Fluorescence lifetime imaging (FLi) could potentially improve exogenous near-infrared (NIR) fluorescence imaging, because it offers the capability of discriminating a signal of interest from background, provides real-time monitoring of a chemical environment, and permits the use of several different fluorescent dyes having the same emission wavelength. We present a high-power, LED-based, NIR light source for the clinical translation of wide-field (larger than 5 cm in diameter) FLi at frequencies up to 35 MHz. Lifetime imaging of indocyanine green (ICG), IRDye 800-CW, and 3,3-diethylthiatricarbocyanine iodide (DTTCI) was performed over a large field of view (10 cm by 7.5 cm) using the LED light source. For comparison, a laser diode light source was employed as a gold standard. Experiments were performed both on the bench by diluting the fluorescent dyes in various chemical environments in Eppendorf tubes, and in vivo by injecting the fluorescent dyes mixed in Matrigel subcutaneously into CD-1 mice. Last, measured fluorescence lifetimes obtained using the LED and the laser diode sources were compared with those obtained using a state-of-the-art time-domain imaging system and with those previously described in the literature. On average, lifetime values obtained using the LED and the laser diode light sources were consistent, exhibiting a mean difference of 3% from the expected values and a coefficient of variation of 12%. Taken together, our study offers an alternative to laser diodes for clinical translation of FLi and explores the use of relatively low frequency modulation for in vivo imaging.
KEYWORDS: Skin, Near infrared, Luminescence, Optical clearing, Image-guided intervention, Tissue optics, In vivo imaging, Statistical analysis, Systems modeling, Cameras
Near-infrared (NIR) light penetrates relatively deep into skin, but its usefulness for biomedical imaging is constrained by high scattering of living tissue. Previous studies have suggested that treatment with hyperosmotic "clearing" agents might change the optical properties of tissue, resulting in improved photon transport and reduced scatter. Since this would have a profound impact on image-guided surgery, we seek to quantify the magnitude of the optical clearing effect in living subjects. A custom NIR imaging system is used to perform sentinel lymph node mapping and superficial perforator angiography in vivo on 35-kg pigs in the presence or absence of glycerol or polypropylene glycol:polyethylene glycol (PPG:PEG) pretreatment of skin. Ex-vivo, NIR fluorescent standards are placed at a fixed distance beneath sections of excised porcine skin, either preserved in saline or stored dry, then treated or not treated with glycerol. Fluorescence intensity through the skin is quantified and analyzed statistically. Surprisingly, the expected increase in intensity is not measurable either in vivo or ex vivo, unless the skin is previously dried. Histological evaluation shows a morphological difference only in stratum corneum, with this difference being negligible in living tissue. In conclusion, topically applied hyperosmotic agents are ineffective for image-guided surgery of living subjects.
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.
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.
KEYWORDS: Diffusion, Monte Carlo methods, Tissue optics, 3D modeling, Scattering, Photon transport, Absorption, Light scattering, Tissues, Chemical elements
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 mfp's 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.
KEYWORDS: Near infrared, Luminescence, Surgery, Cameras, Imaging systems, Zoom lenses, Light emitting diodes, LabVIEW, Electrocardiography, Signal to noise ratio
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 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.
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