Low-frequency wide-field fluorescence lifetime imaging using a high-power near-infrared light-emitting diode light source.

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.

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.

Optical clearing of the skin for near-infrared fluorescence image-guided surgery.

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.

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.

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.

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.

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.