Significance: Changes in interstitial fluid clearance are implicated in many diseases. Using near-infrared (NIR) imaging with properly sized tracers could enhance our understanding of how venous and lymphatic drainage are involved in disease progression or enhance drug delivery strategies.
Aim: We investigated multichromatic NIR imaging with multiple tracers to assess in vivo microvascular clearance kinetics and pathways in different tissue spaces.
Approach: We used a chemically inert IR Dye 800CW (D800) to target venous capillaries and a purified conjugate of IR dye 680RD with 40 kDa PEG (P40D680) to target lymphatic capillaries in vivo. Optical imaging settings were validated and tuned in vitro using tissue phantoms. We investigated multichromatic NIR imaging’s utility in two in vivo tissue beds: the mouse tail and rat knee joint. We then tested the ability of the approach to detect interstitial fluid perturbations due to exercise.
Results: In an in vitro simulated tissue environment, free dye and PEG mixture allowed for simultaneous detection without interference. In the mouse tail, co-injected NIR tracers cleared from the interstitial space via distinct routes, suggestive of lymphatic and venous uptake mechanisms. In the rat knee, we determined that exercise after injection transiently increased lymphatic drainage as measured by lower normalized intensity immediately after exercise, whereas exercise pre-injection exhibited a transient delay in clearance from the joint.
Conclusions: NIR imaging enables simultaneous imaging of lymphatic and venous-mediated fluid clearance with great sensitivity and can be used to measure temporal changes in clearance rates and pathways.
Nearly all dietary lipids are transported from the intestine to venous circulation through the lymphatic system, yet the mechanisms that regulate this process remain unclear. Elucidating the mechanisms involved in the functional response of lymphatics to changes in lipid load would provide valuable insight into recent implications of lymphatic dysfunction in lipid related diseases. Therefore, we sought to develop an in situ imaging system to quantify and correlate lymphatic function as it relates to lipid transport. The imaging platform provides the capability of dual-channel imaging of both high-speed bright-field video and fluorescence simultaneously. Utilizing post-acquisition image processing algorithms, we can quantify correlations between vessel pump function, lymph flow, and lipid concentration of mesenteric lymphatic vessels in situ. All image analysis is automated with customized LabVIEW virtual instruments; local flow is measured through lymphocyte velocity tracking, vessel contraction through measurements of the vessel wall displacement, and lipid uptake through fluorescence intensity tracking of an orally administered fluorescently labelled fatty acid analogue, BODIPY FL C16. This system will prove to be an invaluable tool for scientists studying intestinal lymphatic function in health and disease, and those investigating strategies for targeting the lymphatics with orally delivered drugs to avoid first pass metabolism.
KEYWORDS: Lymphatic system, Luminescence, Near infrared, Tissues, Imaging systems, In vivo imaging, Video, Signal to noise ratio, Scattering, Visualization
Near-infrared imaging of lymphatic drainage of injected indocyanine green (ICG) has emerged as a new technology for clinical imaging of lymphatic architecture and quantification of vessel function, yet the imaging capabilities of this approach have yet to be quantitatively characterized. We seek to quantify its capabilities as a diagnostic tool for lymphatic disease. Imaging is performed in a tissue phantom for sensitivity analysis and in hairless rats for in vivo testing. To demonstrate the efficacy of this imaging approach to quantifying immediate functional changes in lymphatics, we investigate the effects of a topically applied nitric oxide (NO) donor glyceryl trinitrate ointment. Premixing ICG with albumin induces greater fluorescence intensity, with the ideal concentration being 150 μg / mL ICG and 60 g / L albumin. ICG fluorescence can be detected at a concentration of 150 μg / mL as deep as 6 mm with our system, but spatial resolution deteriorates below 3 mm, skewing measurements of vessel geometry. NO treatment slows lymphatic transport, which is reflected in increased transport time, reduced packet frequency, reduced packet velocity, and reduced effective contraction length. NIR imaging may be an alternative to invasive procedures measuring lymphatic function in vivo in real time.
All dietary lipids are transported to venous circulation through the lymphatic system, yet the underlying mechanisms that
regulate this process remain unclear. Understanding how the lymphatics functionally respond to changes in lipid load is
important in the diagnosis and treatment of lipid and lymphatic related diseases such as obesity, hypercholesterolemia,
and lymphedema. Therefore, we sought to develop an in situ imaging system to quantify and correlate lymphatic
function as it relates to lipid transport. A custom-built optical set-up provides us with the capability of dual-channel
imaging of both high-speed bright-field video and fluorescence simultaneously. This is achieved by dividing the light
path into two optical bands. Utilizing high-speed and back-illuminated CCD cameras and post-acquisition image
processing algorithms, we have the potential quantify correlations between vessel contraction, lymph flow and lipid
concentration of mesenteric lymphatic vessels in situ. Local flow velocity is measured through lymphocyte tracking,
vessel contraction through measurements of the vessel walls and lipid uptake through fluorescence intensity tracking of a
fluorescent long chain fatty acid analogue, Bodipy FL C16. This system will prove to be an invaluable tool for both
scientists studying lymphatic function in health and disease, and those investigating strategies for targeting the lymphatic
system with orally delivered drugs.
Background - Near-infrared (NIR) imaging of lymphatic drainage of injected indocyanine green (ICG) has emerged as a
new technology for clinical imaging of lymphatic architecture and quantification of vessel function, offering better
spatial and temporal resolution than competing imaging modalities. While NIR lymphatic imaging has begun to be
reported in the literature, the technology is still in its infancy and its imaging capabilities have yet to be quantitatively
characterized. The objective of this study, therefore, was to characterize the parameters of NIR lymphatic imaging to
quantify its capabilities as a diagnostic tool for evaluating lymphatic disease.
Methods - An NIR imaging system was developed using a laser diode for excitation, ICG as a fluorescent agent, and a
CCD camera to detect emission. A tissue phantom with mock lymphatic vessels of known depths and diameters was
used as an alternative to in vivo lymphatic vessels due to the greater degree of control with the phantom.
Results and Conclusions - When dissolved in an albumin physiological salt solution (APSS) to mimic interstitial fluid,
ICG experiences shifts in the excitation/emission wavelengths such that it is maximally excited at 805nm and produces
peak fluorescence at 840nm. Premixing ICG with albumin induces greater fluorescence intensity, with the ideal
concentration being: 900μM (60g/L) albumin and 193.5μM (150μg/mL) ICG. ICG fluorescence can be detected as deep as 6mm, but spatial resolution deteriorates severely below 3mm, thus skewing vessel geometry measurements. ICG packet travel, a common measure of lymphatic transport, can be detected as deep as 5mm.
Collecting microlymphatics play a vital role in promoting lymph flow from the initial lymphatics in the interstitial spaces to the large transport lymph ducts. In most tissues, the primary mechanism for producing this flow is the spontaneous contractions of the lymphatic wall. Individual units, known as lymphangion, are separated by valves that help prevent backflow when the vessel contracts, thus promoting flow through the lymphatic network. Lymphatic contractile activity is inhibited by flow in isolated lymphatics, however there are virtually no in situ measurements of lymph flow in these vessels. One of the difficulties associated with obtaining such measurements is the time consuming methods of manual particle tracking used previously by our group. Using an in situ preparation with mesenteric microlymphatics (~ 100 μm in diameter) and a high speed imaging system (500 fps), we have developed an image correlation method to measure lymphatic flow with a standard error of prediction of 0.3 mm/sec when compared with manual particle tracking.
Despite advances in the measurement of lymphatic function, little is known about the actual velocities of flow in microlymphatic (~100 µm diam) vessels. In this work, video microscopy and particle tracking methods are adapted and integrated with an ultra-high-speed imaging camera to obtain measurements of lymph velocities throughout the entire lymphatic contraction cycle in the ratmesentery, something that previous systems were incapable of measuring. To determine the system's accuracy, calibration experiments are conducted across the hypothesized physiologically significant range of velocities for microlymphatic flow (up to 15 mm/sec). The system shows high accuracy, less than 2% error, when comparing actual with measured velocities. Microspheres flowing through 140-µm-diam tubing are imaged to demonstrate the system's ability to determine flow rates in these small vessels by measuring particle velocities. To demonstrate biological applicability, mesenteric microlymphatics in loops of the small intestine of three male Sprague-Dawley rats are exteriorized and imaged with the high-speed system at a rate of 500 frames/sec for several contraction sequences. Lymph velocity fluctuates cyclically with the vessel wall contractions, ranging from –1 to 7 mm/sec. These rates are higher than would be possible with standard video microscopy (3.75 mm/sec maximum).
Despite advances in the measurement of lymphatic flow, little is known about the actual velocities of flow in microlymphatic (~100 um diameter) vessels. In this paper, video microscopy and particle tracking methods were adapted and integrated with an ultra high-speed imaging camera to obtain measurements of high-speed lymph velocities that previous systems were incapable of measuring. In this study, a mesenteric microlymphatic vessel in a loop of the small intestine of a male Sprague-Dawley rat was exteriorized and imaged at a rate of 500 frames per second (fps) for several contraction sequences. Lymph velocity was shown to fluctuate cyclically with the vessel wall contractions and ranged from -1 to 4 mm/sec through a ten second sequence.
The increasing prevalence of diabetes in the United States has led many to pursue methods for non-invasive glucose detection using various optical approaches such as NIR absorption spectroscopy, Raman spectroscopy, fluorescence spectroscopy, and polarization. Polarization approaches using the aqueous humor as the sensing site have been previously shown to achieve 5 mg/dl accuracy in vitro, however accuracy in vivo has yet to be obtained due to motion induced birefringence changes in the cornea. A dual-wavelength close-looped system was developed to compensate for motion artifact. This method has shown 15 mg/dl accuracy in the presence of birefringence changes in the optical path in vitro similar to those that occur in the cornea -- something previous systems were not capable of doing.
In a continuing effort to develop a noninvasive means of monitoring glucose levels using the aqueous humor of the eye, a dual-wavelength system is developed to show that varying birefringence, similar to what is seen with a moving cornea, can be compensated. In this work, a dual-wavelength, closed-loop system is designed and a model is developed to extract the glucose concentration information. The system and model are tested using various concentrations of glucose in a birefringent test cell subject to motion artifact. The results show that for a static, nonmoving sample, glucose can be predicted to within 10 mg/dl for the entire physiologic range (0 to 600 mg/dl) for either laser wavelength (523 or 635 nm). In the presence of moving birefringence, each individual wavelength produces standard errors on the order of a few thousand mg/dL. However, when the two wavelengths are combined into the developed model, this error is less than 20 mg/dL. The approach shows that multiple wavelengths can be used to drastically reduce the error in the presence of a moving birefringent sample and thus may have the potential to be used to noninvasively monitor glucose levels in vivo in the presence of moving corneal birefringence.
A Monte Carlo method was developed to model light transport through multi-layered tissue with the application focused on the development of an implantable perfusion monitor. The model was developed and then verified experimentally with a micro perfusion phantom. The program modeled a three-layer (tissue, capillary bed, tissue) scenario to investigate the source-detector separation effects for an implantable sensor. The Monte Carlo code was used specifically to model the effects of absorption and scattering properties of the surrounding tissue, the hemoglobin concentration in the middle layer, the ratio of thickness of the capillary layer to the first layer, and the probe-source separation distance on the propagation of the light through the tissue. The model was verified experimentally, using a simple in vitro system with optical source and detector fibers separated at various distances. The model was also used to investigate fluctuations in luminance as a result of hemoglobin concentrations and the response of the system to various wavelengths. The model was helpful for an ongoing project to develop an implantable perfusion monitor for transplanted organs or skin flaps.
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