the updated scDCT is demonstrated by imaging dynamic changes in forearm blood flow distribution during a cuff-occlusion procedure. The feasibility and safety in clinical use are evidenced by intraoperative imaging of mastectomy skin flaps and comparison with fluorescence angiography

NONINVASIVE MULTIMODAL DIFFUSE OPTICAL IMAGING OF VULNERABLE TISSUE HEMODYNAMICS

Measurement of tissue hemodynamics provides vital information for the assessment of tissue viability. This thesis reports three noninvasive near-infrared diffuse optical systems for spectroscopic measurements and tomographic imaging of tissue hemodynamics in vulnerable tissues with the goal of disease diagnosis and treatment monitoring. A hybrid near-infrared spectroscopy/diffuse correlation spectroscopy (NIRS/DCS) instrument with a contact fiber-optic probe was developed and utilized for simultaneous and continuous monitoring of blood flow (BF), blood oxygenation, and oxidative metabolism in exercising gastrocnemius. Results measured by the hybrid NIRS/DCS instrument in 37 subjects (mean age: 67 ± 6) indicated that vitamin D supplement plus aerobic training improved muscle metabolic function in older population. To reduce the interference and potential infection risk on vulnerable tissues caused by the contact measurement, a noncontact diffuse correlation spectroscopy/tomography (ncDCS/ncDCT) system was then developed. The ncDCS/ncDCT system employed optical lenses to project limited numbers of sources and detectors on the tissue surface. A motor-driven noncontact probe scanned over a region of interest to collect boundary data for three dimensional (3D) tomographic imaging of blood flow distribution. The ncDCS was tested for BF measurements in mastectomy skin flaps. Nineteen (19) patients underwent mastectomy and implant-based breast reconstruction were measured before and immediately after mastectomy. The BF index after mastectomy in each patient was normalized to its baseline value before surgery to get relative BF (rBF). Since rBF values in the patients with necrosis (n = 4) were significantly lower than those without necrosis (n = 15), rBF levels can be used to predict mastectomy skin flap necrosis. The ncDCT was tested for 3D imaging of BF distributions in chronic wounds of 5 patients. Spatial variations in BF contrasts over the wounded tissues were observed, indicating the capability of ncDCT in detecting tissue hemodynamic heterogeneities. To improve temporal/spatial resolution and avoid motion artifacts due to a long mechanical scanning of ncDCT, an electron-multiplying charge-coupled device based noncontact speckle contrast diffuse correlation tomography (scDCT) was developed. Validation of scDCT was done by imaging both high and low BF contrasts in tissue-like phantoms and human forearms. In a wound imaging study using scDCT, significant lower BF values were observed in the burned areas/volumes compared to surrounding normal tissues in two patients with burn. One limitation in this study was the potential influence of other unknown tissue optical properties such as tissue absorption coefficient (µa) on BF measurements. A new algorithm was then developed to extract both µa and BF using light intensities and speckle contrasts measured by scDCT at multiple source-detector distances. The new algorithm was validated using tissue-like liquid phantoms with varied values of µa and BF index. In-vivo validation and application of the innovative scDCT technique with the new algorithm is the subject of future work

NONCONTACT DIFFUSE CORRELATION TOMOGRAPHY OF BREAST TUMOR

Since aggressive cancers are frequently hypermetabolic with angiogenic vessels, quantification of blood flow (BF) can be vital for cancer diagnosis. Our laboratory has developed a noncontact diffuse correlation tomography (ncDCT) system for 3-D imaging of BF distribution in deep tissues (up to centimeters). The ncDCT system employs two sets of optical lenses to project source and detector fibers respectively onto the tissue surface, and applies finite element framework to model light transportation in complex tissue geometries. This thesis reports our first step to adapt the ncDCT system for 3-D imaging of BF contrasts in human breast tumors. A commercial 3-D camera was used to obtain breast surface geometry which was then converted to a solid volume mesh. An ncDCT probe scanned over a region of interest on the breast mesh surface and the measured boundary data were used for 3-D image reconstruction of BF distribution. This technique was tested with computer simulations and in 28 patients with breast tumors. Results from computer simulations suggest that relatively high accuracy can be achieved when the entire tumor was within the sensitive region of diffuse light. Image reconstruction with a priori knowledge of the tumor volume and location can significantly improve the accuracy in recovery of tumor BF contrasts. In vivo ncDCT imaging results from the majority of breast tumors showed higher BF contrasts in the tumor regions compared to the surrounding tissues. Reconstructed tumor depths and dimensions matched ultrasound imaging results when the tumors were within the sensitive region of light propagation. The results demonstrate that ncDCT system has the potential to image BF distributions in soft and vulnerable tissues without distorting tissue hemodynamics. In addition to this primary study, detector fibers with different modes (i.e., single-mode, few-mode, multimode) for photon collection were experimentally explored to improve the signal-to-noise ratio of diffuse correlation spectroscopy flow-oximeter measurements

Time-resolved laser speckle contrast imaging (TR-LSCI) of cerebral blood flow

To address many of the deficiencies in optical neuroimaging technologies such as poor spatial resolution, time-consuming reconstruction, low penetration depth, and contact-based measurement, a novel, noncontact, time-resolved laser speckle contrast imaging (TR-LSCI) technique has been developed for continuous, fast, and high-resolution 2D mapping of cerebral blood flow (CBF) at different depths of the head. TR-LSCI illuminates the head with picosecond-pulsed, coherent, widefield near-infrared light and synchronizes a newly developed, high-resolution, gated single-photon avalanche diode camera (SwissSPAD2) to capture CBF maps at different depths. By selectively collecting diffuse photons with longer pathlengths through the head, TR-LSCI reduces partial volume artifacts from the overlying tissues, thus improving the accuracy of CBF measurement in the deep brain. CBF map reconstruction was dramatically expedited by incorporating highly parallelized computation. The performance of TR-LSCI was evaluated using head-simulating phantoms with known properties and in-vivo rodents with varied hemodynamic challenges to the brain. Results from these pilot studies demonstrated that TR-LSCI enabled mapping CBF variations at different depths with a sampling rate of up to 1 Hz and spatial resolutions ranging from tens of micrometers on the head surface to 1-2 millimeters in the deep brain. With additional improvements and validation in larger populations against established methods, we anticipate offering a noncontact, fast, high-resolution, portable, and affordable brain imager for fundamental neuroscience research in animals and for translational studies in humans.Comment: 22 pages, 7 figures, 4 table

Fluorescence molecular tomography: Principles and potential for pharmaceutical research

Fluorescence microscopic imaging is widely used in biomedical research to study molecular and cellular processes in cell culture or tissue samples. This is motivated by the high inherent sensitivity of fluorescence techniques, the spatial resolution that compares favorably with cellular dimensions, the stability of the fluorescent labels used and the sophisticated labeling strategies that have been developed for selectively labeling target molecules. More recently, two and three-dimensional optical imaging methods have also been applied to monitor biological processes in intact biological organisms such as animals or even humans. These whole body optical imaging approaches have to cope with the fact that biological tissue is a highly scattering and absorbing medium. As a consequence, light propagation in tissue is well described by a diffusion approximation and accurate reconstruction of spatial information is demanding. While in vivo optical imaging is a highly sensitive method, the signal is strongly surface weighted, i.e., the signal detected from the same light source will become weaker the deeper it is embedded in tissue, and strongly depends on the optical properties of the surrounding tissue. Derivation of quantitative information, therefore, requires tomographic techniques such as fluorescence molecular tomography (FMT), which maps the three-dimensional distribution of a fluorescent probe or protein concentration. The combination of FMT with a structural imaging method such as X-ray computed tomography (CT) or Magnetic Resonance Imaging (MRI) will allow mapping molecular information on a high definition anatomical reference and enable the use of prior information on tissue’s optical properties to enhance both resolution and sensitivity. Today many of the fluorescent assays originally developed for studies in cellular systems have been successfully translated for experimental studies in animals. The opportunity of monitoring molecular processes non-invasively in the intact organism is highly attractive from a diagnostic point of view but even more so for the drug developer, who can use the techniques for proof-of-mechanism and proof-of-efficacy studies. This review shall elucidate the current status and potential of fluorescence tomography including recent advances in multimodality imaging approaches for preclinical and clinical drug development

Noncontact Diffuse Optical Assessment of Blood Flow Changes in Head and Neck Free Tissue Transfer Flaps

Knowledge of tissue blood flow (BF) changes after free tissue transfer may enable surgeons to predict the failure of flap thrombosis at an early stage. This study used our recently developed noncontact diffuse correlation spectroscopy to monitor dynamic BF changes in free flaps without getting in contact with the targeted tissue. Eight free flaps were elevated in patients with head and neck cancer; one of the flaps failed. Multiple BF measurements probing the transferred tissue were performed during and post the surgical operation. Postoperative BF values were normalized to the intraoperative baselines (assigning “1”) for the calculation of relative BF change (rBF). The rBF changes over the seven successful flaps were 1.89±0.15, 2.26±0.13, and 2.43±0.13 (mean±standard error), respectively, on postoperative days 2, 4, and 7. These postoperative values were significantly higher than the intraoperative baseline values (