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.
Lung cancer is the leading cause of cancer death in the United States, accounting for 28% of all cancer deaths. Standard of care for potentially curable lung cancer involves preoperative radiographic or invasive staging, followed by surgical resection. With recent adjuvant chemotherapy and radiation studies showing a survival advantage in nodepositive patients, it is crucial to accurately stage these patients surgically in order to identify those who may benefit. However, lymphadenectomy in lung cancer is currently performed without guidance, mainly due to the lack of tools permitting real-time, intraoperative identification of lymph nodes. In this study we report the design and validation of a novel, clinically compatible near-infrared (NIR) fluorescence thoracoscope for real-time intraoperative guidance during lymphadenectomy. A novel, NIR-compatible, clinical rigid endoscope has been designed and fabricated, and coupled to a custom source and a dual channel camera to provide simultaneous color and NIR fluorescence information to the surgeon. The device has been successfully used in conjunction with a safe, FDA-approved fluorescent tracer to detect and resect mediastinal lymph nodes during thoracic surgery on Yorkshire pigs. Taken together, this study lays the foundation for the clinical translation of endoscopic NIR fluorescence intraoperative guidance and has the potential to profoundly impact the management of lung cancer patients.
There is a pressing clinical need to provide image guidance during surgery. Currently, assessment of tissue that needs to be resected or avoided is performed subjectively, leading to a large number of failures, patient morbidity, and increased healthcare costs. Because near-infrared (NIR) optical imaging is safe, noncontact, inexpensive, and can provide relatively deep information (several mm), it offers unparalleled capabilities for providing image guidance during surgery. These capabilities are well illustrated through the clinical translation of fluorescence imaging during oncologic surgery. In this work, we introduce a novel imaging platform that combines two complementary NIR optical modalities: oxygenation imaging and fluorescence imaging. We validated this platform during facial reconstructive surgery on large animals approaching the size of humans. We demonstrate that NIR fluorescence imaging provides identification of perforator arteries, assesses arterial perfusion, and can detect thrombosis, while oxygenation imaging permits the passive monitoring of tissue vital status, as well as the detection and origin of vascular compromise simultaneously. Together, the two methods provide a comprehensive approach to identifying problems and intervening in real time during surgery before irreparable damage occurs. Taken together, this novel platform provides fully integrated and clinically friendly endogenous and exogenous NIR optical imaging for improved image-guided intervention during surgery.
Over the last few years, fluorescence imaging for biomedical applications has experienced very rapid growth. An application triggering significant interest is the use of fluorescence for image guidance during surgical interventions. A custom 15x broadband (400-900 nm) macro-zoom objective has been designed, manufactured, and tested for use in image-guided surgery that employs near-infrared (NIR) fluorescence imaging. The lens has been incorporated into the novel FLARE™ imaging system for NIR fluorescence image-guided surgery.
Oxygenation measurements are widely used in patient care. However, most clinically available instruments currently consist of contact probes that only provide global monitoring of the patient (e.g., pulse oximetry probes) or local monitoring of small areas (e.g., spectroscopy-based probes). Visualization of oxygenation over large areas of tissue, without a priori knowledge of the location of defects, has the potential to improve patient management in many surgical and critical care applications. In this study, we present a clinically compatible multispectral spatial frequency domain imaging (SFDI) system optimized for surgical oxygenation imaging. This system was used to image tissue oxygenation over a large area (16×12 cm) and was validated during preclinical studies by comparing results obtained with an FDA-approved clinical oxygenation probe. Skin flap, bowel, and liver vascular occlusion experiments were performed on Yorkshire pigs and demonstrated that over the course of the experiment, relative changes in oxygen saturation measured using SFDI had an accuracy within 10% of those made using the FDA-approved device. Finally, the new SFDI system was translated to the clinic in a first-in-human pilot study that imaged skin flap oxygenation during reconstructive breast surgery. Overall, this study lays the foundation for clinical translation of endogenous contrast imaging using SFDI.
Introduction: Two major disadvantages of currently available oxygenation probes are the need for contact with the skin
and long measurement stabilization times. A novel oxygenation imaging device based on spatial frequency domain and
spectral principles has been designed, validated preclinically on pigs, and validated clinically on humans. Importantly,
this imaging system has been designed to operate under the rigorous conditions of an operating room. Materials and
Methods: Optical properties reconstruction and wavelength selection have been optimized to allow fast and reliable
oxyhemoglobin and deoxyhemoglobin imaging under realistic conditions. In vivo preclinical validation against
commercially available contact oxygenation probes was performed on pigs undergoing arterial and venous occlusions.
Finally, the device was used clinically to image skin flap oxygenation during a pilot study on women undergoing breast
reconstruction after mastectomy. Results: A novel illumination head containing a spatial light modulator (SLM) and a
novel fiber-coupled high power light source were constructed. Preclinical experiments showed similar values between
local probes and the oxygenation imaging system, with measurement times of the new system being < 500 msec. During
pilot clinical studies, the imaging system was able to provide near real-time oxyHb, deoxyHb, and saturation
measurements over large fields of view (> 300 cm2). Conclusion: A novel optical-based oxygenation imaging system has
the potential to replace contact probes during human surgery and to provide quantitative, wide-field measurements in
near real-time.
Spatial frequency-domain imaging (SFDI) utilizes multiple-frequency structured illumination and model-based computation to generate two-dimensional maps of tissue absorption and scattering properties. SFDI absorption data are measured at multiple wavelengths and used to fit for the tissue concentration of intrinsic chromophores in each pixel. This is done with a priori knowledge of the basis spectra of common tissue chromophores, such as oxyhemoglobin (ctO2Hb), deoxyhemoglobin (ctHHb), water (ctH2O), and bulk lipid. The quality of in vivo SFDI fits for the hemoglobin parameters ctO2Hb and ctHHb is dependent on wavelength selection, fitting parameters, and acquisition rate. The latter is critical because SFDI acquisition time is up to six times longer than planar two-wavelength multispectral imaging due to projection of multiple-frequency spatial patterns. Thus, motion artifact during in vivo measurements compromises the quality of the reconstruction. Optimal wavelength selection is examined through matrix decomposition of basis spectra, simulation of data, and dynamic in vivo measurements of a human forearm during cuff occlusion. Fitting parameters that minimize cross-talk from additional tissue chromophores, such as water and lipid, are determined. On the basis of this work, a wavelength pair of 670 nm/850 nm is determined to be the optimal two-wavelength combination for in vivo hemodynamic tissue measurements provided that assumptions for water and lipid fractions are made in the fitting process. In our SFDI case study, wavelength optimization reduces acquisition time over 30-fold to 1.5s compared to 50s for a full 34-wavelength acquisition. The wavelength optimization enables dynamic imaging of arterial occlusions with improved spatial resolution due to reduction of motion artifacts.
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.
We introduce a noncontact imaging method utilizing multifrequency structured illumination for improving lateral and axial resolution and contrast of fluorescent molecular probes in thick, multiple-scattering tissue phantoms. The method can be implemented rapidly using a spatial light modulator and a simple image demodulation scheme similar to structured light microscopy in the diffraction regime. However, imaging is performed in the multiple-scattering regime utilizing spatially modulated scalar photon density waves. We demonstrate that by increasing the structured light spatial frequency, fluorescence from deeper structures is suppressed and signals from more superficial objects enhanced. By measuring the spatial frequency dependence of fluorescence, background can be reduced by localizing the signal to a buried fluorescent object. Overall, signal-to-background ratio (SBR) and resolution improvements are dependent on spatial frequency and object depth/dimension with as much as sevenfold improvement in SBR and 33% improvement in resolution for ~1-mm objects buried 3 mm below the surface in tissue-like media with fluorescent background.
KEYWORDS: Luminescence, Near infrared, In vivo imaging, Optical imaging, Cameras, Signal detection, Image acquisition, Heart, Video, Electrocardiography
Wide-field continuous wave fluorescence imaging, fluorescence lifetime imaging, frequency domain photon migration, and spatially modulated imaging have the potential to provide quantitative measurements in vivo. However, most of these techniques have not yet been successfully translated to the clinic due to challenging environmental constraints. In many circumstances, cardiac and respiratory motion greatly impair image quality and/or quantitative processing. To address this fundamental problem, we have developed a low-cost, field-programmable gate array-based, hardware-only gating device that delivers a phase-locked acquisition window of arbitrary delay and width that is derived from an unlimited number of pseudo-periodic and nonperiodic input signals. All device features can be controlled manually or via USB serial commands. The working range of the device spans the extremes of mouse electrocardiogram (1000 beats per minute) to human respiration (4 breaths per minute), with timing resolution 0.06%, and jitter 0.008%, of the input signal period. We demonstrate the performance of the gating device, including dramatic improvements in quantitative measurements, in vitro using a motion simulator and in vivo using near-infrared fluorescence angiography of beating pig heart. This gating device should help to enable the clinical translation of promising new optical imaging technologies.
We describe a noncontact profile correction technique for quantitative, wide-field optical measurement of tissue absorption (µa) and reduced scattering (µ) coefficients, based on geometric correction of the sample's Lambertian (diffuse) reflectance intensity. Because the projection of structured light onto an object is the basis for both phase-shifting profilometry and modulated imaging, we were able to develop a single instrument capable of performing both techniques. In so doing, the surface of the three-dimensional object could be acquired and used to extract the object's optical properties. The optical properties of flat polydimethylsiloxane (silicone) phantoms with homogenous tissue-like optical properties were extracted, with and without profilometry correction, after vertical translation and tilting of the phantoms at various angles. Objects having a complex shape, including a hemispheric silicone phantom and human fingers, were acquired and similarly processed, with vascular constriction of a finger being readily detectable through changes in its optical properties. Using profilometry correction, the accuracy of extracted absorption and reduced scattering coefficients improved from two- to ten-fold for surfaces having height variations as much as 3 cm and tilt angles as high as 40 deg. These data lay the foundation for employing structured light for quantitative imaging during surgery.
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.
Image-assisted diagnosis and therapy is becoming more commonplace in medicine. However, most imaging
techniques suffer from voluntary or involuntary motion artifacts, especially cardiac and respiratory motions, which
degrade image quality. Current software solutions either induce computational overhead or reject out-of-focus images
after acquisition. In this study we demonstrate a hardware-only gating circuit that accepts multiple, pseudo-periodic
signals and produces a single TTL (0-5 V) imaging window of accurate phase and period. The electronic circuit Gerber
files described in this article and the list of components are available online at www.frangionilab.org.
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.
Approximately 12,000 people are diagnosed with invasive transitional cell carcinoma of the urinary bladder (InvTCC) each year in the United States. Surgical removal of the bladder (cystectomy) and regional lymph node dissection are considered frontline therapy. Cystectomy causes extensive acute morbidity, and 50% of patients with InvTCC have occult metastases at the time of diagnosis. Better staging procedures for InvTCC are greatly needed. This study was performed to evaluate an intra-operative near infrared fluorescence imaging (NIRF) system (Frangioni laboratory) for identifying sentinel lymph nodes draining InvTCC. NIRF imaging was used to map lymph node drainage from specific quadrants of the urinary bladder in normal dogs and pigs, and to map lymph node drainage from naturally-occurring InvTCC in pet dogs where the disease closely mimics the human condition. Briefly, during surgery NIR fluorophores (human serum albumen-fluorophore complex, or quantum dots) were injected directly into the bladder wall, and fluorescence observed in lymphatics and regional nodes. Conditions studied to optimize the procedure including: type of fluorophore, depth of injection, volume of fluorophore injected, and degree of bladder distention at the time of injection. Optimal imaging occurred with very superficial injection of the fluorophore in the serosal surface of the moderately distended bladder. Considerable variability was noted from dog to dog in the pattern of lymph node drainage. NIR fluorescence was noted in lymph nodes with metastases in dogs with InvTCC. In conclusion, intra-operative NIRF imaging is a promising approach to improve sentinel lymph node mapping in invasive urinary bladder cancer.
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: 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.
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