Functional lymphatics are essential for removal and transport of cellular waste and excess fluid from regional tissues and are dependent upon contractile lymphangion activity for proximal drainage into the venous blood stream. Lymphatic insufficiency in patients with cancer-acquired lymphedema is manifested by progressive dermal backflow, or retrograde flow of lymph into dermal lymphatic capillaries. Prior studies using near-infrared fluorescence (NIRF) lymphatic imaging with ICG as a contrast agent, found that dermal backflow provides early indication of lymphedema onset which, when untreated, persists over months and years, but, with two weeks of physiotherapy, dermal backflow could be resolved or reduced in early head and neck cancer survivorship. Thus, the extent or area of dermal backflow may provide an accurate, longitudinal measure of progressing/improving lymphatic dysfunction. In this work, we develop hardware and software solutions to automate determination of dermal backflow area on 3-dimensional tissue surface profiles for entry into the medical record. Specifically, we incorporate a stereo depth module into our custom NIRF lymphatic imaging system for simultaneous acquisition of depth, color, and NIRF images. Using camera calibration techniques, NIRF images are mapped onto point clouds derived from depth images. Algorithms for image segmentation of dermal backflow and stitching of multiple point clouds for more complete representation of dermal backflow across complex 3-dimensional tissue surfaces are described. Non-clinical testing demonstrates ±3% errors in dermal backflow area determination, with clinical testing on head and neck cancer survivors underway to assess efficacy of physiotherapies provided early after cancer treatment.
Lymphatics are crucial in maintaining cardiovascular health and facilitating immune surveillance, yet their significance is often overlooked in medical practice. One notable consequence of cancer treatment, affecting a growing population of survivors, is cancer-acquired lymphedema (LE), a prevalent and incurable condition diagnosed by increased tissue volumes. A recent diagnostic finding, using Near-Infrared Fluorescence (NIRF) lymphatic imaging, indicates that dermal backflow, the retrograde flow of lymphatic fluid from collecting lymphatic vessels into the lymphatic capillaries, is predictive of LE. Dermal backflow contributes to the development of irreversible tissue changes associated with LE, including tissue swelling, accumulation of subcutaneous adipose tissue, and ultimately fibrosis. Evidence suggests that early intervention, prior to tissue swelling, may ameliorate LE, unfortunately, diagnostic methods for detecting early lymphatic dysfunction and monitoring the effectiveness of early interventions on the onset of LE are limited. In this work, we build a dedicated, quantitative NIRF lymphatic imaging system to assess dermal backflow and the impact of early physiotherapy on the progression of LE in head and neck cancer survivors. This system integrates NIRF and RGB-D stereo camera hardware and software for image acquisition, 3D rendering, stereo calibration, registration, and visualization of dermal backflow. Additionally, we develop software solutions to automate the segmentation and quantification of lymphatic dysfunction over complex 3D surface profiles. Our preliminary results demonstrate the accurate reconstruction of 3D models with a NIRF texture overlay using data from our NIRF and RGB-D stereo camera device. Furthermore, dermal backflow segmentation automation was accomplished in 2D NIRF images and 3D reconstructions of clinically relevant surfaces and then incorporated into the process of dermal backflow quantification.
Head and neck (HN) cancer survivors are particularly susceptible to lymphedema with a reported incidence of 75%. Treatment of HN lymphedema (HNL) consists of manual lymphatic drainage and compression. In this FDA-approved study, we used near-infrared fluorescence lymphatic imaging (NIRFLI) to assess the response of HNL to a single session of sequential, pneumatic compression therapy (PCT) and after two weeks of daily PCT.
Following informed consent, 5 HNL subjects were enrolled into this study which consisted of two imaging sessions where each subject received six intradermal injections of 25µg of ICG in 0.1ml saline. Immediately after injection, NIRFLI was performed by illuminating the HN with excitation light and collecting the resultant fluorescent signal using a custom imaging system. After initial imaging, each subject underwent a single PCT session after which NIRFLI was again performed. Subjects were provided a pneumatic compression device for daily use over two weeks, after which imaging was repeated.
Abnormal dermal lymphatic backflow was observed in all subjects. Enhanced lymphatic uptake was observed in all subjects following a single PCT session. After two weeks of PCT, NIRFLI revealed the complete resolution of dermal backflow in one subject, the reduction of dermal backflow in two subjects, and no apparent lymphatic change in two subjects though both reported subjective improvements in swallowing or speaking.
PCT therapy may be an effective method to treat HNL. Additional studies are needed to assess long term clinical and quality of life impact.
This study was funded in part by Tactile Medical.
John Rasmussen, Banghe Zhu, Aaron Sahihi, Melissa Aldrich, Susan Pouliot, Stuart Harlin, Kristofer Charlton-Ouw, Caroline Fife, Thomas O'Donnell, Eva Sevick-Muraca
Emerging evidence suggests that the lymphatics play an important role in the pathogenesis and progression of peripheral arterial and venous diseases. In two pilot studies, we sought to evaluate lymphatic function of patients with (i) late stage chronic venous disease (CVD) with active venous leg ulcers (VLUs) and (ii) early CVD and mild to moderate peripheral arterial disease (PAD) using near‐infrared fluorescence lymphatic imaging (NIRFLI).
After informed consent and under an FDA-approved IND for the off‐label administration of indocyanine green (ICG), each subjects received intradermal injections of ICG on the feet and legs. Imaging of lymphatic anatomy and pumping activity was performed by illuminating the legs with near-infrared light and collecting the resultant fluorescent light emanating from the ICG‐laden lymph using a custom imaging system. Data collection occurred in an outpatient setting between the dates of 2009 until present, with the study of early CVD and PAD subjects underway.
We observed abnormal lymphatic anatomy and reduced lymphatic pumping in all subjects enrolled in these two pilot studies. Observed abnormal lymphatic anatomy, as compared to previously imaged healthy subjects, included dermal lymphatic backflow as well as segmented, dilated and/or tortuous lymphatic vessels. Reduced lymphatic pumping was also observed in all subjects, and lymphatic reflux was noted in those subjects with an arterial component to their disease.
While these studies continue, evidence is mounting that lymphatic dysfunction is associated with the etiology of peripheral arterial and venous diseases.
Supported in parts by the National Institutes of Health R21 HL132598‐01 and Tactile Medical.
All medical devices for Food and Drug market approval require specifications of performance based upon International System of Units (SI) or units derived from SI for reasons of traceability. Recently, near-infrared fluorescence (NIRF) imaging devices of a variety of designs have emerged on the market and in investigational clinical studies. Yet the design of devices used in the clinical studies vary widely, suggesting variable device performance. Device performance depends upon optimal excitation of NIRF imaging agents, rejection of backscattered excitation and ambient light, and selective collection of fluorescence emanating from the fluorophore. There remains no traceable working standards with SI units of radiance to enable prediction that a given molecular imaging agent can be detected in humans by a given NIRF imaging device. Furthermore, as technologies evolve and as NIRF imaging device components change, there remains no standardized means to track device improvements over time and establish clinical performance without involving clinical trials, often costly. In this study, we deployed a methodology to calibrate luminescent radiance of a stable, solid phantom in SI units of mW/cm2/sr for characterizing the measurement performance of ICCD and IsCMOS camera based NIRF imaging devices, such as signal-to-noise ratio (SNR) and contrast. The methodology allowed determination of superior SNR of the ICCD over the IsCMOS system; comparable contrast of ICCD and IsCMOS depending upon binning strategies.
Primary lymphedema and lymphatic malformations in the pediatric population remains poorly diagnosed and misunderstood due to a lack of information on the underlying anatomy and function of the lymphatic system. Diagnostics for the lymphatic vasculature are limited, consisting of lymphoscintigraphy or invasive lymphangiography, both of which require sedation that can restrict use in infants and children. As a result, therapeutic protocols for pediatric patients with lymphatic disorders remain sparse and with little evidence to support them. Because near-infrared fluorescence (NIRF) imaging enables image acquisition on the order of tenths of seconds with trace administration of fluorescent dye, sedation is not necessary. The lack of harmful radiation and radioactive contrast agents further facilitates imaging. Herein we summarize our experiences in imaging infants and children who are suspected to have disorders of the lymphatic vascular system using indocyanine green (ICG) and who have developed chylothorax following surgery for congenital heart defects. The results show both anatomical as well as functional lymphatic deficits in children with congenital disease. In the future, NIRF lymphatic imaging could provide new opportunities to tailor effective therapies and monitor responses. The opportunity to use expand NIRF imaging for pediatric diagnostics beyond the lymphatic vasculature is also afforded by the rapid acquisition following trace administration of NIRF contrast agent.
Radiation therapy (RT) can promote anti-tumoral responses, but is also known to cause lymphatic endothelial cell apoptosis, loss of dermal lymphatics, and reduction in lymph transport to draining lymph node basins. When combined with lymph node dissection (LND), the radiogenic lymphatic disruption may possibly result in lymph stasis and dermal backflow. If not resolved, this disruption may lead to chronic inflammation, edema, fibrosis, adipose tissue deposition, and ultimately to functional deficits and disfigurement. Because the head and neck (HN) region contains 1/3 of the body’s lymph nodes, lymphatic responses to cancer progression and therapy may be significant. Furthermore, it may not be surprising that lymphedema has been estimated to impact as many as 75% of HN cancer survivors three months or more after LND and RT.
In this study, we used near-infrared fluorescence imaging to longitudinally assess the lymphatics of 18 patients undergoing treatment for cancer of the oral cavity, oropharynx, and/or larynx following intraoral and intradermal injections of ICG. Patients were imaged before and after surgery, before and after fractionated RT for up to 100 weeks after treatments. Patients who underwent both LND and RT developed lymphatic dermal backflow on treated sides ranging from days after the start of RT to weeks after its completion, while contralateral regions that were not associated with LND but also treated with RT, experienced no such changes in functional lymphatic anatomies. The results show for the first time, the striking reorganization of the lymphatic vasculature and may enable early diagnosis of HN lymphedema.
The success of optical surgical navigation depends upon being able to intraoperatively employ a contrast agent and an imaging device to successfully guide surgery. Development of devices and contrast agents typically occur separately even though it is their combined performance that ultimately determines success and clinical adoption. Herein, we review critical issues and summarize our strategies and approaches for validating molecularly-targeted, near-infrared fluorescent contrast agents and the devices sufficiently sensitive enough for their detection in order to guide lymph node dissection.
A tri-modal (PET/CT/Optical) small animal tomographic imaging system was developed by
integrating our advanced non-contact intensified CCD (ICCD) frequency-domain fluorescence
imaging components into a Siemens Inveon scanner. We performed a performance evaluation
of the developed imaging system by using the developed regularization-free high-order
radiative-transfer-based reconstruction algorithm and custom solid phantoms. Our results show
that frequency-domain photon migration (FDPM) fluorescence tomography can achieve better
tomographic images with less artifacts and more precise fluorescent source localization
compared to the continuous-wave counterpart. The developed multimodal tomographic imaging
system provides a powerful tool for translational biomedical research.
Although there has been a plethora of devices advanced for clinical translation, there has been no standards to
compare and determine the optical device for fluorescence molecular imaging. In this work, we compare different
CCD configurations using a solid phantom developed to mimic pM - fM concentrations of near-infrared fluorescent
dyes in tissues. Our results show that intensified CCD systems (ICCDs) offer greater contrast at larger signal-tonoise
ratios (SNRs) in comparison to their un-intensified CCD systems operated at clinically reasonable, sub-second
acquisition times. Furthermore, we compared our investigational ICCD device to the commercial NOVADAQ SPY
system, demonstrating different performance in both SNR and contrast.
In this proof-of-concept study we seek to demonstrate the delivery of fluorescent contrast agent to the tumor-draining
lymph node basin following intraparenchymal breast injections and intradermal arm injection of micrograms of
indocyanine green in 20 breast cancer patients undergoing complete axillary lymph node dissection. Individual lymph
nodes were assessed ex vivo for presence of fluorescent signal. In all, 88% of tumor-negative lymph nodes and 81% of
tumor-positive lymph nodes were fluorescent. These results indicate that future studies utilizing targeted fluorescent
contrast agents may demonstrate improved surgical and therapeutic intervention.
Care for head and neck (HN) cancer could be improved with better mapping of lymphatic drainage pathways in HN
region as well as understanding the effect of the cancer treatments on lymphatics. In this study, near-infrared
fluorescence imaging is being used to visualize the lymphatics in human subjects diagnosed with HN cancer before and
after treatments. Imaging results show the lymphatic architecture and contractile function in HN. Reformation of
lymphatics during the course of cancer care was also seen in the longitudinal imaging. This allows us to better
understand the lymphatics in HN cancer patients.
Fluorescence gene reporters have recently become available for excitation at far-red wavelengths, enabling opportunities for small animal in vivo gene reporter fluorescence tomography (GRFT). We employed multiple projections of the far-red fluorescence gene reporters IFP1.4 and iRFP, excited by a point source in transillumination geometry in order to reconstruct the location of orthotopically implanted human prostate cancer (PC3), which stably expresses the reporter. Reconstruction was performed using a linear radiative-transfer-based regularization-free tomographic method. Positron emission tomography (PET) imaging of a radiolabeled antibody-based agent that targeted epithelial cell adhesion molecule overexpressed on PC3 cells was used to confirm in vivo GRFT results. Validation of GRFT results was also conducted from ex vivo fluorescence imaging of resected prostate tumor. In addition, in mice with large primary prostate tumors, a combination of GRFT and PET showed that the radiolabeled antibody did not penetrate the tumor, consistent with known tumor transport limitations of large (∼150 kDa ) molecules. These results represent the first tomography of a living animal using far-red gene reporters.
Near-infrared (NIR) fluorescence is an alternative modality for molecular imaging that has been demonstrated in animals
and recently in humans. Fluorescence-enhanced optical tomography (FEOT) using continuous wave or frequency
domain photon migration techniques could be used to provide quantitative molecular imaging in vivo if it could be
validated against "gold-standard," nuclear imaging modalities, using dual-labeled imaging agents. Unfortunately,
developed FEOT systems are not suitable for incorporation with CT/PET/SPECT scanners because they utilize benchtop
devices and require a large footprint. In this work, we developed a miniaturized fluorescence imaging system installed in
the gantry of the Siemens Inveon PET/CT scanner to enable NIR transillumination measurements. The system consists
of a CCD camera equipped with NIR sensitive intensifier, a diode laser controlled by a single board compact controller,
a 2-axis galvanometer, and RF circuit modules for homodyne detection of the phase and amplitude of fluorescence
signals. The performance of the FEOT system was tested and characterized. A mouse-shaped solid phantom of uniform
optical properties with a fluorescent inclusion was scanned using CT, and NIR fluorescence images at several
projections were collected. The method of high-order approximation to the radioactive transfer equation was then used to
reconstruct the optical images. Dual-labeled agents were also used on a tumor bearing mouse to validate the results of
the FEOT against PET/CT image. The results showed that the location of the fluorophore obtained from the FEOT
matches the location of tumor obtained from the PET/CT images. Besides validation of FEOT, this hybrid system could
allow multimodal molecular imaging (FEOT/PET/CT) for small animal imaging.
The goal of preclinical fluorescence-enhanced optical tomography (FEOT) is to provide three-dimensional fluorophore distribution for a myriad of drug and disease discovery studies in small animals. Effective measurements, as well as fast and robust image reconstruction, are necessary for extensive applications. Compared to bioluminescence tomography (BLT), FEOT may result in improved image quality through higher detected photon count rates. However, background signals that arise from excitation illumination affect the reconstruction quality, especially when tissue fluorophore concentration is low and/or fluorescent target is located deeply in tissues. We show that near-infrared fluorescence (NIRF) imaging with an optimized filter configuration significantly reduces the background noise. Model-based reconstruction with a high-order approximation to the radiative transfer equation further improves the reconstruction quality compared to the diffusion approximation. Improvements in FEOT are demonstrated experimentally using a mouse-shaped phantom with targets of pico- and subpico-mole NIR fluorescent dye.
Quantitative analysis of lymphatic function is crucial for understanding the lymphatic system and diagnosing
the associated diseases. Recently, a near-infrared (NIR) fluorescence imaging system is developed for real-time
imaging lymphatic propulsion by intradermal injection of microdose of a NIR fluorophore distal to the
lymphatics of interest. However, the previous analysis software3, 4 is underdeveloped, requiring extensive time
and effort to analyze a NIR image sequence. In this paper, we develop a number of image processing techniques to
automate the data analysis workflow, including an object tracking algorithm to stabilize the subject and remove
the motion artifacts, an image representation named flow map to characterize lymphatic flow more reliably,
and an automatic algorithm to compute lymph velocity and frequency of propulsion. By integrating all these
techniques to a system, the analysis workflow significantly reduces the amount of required user interaction and
improves the reliability of the measurement.
Near infrared fluorescence (NIRF) optical imaging has been successfully demonstrated to offer a high specificity and
sensitivity in detecting various diseases. However, the measurement sensitivity of NIRF optical imaging system is
limited by strong backscattered excitation light leakage. Herein, appropriate filter combination and collimation optics
was adapted to the NIRF optical imager. The sensitivity of near-infrared fluorescence imaging instrumentation can be
dramatically improved upon using the appropriate filter combination and collimation optics. This validation and
qualification approach to reduce the noise floor and improve sensitivity is presented a standardized metric for all
fluorescence based imaging systems proposed.
Treatment of lymphatic disease is complicated and controversial, due in part to the limited understanding of the
lymphatic system. Lymphedema (LE) is a frequent complication after surgical resection and radiation treatment in
cancer survivors, and is especially debilitating in regions where treatment options are limited. Although some extremity
LE can be effectively treated with manual lymphatic drainage (MLD) therapy or compression devices to direct proximal
lymph transport, head and neck LE is more challenging, due to complicated geometry and complex lymphatic structure
in head and neck region.
Herein, we describe the compassionate use of an investigatory technique of near-infrared (NIR) fluorescence imaging to
understand the lymphatic anatomy and function, and to help direct MLD in a patient with head and neck LE.
Immediately after 9 intradermal injections of 25 μg indocyanine green each around the face and neck region, NIR
fluorescence images were collected using a custom-built imaging system with diffused excitation light illumination.
These images were then used to direct MLD therapy. In addition, 3-dimensional (3D) surface profilometry was used to
monitor response to therapy.
NIR fluorescence images of functioning lymphatic vessels and abnormal structures were obtained. Precise geometries of
facial structures were obtained using 3D profilometry, and detection of small changes in edema between therapy sessions
was achieved.
NIR fluorescence imaging provides a mapping of lymphatic architecture to direct MLD therapy and thus improve
treatment efficacy in the head and neck LE, while 3D profilometry allowed longitudinal assessment of edema to evaluate
the efficacy of therapy.
KEYWORDS: Near infrared, Charge-coupled devices, Luminescence, Optical tomography, Signal to noise ratio, Oscillators, Modulation, Tissues, Data acquisition, Signal detection
Herein we report on hardware development and evaluation for frequency-domain photon migration (FDPM) technique
that is miniaturized for incorporation into a micro-CT gantry for hybrid CT/NIR/PET imaging. Immunity to endogenous
optical properties and enhanced contrast associated with fluorophore lifetime is inherent to the FDPM measurements and
enables unique opportunities for quantitative tomography when compared to the time independent (continuous wave)
approach. A miniaturized radiofrequency (rf) circuitry has been developed in our laboratory for homodyne FDPM
measurements that makes use of a single 100MHz oscillator to simultaneously launch optically modulated excitation
light into a small animal as well as to modulate an NIR sensitive image intensifier for collection of fluorescent signals.
The use of a single oscillator not only eliminates signal drift that otherwise results from the use of multiple oscillators
individually driving both source and detector, but also reduces the circuit footprint for incorporation into the CT gantry.
Herein, overall system performance parameters of signal-to-noise ratio, measurement precision, spatial resolution,
modulation depth (ac/dc), excitation light rejection, and clinically relevant data acquisition times are presented for mouse
phantom data. Image reconstruction of phantom data and integration of circuitry for hybrid CT/NIR/PET imaging is also
presented towards the ultimate validation of NIR optical tomography using PET imaging as a "gold-standard" for
quantification.
Recently, we demonstrated near-infrared (NIR) fluorescence imaging for quantifying real-time lymphatic propulsion in
humans following intradermal injections of microdose amounts of indocyanine green. However computational methods
for image analysis are underdeveloped, hindering the translation and clinical adaptation of NIR fluorescent lymphatic
imaging. In our initial work we used ImageJ and custom MatLab programs to manually identify lymphatic vessels and
individual propulsion events using the temporal transit of the fluorescent dye. In addition, we extracted the apparent
velocities of contractile propagation and time periods between propulsion events. Extensive time and effort were
required to analyze the 6-8 gigabytes of NIR fluorescent images obtained for each subject. To alleviate this bottleneck,
we commenced development of ALFIA, an integrated software platform which will permit automated, near real-time
analysis of lymphatic function using NIR fluorescent imaging. However, prior to automation, the base algorithms
calculating the apparent velocity and period must be validated to verify that they produce results consistent with the
proof-of-concept programs. To do this, both methods were used to analyze NIR fluorescent images of two subjects and
the number of propulsive events identified, the average apparent velocities, and the average periods for each subject were
compared. Paired Student's t-tests indicate that the differences between their average results are not significant. With
the base algorithms validated, further development and automation of ALFIA can be realized, significantly reducing the
amount of user interaction required, and potentially enabling the near real-time, clinical evaluation of NIR fluorescent
lymphatic imaging.
Fluorescence-enhanced optical imaging/tomography may play an important role in preclinical research and
clinical diagnostics as a type of optical molecular. Time- and frequency-domain measurement can acquire more
measurement information, reducing the ill-posedness and improving the reconstruction quality of fluorescence-enhanced
optical tomography. Although the diffusion approximation (DA) theory has been extensively in optical
imaging, high-order photon migration models must be further investigated for application to complex and small
tissue volumes. In this paper, a frequency-domain fully parallel adaptive finite element solver is developed with
the simplified spherical harmonics (SPN) approximations. To fully evaluate the performance of the SPN approximations,
a fast tetrahedron-based Monte Carlo simulator suitable for complex heterogeneous geometries
is developed using the convolution strategy to realize the simulation of the fluorescence excitation and emission.
With simple and real digital mouse phantoms, the results show that the significant precision and speed
improvements are obtained from the parallel adaptive mesh evolution strategy.
We compare and contrast the development of optical molecular imaging techniques with nuclear medicine with a didactic emphasis for initiating readers into the field of molecular imaging. The nuclear imaging techniques of gamma scintigraphy, single-photon emission computed tomography, and positron emission tomography are first briefly reviewed. The molecular optical imaging techniques of bioluminescence and fluorescence using gene reporter/probes and gene reporters are described prior to introducing the governing factors of autofluorescence and excitation light leakage. The use of dual-labeled, near-infrared excitable and radio-labeled agents are described with comparative measurements between planar fluorescence and nuclear molecular imaging. The concept of time-independent and -dependent measurements is described with emphasis on integrating time-dependent measurements made in the frequency domain for 3-D tomography. Finally, we comment on the challenges and progress for translating near-infrared (NIR) molecular imaging agents for personalized medicine.
Several phantom and in vivo small animal imaging studies have been performed to detect the re-emitted fluorescence signal arising from micro to pico molar concentrations of fluorophore by employing band-pass and band-rejection filters. However, elimination of the back-reflected excitation light still remains a major challenge for further reducing the noise floor in fluorescence imaging. Furthermore, despite the well-known deterioration of interference filter performance as the angle of incidence deviates from zero degrees, most studies do not employ collimated light optical design required for efficient excitation light rejection using interference filters. In this study, we measured quantities in frequency domain data for the combination of three-cavity interference and holographic super notch filters. To assess excitation leakage, the “out-of-band (S (λx ) )” to “in-band (S (λm ) - S (λx ) )” signal ratio, AC amplitude (IAC ), and phase delay (δ-δ*) measured from a gain modulated, intensified CCD imaging system with and without collimating optics was evaluated. The addition of collimating optics resulted in a reduction of 82% to 91% of the out-of-band to in-band ratio for the phantom studies and an increase of 1.4 to 3.7 times of the target-to-background ratio (T:B) for small animal studies.
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