Vivek Venugopal

Full-field time-resolved fluorescence tomography of small animals

In this experimental investigation, we explore the feasibility of using wide-field illumination for time-resolved fluorescence molecular tomography. The performance of wide-field patterns with a time-resolved imaging platform is investigated in vitro and in a small animal model. A Monte Carlo-based forward model is employed to reconstruct fluorescence yield based on time-gated datasets. An improvement in resolution and quantification when using the time-gate data type compared to the commonly used cw data type is demonstrated in vitro. Furthermore, the feasibility of wide-field strategies for fluorescence preclinical applications is established by an accurate localization of a fluorescent inclusion implanted in the chest cavity of a murine model.

Monte Carlo based method for fluorescence tomographic imaging with lifetime multiplexing using time gates

Time-resolved fluorescence optical tomography allows 3-dimensional localization of multiple fluorophores based on lifetime contrast while providing a unique data set for improved resolution. However, to employ the full fluorescence time measurements, a light propagation model that accurately simulates weakly diffused and multiple scattered photons is required. In this article, we derive a computationally efficient Monte Carlo based method to compute time-gated fluorescence Jacobians for the simultaneous imaging of two fluorophores with lifetime contrast. The Monte Carlo based formulation is validated on a synthetic murine model simulating the uptake in the kidneys of two distinct fluorophores with lifetime contrast. Experimentally, the method is validated using capillaries filled with 2.5nmol of ICG and IRDye™800CW respectively embedded in a diffuse media mimicking the average optical properties of mice. Combining multiple time gates in one inverse problem allows the simultaneous reconstruction of multiple fluorophores with increased resolution and minimal crosstalk using the proposed formulation.

Development of an optical imaging platform for functional imaging of small animals using wide-field excitation.

The design and characterization of a time-resolved functional imager using a wide-field excitation scheme for small animal imaging is described. The optimal operation parameters are established based on phantom studies. The performance of the platform for functional imaging and the simultaneous 3D reconstruction of absorption and scattering coefficients is investigated in vitro.

Time-resolved diffuse optical tomography with patterned-light illumination and detection

This investigation explores the feasibility of performing diffuse optical tomography based on time-domain wide-field illumination and detection strategies. Wide-field patterned excitation and detection schemes are investigated in transmittance geometry with time-gated detection channels. A Monte Carlo forward model is employed to compute the time-resolved Jacobians for rigorous light propagation modeling. We demonstrate both in silico and experimentally that reconstructions of absorption structures based on wide-field patterned-light strategies are feasible and outperform classical point excitation schemes for similar data set sizes. Moreover, we demonstrate that time-domain information is retained even though large spatial areas are illuminated. The enhanced time-domain data set allows for quantitative three-dimensional imaging in thick tissue based on relatively small data sets associated with much shorter acquisition times.

Quantitative tomographic imaging of intermolecular FRET in small animals

Forster resonance energy transfer (FRET) is a nonradiative transfer of energy between two fluorescent molecules (a donor and an acceptor) in nanometer range proximity. FRET imaging methods have been applied to proteomic studies and drug discovery applications based on intermolecular FRET efficiency measurements and stoichiometric measurements of FRET interaction as quantitative parameters of interest. Importantly, FRET provides information about biomolecular interactions at a molecular level, well beyond the diffraction limits of standard microscopy techniques. The application of FRET to small animal imaging will allow biomedical researchers to investigate physiological processes occurring at nanometer range in vivo as well as in situ. In this work a new method for the quantitative reconstruction of FRET measurements in small animals, incorporating a full-field tomographic acquisition system with a Monte Carlo based hierarchical reconstruction scheme, is described and validated in murine models. Our main objective is to estimate the relative concentration of two forms of donor species, i.e., a donor molecule involved in FRETing to an acceptor close by and a nonFRETing donor molecule.

Design and characterization of an optimized simultaneous color and near-infrared fluorescence rigid endoscopic imaging system

Abstract. We report the design, characterization, and validation of an optimized simultaneous color and near-infrared (NIR) fluorescence rigid endoscopic imaging system for minimally invasive surgery. This system is optimized for illumination and collection of NIR wavelengths allowing the simultaneous acquisition of both color and NIR fluorescence at frame rates higher than 6.8 fps with high sensitivity. The system employs a custom 10-mm diameter rigid endoscope optimized for NIR transmission. A dual-channel light source compatible with the constraints of an endoscope was built and includes a plasma source for white light illumination and NIR laser diodes for fluorescence excitation. A prism-based 2-CCD camera was customized for simultaneous color and NIR detection with a highly efficient filtration scheme for fluorescence imaging of both 700- and 800-nm emission dyes. The performance characterization studies indicate that the endoscope can efficiently detect fluorescence signal from both indocyanine green and methylene blue in dimethyl sulfoxide at the concentrations of 100 to 185 nM depending on the background optical properties. Finally, we performed the validation of this imaging system in vivo during a minimally invasive procedure for thoracic sentinel lymph node mapping in a porcine model.

Adaptive wide-field optical tomography

Abstract. We describe a wide-field optical tomography technique, which allows the measurement-guided optimization of illumination patterns for enhanced reconstruction performances. The iterative optimization of the excitation pattern aims at reducing the dynamic range in photons transmitted through biological tissue. It increases the number of measurements collected with high photon counts resulting in a dataset with improved tomographic information. Herein, this imaging technique is applied to time-resolved fluorescence molecular tomography for preclinical studies. First, the merit of this approach is tested by in silico studies in a synthetic small animal model for typical illumination patterns. Second, the applicability of this approach in tomographic imaging is validated in vitro using a small animal phantom with two fluorescent capillaries occluded by a highly absorbing inclusion. The simulation study demonstrates an improvement of signal transmitted (∼2 orders of magnitude) through the central portion of the small animal model for all patterns considered. A corresponding improvement in the signal at the emission wavelength by 1.6 orders of magnitude demonstrates the applicability of this technique for fluorescence molecular tomography. The successful discrimination and localization (∼1  mm error) of the two objects with higher resolution using the optimized patterns compared with nonoptimized illumination establishes the improvement in reconstruction performance when using this technique.

Ex vivo fluorescence molecular tomography of the spine

We investigated the potential of fluorescence molecular tomography to image ex vivo samples collected from a large animal model, in this case, a dog spine. Wide-field time-gated fluorescence tomography was employed to assess the impact of multiview acquisition, data type, and intrinsic optical properties on the localization and quantification accuracy in imaging a fluorescent inclusion in the intervertebral disk. As expected, the TG data sets, when combining early and late gates, provide significantly better performances than the CW data sets in terms of localization and quantification. Moreover, the use of multiview imaging protocols led to more accurate localization. Additionally, the incorporation of the heterogeneous nature of the tissue in the model to compute the Jacobians led to improved imaging performances. This preliminary imaging study provides a proof of concept of the feasibility of quantitatively imaging complex ex vivo samples nondestructively and with short acquisition times. This work is the first step towards employing optical molecular imaging of the spine to detect and characterize disc degeneration based on targeted fluorescent probes.

Incidence, Degree, and Clinical Effect of Subchondral Bone Overgrowth After Microfracture in the Knee

Background: Subchondral bone overgrowth has been described with variable frequency after microfracture, but little systematic information is available about the clinical incidence, risk factors, or clinical relevance of this phenomenon. Hypothesis: Subchondral overgrowth (1) occurs with high incidence and various degrees after microfracture, (2) is associated with risk factors, and (3) affects clinical outcome score and failure rate. Study Design: Cohort study; Level of evidence, 3. Methods: A total of 84 patients undergoing microfracture in the knee were prospectively followed up to 6 years postoperatively through use of cartilage-sensitive magnetic resonance imaging to detect the incidence of subchondral bone overgrowth. The quantity of the overgrowth was graded systematically and was then correlated to patient and lesion characteristics, surgical technique, validated functional outcome scores, and failure rate. Results: Subchondral bone overgrowth was observed in 62% of patients at a mean of 22 months after surgery. Most bone overgrowth (64%) was low grade and developed during the first 12 months after microfracture. Risk factors for bone overgrowth included high body mass index, defects on the lateral femoral condyle, and aggressive debridement of the calcified cartilage layer. Knee injury and Osteoarthritis Outcome Score results were not significantly different between patients with or without subchondral bone overgrowth. However, 93% of patients who failed microfracture demonstrated osseous overgrowth, and patients with osseous overgrowth showed a significantly higher failure rate (25%) than patients without overgrowth (3.1%; P < .01). Conclusion: Subchondral bone overgrowth is frequently observed after microfracture surgery but is mostly of low grade. Several risk factors were identified that can affect the incidence of this phenomenon. Importantly, subchondral overgrowth is associated with an increased rate of postoperative failure after microfracture.

Recent Advances in Optical Mammography

Diffuse optical imaging (DOI) techniques provide a non-invasive, low cost and sensitive method for assessing the functional state of the human breast. Several studies undertaken in recent years with the objective of investigating the clinical applications of diffuse optical methods, demonstrate the potential of the technique for screening for breast cancer, associated risk factors and for monitoring the efficacy of therapeutic regimen post-diagnosis. In this review, the different diffuse optical methods and instrumentation used in the functional imaging of the breast are described and the results of the clinical studies of the healthy and diseased breast are provided. Moreover, the application of optical imaging in moni- toring neo-adjuvant chemotherapy is summarized, the role of alternate contrast mechanisms in optical imaging is outlined and lastly, the role of optical imaging in multimodal imaging - wherein optical imaging is combined with existing clinical imaging modalities providing complementary diagnostic information - is discussed.