Near-infrared fluorescent digital pathology for the automation of disease diagnosis and biomarker assessment.

Hematoxylin-eosin (H&E) staining of tissue has been the mainstay of pathology for more than a century. However, the learning curve for H&E tissue interpretation is long, whereas intra- and interobserver variability remain high. Computer-assisted image analysis of H&E sections holds promise for increased throughput and decreased variability but has yet to demonstrate significant improvement in diagnostic accuracy. Addition of biomarkers to H&E staining can improve diagnostic accuracy; however, coregistration of immunohistochemical staining with H&E is problematic as immunostaining is completed on slides that are at best 4 µm apart. Simultaneous H&E and immunostaining would alleviate coregistration problems; however, current opaque pigments used for immunostaining obscure H&E. In this study, we demonstrate that diagnostic information provided by two or more independent wavelengths of near-infrared (NIR) fluorescence leave the H&E stain unchanged while enabling computer-assisted diagnosis and assessment of human disease. Using prostate cancer as a model system, we introduce NIR digital pathology and demonstrate its utility along the spectrum from prostate biopsy to whole mount analysis of H&E-stained tissue.

The FLARE intraoperative near-infrared fluorescence imaging system: a first-in-human clinical trial in breast cancer sentinel lymph node mapping.

BACKGROUND: Invisible NIR fluorescent light can provide high sensitivity, high-resolution, and real-time image-guidance during oncologic surgery, but imaging systems that are presently available do not display this invisible light in the context of surgical anatomy. The FLARE imaging system overcomes this major obstacle. METHODS: Color video was acquired simultaneously, and in real-time, along with two independent channels of NIR fluorescence. Grayscale NIR fluorescence images were converted to visible "pseudo-colors" and overlaid onto the color video image. Yorkshire pigs weighing 35 kg (n = 5) were used for final preclinical validation of the imaging system. A six-patient pilot study was conducted in women undergoing sentinel lymph node (SLN) mapping for breast cancer. Subjects received (99m)Tc-sulfur colloid lymphoscintigraphy. In addition, 12.5 microg of indocyanine green (ICG) diluted in human serum albumin (HSA) was used as an NIR fluorescent lymphatic tracer. RESULTS: The FLARE system permitted facile positioning in the operating room. NIR light did not change the look of the surgical field. Simultaneous pan-lymphatic and SLN mapping was demonstrated in swine using clinically available NIR fluorophores and the dual NIR capabilities of the system. In the pilot clinical trial, a total of nine SLNs were identified by (99m)Tc- lymphoscintigraphy and nine SLNs were identified by NIR fluorescence, although results differed in two patients. No adverse events were encountered. CONCLUSIONS: We describe the successful clinical translation of a new NIR fluorescence imaging system for image-guided oncologic surgery.

Image-guided perforator flap design using invisible near-infrared light and validation with x-ray angiography.

Although perforator flaps mark an important conceptual change in reconstructive surgery, individual perforator vessels show a high degree of variability with respect to anatomic landmarks. We have developed an intraoperative imaging system that simultaneously displays surgical anatomy and otherwise invisible near-infrared images. In 22 adult pigs, perforating vessels were identified within seconds using this optical imaging system and systemic injection of indocyanine green. Perforator flaps were then designed based on these results, and vessel location confirmed by direct visualization and anatomic dissection. Since x-ray angiography remains the gold standard for identification of underlying vessels, conventional x-ray angiography was also performed in 8 pigs to verify the location of perforators. There was full correlation of all the perforators identified among near-infrared fluorescence angiography, x-ray angiography, and anatomic dissection. The technology we describe provides high-sensitivity real-time image guidance throughout perforator dissection, and permits patient-specific flap design.

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.

Real-time intra-operative near-infrared fluorescence identification of the extrahepatic bile ducts using clinically available contrast agents.

BACKGROUND: Iatrogenic bile duct injuries are serious complications with patient morbidity. We hypothesized that the invisible near-infrared (NIR) fluorescence properties of methylene blue (MB) and indocyanine green (ICG) could be exploited for real-time, intraoperative imaging of the extrahepatic bile ducts during open and laparoscopic surgeries. METHODS: In all, 2.0 mg/kg of MB and 0.05 mg/kg of ICG were injected intravenously into 35-kg female Yorkshire pigs and the extrahepatic bile ducts were imaged over time using either the Fluorescence-Assisted Resection and Exploration (FLARE) image-guided surgery system (open surgery) or a custom NIR fluorescence laparoscopy system. Surgical anatomy was confirmed using x-ray cholangiography. The contrast-to-background ratio (CBR), contrast-to-liver ratio (CLR), and chemical concentrations in the cystic duct (CD) and common bile duct (CBD) were measured, and the performance of each agent was quantified. RESULTS: Using NIR fluorescence of MB, the CD and CBD could be identified with good sensitivity (CBR and CLR > or =4), during both open and laparoscopic surgeries, from 10 to 120 min postinjection. Functional impairment of the ducts, including constriction and injury were immediately identifiable. Using NIR fluorescence of ICG, extrahepatic bile ducts did not become visible until 90 min postinjection because of strong residual liver retention; however, between 90 and 240 min, ICG provided exquisitely high sensitivity for both CD and CBD, with CBR > or =8 and CLR > or =4. CONCLUSION: We demonstrate that 2 clinically available NIR fluorophores, MB fluorescing at 700 nm and ICG fluorescing at 800 nm, provide sensitive, prolonged identification of the extrahepatic bile ducts and assessment of their functional status.

High-power, computer-controlled, light-emitting diode-based light sources for fluorescence imaging and image-guided surgery.

Optical imaging requires appropriate light sources. For image-guided surgery, in particular fluorescence-guided surgery, a high fluence rate, a long working distance, computer control, and precise control of wavelength are required. In this article, we describe the development of light-emitting diode (LED)-based light sources that meet these criteria. These light sources are enabled by a compact LED module that includes an integrated linear driver, heat dissipation technology, and real-time temperature monitoring. Measuring only 27 mm wide by 29 mm high and weighing only 14.7 g, each module provides up to 6,500 lx of white (400-650 nm) light and up to 157 mW of filtered fluorescence excitation light while maintaining an operating temperature < or = 50 degrees C. We also describe software that can be used to design multimodule light housings and an embedded processor that permits computer control and temperature monitoring. With these tools, we constructed a 76-module, sterilizable, three-wavelength surgical light source capable of providing up to 40,000 lx of white light, 4.0 mW/cm2 of 670 nm near-infrared (NIR) fluorescence excitation light, and 14.0 mW/cm2 of 760 nm NIR fluorescence excitation light over a 15 cm diameter field of view. Using this light source, we demonstrated NIR fluorescence-guided surgery in a large-animal model.

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.