Stephen Johnson Lomnes

Organic alternatives to quantum dots for intraoperative near-infrared fluorescent sentinel lymph node mapping.

Intraoperative near-infrared (NIR) fluorescence imaging provides the surgeon with real-time image guidance during cancer and other surgeries. We have previously reported the use of NIR fluorescent quantum dots (QDs) for sentinel lymph node (SLN) mapping. However, because of concerns over potential toxicity, organic alternatives to QDs will be required for initial clinical studies. We describe a family of 800 nm organic heptamethine indocyanine-based contrast agents for SLN mapping spanning a spectrum from 775 Da small molecules to 7 MDa nanocolloids. We provide a detailed characterization of the optical and physical properties of these contrast agents and discuss the advantages and disadvantages of each. We present robust methods for the covalent conjugation, purification, and characterization of proteins with tetra-sulfonated heptamethine indocyanines, including mass spectroscopic site mapping of highly substituted molecules. One contrast agent, NIR fluorescent human serum albumin (HSA800), emerged as the molecule with the best overall performance with respect to entry to lymphatics, flow to the SLN, retention in the SLN, fluorescence yield and reproducibility. This preclinical study, performed on large animals approaching the size of humans, should serve as a foundation for future clinical studies.

Tissue-like phantoms for near-infrared fluorescence imaging system assessment and the training of surgeons

We demonstrate how to construct calibrated, stable, and inexpensive tissue-like phantoms for near-IR (NIR) fluorescence imaging applications. The bulk phantom material is composed of gelatin, intralipid, hemoglobin, and indocyanine green (ICG). Absorbance, scatter, background fluorescence, and texture can be tuned as desired. NIR fluorescent inclusions are comprised of ICG-labeled polystyrene divinylbenzene beads and Pam78-labeled hydroxyapatite crystals. The former mimic tumor masses of controllable size and contrast agent concentration, and the latter mimic microcalcifications in breast cancer. NIR-fluorescent inclusions can be positioned precisely in phantoms, with one or more regions having different optical properties, and their position can be verified independently using microcomputed tomography. We demonstrate how these phantoms can be used to calibrate and compare imaging systems, and to train surgeons to operate under NIR fluorescence image guidance.

Real-time, near-infrared, fluorescence-guided identification of the ureters using methylene blue

BACKGROUND The aim of this study was to determine whether the invisible near-infrared (NIR) fluorescence properties of methylene blue (MB), a dye already approved by the U.S. Food and Drug Administration for other indications, could be exploited for real-time, intra-operative identification of the ureters. METHODS The optical properties of MB were quantified in vitro. Open surgery and laparoscopic NIR fluorescence imaging systems were employed. Yorkshire pigs were injected intravenously with 0.1-mg/kg MB (n = 8), 10-mg furosemide followed by 0.1-mg/kg MB (n = 6), or 0.5-mg/kg MB (n = 6). The contrast-to-background ratio (CBR) of the kidney and ureters, and the MB concentration in the urine, were quantified. RESULTS Peak MB absorbance, emission, and intensity in urine occurred at 668 nm, 688 nm, and 20 mumol/L, respectively. After intravenous injection, doses as low as 0.1-mg/kg MB provided prolonged imaging of the ureters, and a dose of 0.5 mg/kg provided statistically significant improvement of CBR. The preinjection of furosemide increased urine volume but did not improve CBR. Laparoscopic identification of the ureter using MB NIR fluorescence was demonstrated. CONCLUSION Ureteral imaging using MB NIR fluorescence provides sensitive, real-time, intra-operative identification of the ureters during open and laparoscopic surgeries.

A Multimodal Contrast Agent for Preoperative MR Imaging and Intraoperative Tumor Margin Delineation

We have constructed a multimodal contrast agent suitable for near-infrared, NIR, fluorescent imaging as well as magnetic resonance imaging, MRI. This class of agents may be useful for preoperative tumor localization and tumor functional evaluation and for intraoperative delineation of tumor margins. We have covalently attached dyes of the cyanine family to a previously described polymeric contrast agent, Gd-DTPA-polylysine, of an extended, uncoiled conformation. The dual modality agent is as effective in imaging tumors by MRI as the parent compound provided that the dye loading on the polymer is such that it does not eliminate all the available free-lysine groups on the parent Gd-DTPA-polylysine polymers. NIR fluorescence from preclinical subcutaneous and orthotopic mammary gland tumors could be detected with a signal to background ratio of as high as 4.5 at 12 hours post agent injection at a dye dose of 125 nmole/kg. For intraoperative delineation of tumor margins, a wide-field illumination camera system was devised giving high signal to background NIR fluorescent images of surgically exposed orthotopic mammary gland tumors. Histologic microscopy confirmed the location of the dual modality agent at the boundary of the tumor with a margin distance of about 0.3 mm from labeled tumor cells.

Improved optical sub-systems for intraoperative near-infrared fluorescence imaging

Near-infrared light propagation through living tissue provides promising opportunities for the development of non-invasive imaging techniques for human care. We have developed a Fluorescence-Assisted Resection and Exploration (FLARE) imaging system for surgery. The FLARE system uses invisible near-infrared light to help the surgeon visualize critical structures intraoperatively and in real-time. We present here the continued optimization of our imaging system from a research prototype to an efficient and ergonomic tool to be used during human surgery. New, hands-free operation enables the surgeon to zoom, focus, recall and save images through a footswitch. A LabVIEW curve-fitting algorithm, in combination with stepper motor control, provides auto-focus capability. Cardiac and/or respiratory gating minimizes motion artifacts of moving objects in the surgical field, and permits in-focus imaging during long fluorescence integration times. Automated subtraction of the near-infrared fluorescence signal from background reflections minimizes the effect of ambient illumination and improves the contrast to noise ratio with only moderate effects on intensity precision. Taken together, this study improves several optical components of the FLARE system, and helps ready it for human clinical testing.

A theory for the ultrasonic modulation of incoherent light in turbid medium

A diffusion approximation to the radiative transfer in a medium with varying refractive index has been proposed as a theoretical model for the ultrasonic tagging of fluorescence or FluoroSound, in a scattering medium. It has been found that the diffuse modulation is a defocusing effect. Defocusing is related to scatter - more the scatter, more the defocusing and there exists a component of the defocusing effect of scatter at the ultrasonic frequency. This is in contrast to the modulation for ballistic photons that originates in the focusing effect of the acoustic lens created by the ultrasonic wave. Simulations with circular phantoms of 1.5 and 2.0cm radius have shown that defocusing is minimum when the acoustic lens is midway between the source and the detector. These results are consistent with physics and demonstrate the capability of the model to function as a predictive tool for FluoroSound instrument design. Both ballistic and diffuse FluoroSound signatures can help in the simultaneous localization of the anomaly and determination of its optical properties. As an adjunct, optimally designed ultrasound beams can be also used to enhance diffuse photon modulation signal through acoustic guidance. Optical properties provide a way to discriminate between normal and diseased tissue. FluoroSound could therefore potentially achieve a fusion of anatomical and functional information non-invasively in a single measurement. The additional information made available by this method will improve the speed and accuracy of optical imaging as a tool in the identification and validation of targets.

A low-cost linear DC - 35 MHz high-power LED driver for continuous wave (CW) and fluorescence lifetime imaging (FLIM)

Near-infrared (NIR) fluorescence has the potential to provide surgeons with real-time intraoperative image-guidance. Increasing the signal-to-background ratio of fluorescent agents involves delivering a controllable excitation fluence rate of proper wavelength and/or using complementary imaging techniques such as FLIM. In this study we describe a low-cost linear driver circuit capable of driving Light Emitting Diodes (LEDs) from DC to 35 MHz, at high power, and which permit fluorescence CW and lifetime measurements. The electronic circuit Gerber files described in this article and the list of components are available online at www.frangionilab.org.

Photon transport models for predictive assessment of imageability

Accurate calculation of internal fluence excited in tissue from an optical source can be used for predicting the performance of fluorescent contrast agents for clinical applications. Solutions of excitation fluence for a steady-state Monte Carlo model and a finite element implementation of the 3d diffusion equation have been compared up to depths of 20mm from a point source located on top of a homogeneous cylindrical phantom for a range of reduced scattering-to-absorption ratios. Differences between the fluence calculated by Monte Carlo and diffusion model is found to be dependent on the transport mean free path (mfp), size of the phantom in relation to the penetration depth, distance from the source and mesh resolution. The differences are small at depths ~ mfp and peak at depths ~2mfp. The differences should ideally reduce to zero at large depths but the magnitude of the differences tend to increase due to the finite boundary in the diffusion model. As an example, for a mfp = 0.817mm similar in magnitude to mesh resolution, diffusion fluence at 1mm, 2mm, 10mm and 14mm is 76%, 59%, 66% and 63% respectively of Monte Carlo fluence. For large mfps characteristic of non- diffusive regimes, diffusion model overestimates fluence at distances less than one mfp. This work demonstrates that mean free path and mesh resolution are the critical parameters that distinguish the performance of Monte Carlo and diffusion models to define error margins that could be utilized for predictive assessment of imageability of fluorescent agents using the diffusion model.

A virtual study of shape-based optical reconstruction

We demonstrate the use of a priori knowledge of lesion shape from ultrasound to improve quantitative accuracy of 2D optical tomography. Mean absorption in the lesion can be estimated within 20% of actual value.

Intraoperative near-infrared fluorescence imaging

We have developed a low-cost, safe, and easy to use NIR fluorescence intraoperative imaging system that permits the surgeon to see, in real time, surgical anatomy and invisible NIR fluorescence simultaneously.