ABSTRACT
Fiber optic bundles are used in narrow-diameter medical and industrial instruments for acquiring images from confined locations. Images transmitted through these bundles contain only one pixel of information per fiber core and fail to capture information from the cladding region between cores. Both factors limit the spatial resolution attainable with fiber bundles. We show here that computational imaging (CI) can be combined with spectral coding to overcome these two fundamental limitations and improve spatial resolution in fiber bundle imaging. By acquiring multiple images of a scene with a high-resolution mask pattern imposed, up to 17 pixels of information can be recovered from each fiber core. A dispersive element at the distal end of the bundle imparts a wavelength-dependent lateral shift on light from the object. This enables light that would otherwise be lost at the inter-fiber cladding to be transmitted through adjacent fiber cores. We experimentally demonstrate this approach using synthetic and real objects. Using CI with spectral coding, object features 5× smaller than individual fiber cores were resolved, whereas conventional imaging could only resolve features at least 1.5× larger than each core. In summary, CI combined with spectral coding provides an approach for overcoming the two fundamental limitations of fiber optic bundle imaging.
ABSTRACT
Water and lipids are key participants in many biological processes, but there are few non-invasive methods that provide quantification of these components in vivo, and none that can isolate and quantify lipids in the blood. Here we develop a new imaging modality termed shortwave infrared meso-patterned imaging (SWIR-MPI) to provide label-free, non-contact, spatial mapping of water and lipid concentrations in tissue. The method utilizes patterned hyperspectral illumination to target chromophore absorption bands in the 900-1,300 nm wavelength range. We use SWIR-MPI to monitor clinically important physiological processes including edema, inflammation, and tumor lipid heterogeneity in preclinical models. We also show that SWIR-MPI can spatially map blood-lipids in humans, representing an example of non-invasive and contact-free measurements of in vivo blood lipids. Together, these results highlight the potential of SWIR-MPI to enable new capabilities in fundamental studies and clinical monitoring of major conditions including obesity, cancer, and cardiovascular disease.
Subject(s)
Infrared Rays , Lipids/blood , Optical Imaging/methods , Radio Waves , Spectroscopy, Near-Infrared/methods , Water/analysis , Adipose Tissue, Brown/diagnostic imaging , Adipose Tissue, Brown/pathology , Adult , Animals , Biomarkers/blood , Cardiovascular Diseases/diagnostic imaging , Edema/diagnostic imaging , Edema/pathology , Female , Heterografts , Humans , Inflammation/diagnostic imaging , Inflammation/pathology , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Middle Aged , Neoplasms/diagnostic imaging , Neoplasms/pathology , Obesity/diagnostic imaging , Optical Imaging/instrumentation , Prostatic Neoplasms/diagnostic imaging , Prostatic Neoplasms/pathology , Spectroscopy, Near-Infrared/instrumentationABSTRACT
BACKGROUND: The incidence of acute kidney injury (AKI) during pregnancy precedes a high maternal mortality rate of 20-40%. AKI during pregnancy has multiple etiologies; however, the more common are maternal hypertensive disorders, which include preeclampsia and HELLP (hemolysis, elevated liver enzyme, low platelet) syndrome. Therefore, we sought to assess the impact of AKI on blood pressure, kidney injury, and anti-angiogenic factors during pregnancies with and without HELLP syndrome. METHODS: On gestational day (GD) 12, mini-osmotic pumps were inserted into a subset of normal pregnant (NP) rats infusing 4.7 µg/kg soluble fms-like tyrosine kinase-1 (sFlt-1) and 7 µg/kg soluble endoglin (sEng) to induce HELLP syndrome. On GD18, the renal pedicles were occluded for 45 min to induce AKI via bilateral ischemia reperfusion in a subset of NP (n = 18) or HELLP (n = 20) rats. Control NP (n = 20) and HELLP (n = 20) rats underwent a SHAM surgery on GD18. Plasma, urine, and maternal organs were saved for further analysis. Renal injury was assessed via renal histopathology, glomerular filtration rate (GFR), T cell infiltration, and assessment of kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL). Data was measured via two-way analysis of variance with Tukey's test for post hoc analysis. RESULTS: Blood pressures were increased in HELLP+AKI rats (p = 0.0001); both NP+AKI and HELLP+AKI rats had increased lactate dehydrogenase (p < 0.0001) and aspartate aminotransferase levels (p < 0.0001), and decreased platelet levels (p < 0.001) vs. NP rats. HELLP+AKI (p = 0.002) and HELLP rats (p = 0.0002) had evidence of renal fibrosis vs. NP rats. GFR was decreased in HELLP+AKI (p = 0.01) rats vs. NP rats. Urinary KIM-1 was increased in NP+AKI rats vs. NP (p = 0.003) and HELLP rats (p = 0.01). HELLP+AKI rats had increased urinary KIM-1 vs. NP (p = 0.0008) and HELLP rats (p = 0.004) and increased NGAL vs. HELLP rats (p = 0.002). HELLP+AKI rats had increased sFlt-1 (p = 0.009) vs. NP rats. NP+AKI (p = 0.02) and HELLP+AKI (p = 0.007) rats had increased sEng vs. NP rats. CD3+CD4+ T cells were significantly increased in HELLP+AKI rats vs. NP (p = 0.0002) and NP+AKI (p = 0.05) rats. T regulatory cells were significantly decreased in HELLP+AKI (p = 0.03) and NP+AKI (p = 0.02) rats vs. NP rats; there were no changes between groups in T helper 17 cells (p = 0.34). CONCLUSION: The findings in this study suggest that AKI during pregnancy contributes to increased blood pressure and biochemical markers for HELLP syndrome, creates an anti-angiogenic imbalance, and exacerbates kidney injury as shown on histopathology, GFR, and kidney injury markers.
Subject(s)
Endoglin/metabolism , HELLP Syndrome , Kidney Diseases/etiology , Kidney/cytology , T-Lymphocytes, Regulatory , Vascular Endothelial Growth Factor Receptor-1/metabolism , Animals , Animals, Newborn , Biomarkers/blood , Biomarkers/urine , Birth Weight , Blood Pressure , Endoglin/genetics , Female , Kidney Diseases/pathology , Pregnancy , Rats , Rats, Sprague-Dawley , Vascular Endothelial Growth Factor Receptor-1/geneticsABSTRACT
Neutralization of FasL is linked to suppression of hypertension, placental inflammation, and endothelin system activation in an animal model of hemolysis, elevated liver enzymes, low platelets (HELLP) syndrome. During HELLP syndrome the placenta has been reported to serve as the primary source of Fas ligand (FasL), which has an impact on inflammation and hypertension during pregnancy and is dysregulated in women with severe preeclampsia and HELLP syndrome. We hypothesize that neutralization of FasL during pregnancy in an animal model of HELLP syndrome decreases inflammation and placental apoptosis, improves endothelial damage, and improves hypertension. On gestational day (GD) 12, rats were chronically infused with placental antiangiogenic factors sFlt-1 and sEng to induce HELLP syndrome. To neutralize FasL, MFL4 or FasL antibody was infused into a subset of HELLP or normal pregnant rats on GD13. IgG infusion into another group of NP and HELLP rats on GD13 was used as a control for FasL antibody, and all rats were euthanized on GD19 after blood pressure measurement. Plasma and placentas were collected to assess inflammation, apoptosis, and the degree of placental debris activation of endothelial cells. Administration of MFL4 to HELLP rats significantly decreased blood pressure compared with untreated HELLP rats and HELLP rats infused with IgG and improved the biochemistry of HELLP syndrome. Both circulating and placental FasL were significantly attenuated in response to MFL4 infusion, as were levels of placental and circulating TNFα when compared with untreated HELLP rats and HELLP rats infused with IgG. Endothelial cells exposed to placental debris and media from HP + MFL4 rats secreted significantly less endothelin-1 compared with stimulated endothelial cells from HELLP placentas. Neutralization of FasL is associated with decreased MAP and improvement in placental inflammation and endothelial damage in an animal model of HELLP syndrome.
Subject(s)
Antibodies, Neutralizing/therapeutic use , Endothelin-1/blood , Fas Ligand Protein/immunology , HELLP Syndrome/drug therapy , Placenta/physiopathology , Animals , Disease Models, Animal , Fas Ligand Protein/blood , Female , HELLP Syndrome/blood , HELLP Syndrome/immunology , HELLP Syndrome/physiopathology , Immunoglobulin G , Placenta/immunology , Pregnancy , Rats , Rats, Sprague-Dawley , Treatment Outcome , Tumor Necrosis Factor-alpha/blood , Vascular Endothelial Growth Factor Receptor-1/bloodABSTRACT
We demonstrate the possibility of measuring FRET efficiency with a low-cost frequency-domain fluorescence lifetime imaging microscope (FD-FLIM). The system utilizes single-frequency-modulated excitation, which enables the use of cost-effective laser sources and electronics, simplification of data acquisition and analysis, and a dual-channel detection capability. Following calibration with coumarin 6, we measured the apparent donor lifetime in mTFP1-mVenus FRET standards expressed in living cells. We evaluated the system's sensitivity by differentiating the short and long lifetimes of mTFP1 corresponding to the known standards' high and low FRET efficiency, respectively. Furthermore, we show that the lifetime of the vinculin tension sensor, VinTS, at focal adhesions (2.30 ± 0.16 ns) is significantly (p < 10 - 6) longer than the lifetime of the unloaded TSMod probe (2.02 ± 0.16 ns). The pixel dwell time was 6.8 µs for samples expressing the FRET standards, with signal typically an order of magnitude higher than VinTS. The apparent FRET efficiency (
Subject(s)
Fluorescence Resonance Energy Transfer/methods , Microscopy, Fluorescence/methods , Molecular Probes/chemistry , Animals , Cell Line , Equipment Design , Fluorescence Resonance Energy Transfer/instrumentation , Focal Adhesions/physiology , Image Processing, Computer-Assisted , Mice , Microscopy, Fluorescence/instrumentation , Signal Processing, Computer-Assisted , Vinculin/chemistryABSTRACT
This Letter presents a framework for computational imaging (CI) in fiber-bundle-based endoscopy systems. Multiple observations are acquired of objects spatially modulated with different random binary masks. Sparse-recovery algorithms then reconstruct images with more resolved pixels than individual fibers in the bundle. Object details lying within the diameter of single fibers are resolved, allowing images with 41,663 resolvable points to be generated through a bundle with 2,420 fibers. Computational fiber bundle imaging of micro- and macro-scale objects is demonstrated using fluorescent standards and biological tissues, including in vivo imaging of a human fingertip. In each case, CI recovers details that conventional endoscopy does not provide.
ABSTRACT
BACKGROUND: Myostatin antagonists are being developed as therapies for Duchenne muscular dystrophy due to their strong hypertrophic effects on skeletal muscle. Engineered follistatin has the potential to combine the hypertrophy of myostatin antagonism with the anti-inflammatory and anti-fibrotic effects of activin A antagonism. METHODS: Engineered follistatin was administered to C57BL/6 mice for 4 weeks, and muscle mass and myofiber size was measured. In the mdx model, engineered follistatin was dosed for 12 weeks in two studies comparing to an Fc fusion of the activin IIB receptor or an anti-myostatin antibody. Functional measurements of grip strength and tetanic force were combined with tissue analysis for markers of necrosis, inflammation, and fibrosis to evaluate improvement in dystrophic pathology. RESULTS: In wild-type and mdx mice, dose-dependent increases in muscle mass and quadriceps myofiber size were observed for engineered follistatin. In mdx, increases in grip strength and tetanic force were combined with improvements in muscle markers for necrosis, inflammation, and fibrosis. Improvements in dystrophic pathology were greater for engineered follistatin than the anti-myostatin antibody. CONCLUSIONS: Engineered follistatin generated hypertrophy and anti-fibrotic effects in the mdx model.
Subject(s)
Activins/antagonists & inhibitors , Follistatin/therapeutic use , Muscular Dystrophies/drug therapy , Myostatin/antagonists & inhibitors , Animals , Follistatin/administration & dosage , Hand Strength , Male , Mice , Mice, Inbred C57BL , Mice, Inbred mdx , Muscle Contraction , Recombinant Proteins/administration & dosage , Recombinant Proteins/therapeutic useABSTRACT
This paper investigates a highly parallel extension of the single-pixel camera based on a focal plane array. It discusses the practical challenges that arise when implementing such an architecture and demonstrates that system-specific optical effects must be measured and integrated within the system model for accurate image reconstruction. Three different projection lenses were used to evaluate the ability of the system to accommodate varying degrees of optical imperfection. Reconstruction of binary and grayscale objects using system-specific models and Nesterov's proximal gradient method produced images with higher spatial resolution and lower reconstruction error than using either bicubic interpolation or a theoretical system model that assumes ideal optical behavior. The high-quality images produced using relatively few observations suggest that higher throughput imaging may be achieved with such architectures than with conventional single-pixel cameras. The optical design considerations and quantitative performance metrics proposed here may lead to improved image reconstruction for similar highly parallel systems.
ABSTRACT
Failure of cardiac antiarrhythmic ablation to block action potential conduction produces poor outcomes which lead to repeat procedures. To overcome this, an intraoperative index of the quality of an ablation lesion is needed. We hypothesized that a rise in the translesion stimulus-excitation delay (TED) can indicate a continuous, transmural, linear lesion, and that the TED is related to the path length in the viable tissue around the lesion. Rabbit hearts were isolated, perfused with a warm physiological solution and stained with transmembrane potential-sensitive fluorescent dye. Radiofrequency (RF) ablation was performed on ventricular epicardium with a vacuum-assisted coagulation device to produce either a complete or incomplete lesion. Complete lesions were both transmural and continuous. Incomplete lesions were noncontinuous or nontransmural. The TED was determined with bipolar stimulation at one side of the lesion and either a bipolar electrogram at the other side or optical mapping on both sides. Hearts were then stained with tetrazolium chloride and examined histologically to estimate minimum path lengths of viable tissue from the stimulation site to the recording site. Complete lesions increased the TED by factors of 2.6-3.1 (p < 0.05), whereas incomplete lesions did not significantly increase the TED. Larger minimum path lengths were found for cases that had an increased TED. The TED was quantitatively predictable based on a conduction velocity of 0.38-0.49 m s(-1), which is typical of rabbit hearts. The TED significantly increases when a linear lesion is complete, suggesting that an intraoperative measurement of the TED may help to improve ablation lesions and outcomes. Predictability of the TED based on the viable tissue path suggests that quantitative TEDs for clinical lesions may be anticipated provided that the conduction velocity is considered.
Subject(s)
Catheter Ablation , Electrophysiologic Techniques, Cardiac/methods , Heart/physiopathology , Action Potentials , Animals , Electrocardiography , Electrodes , RabbitsABSTRACT
Cardiac action potentials have been measured with single-photon excitation (SPE) of transmembrane voltage-sensitive fluorescent dye. Two-photon excitation (TPE) may have advantages for localization and depth of the tissue region from which the action potential is measured. However measurements of action potentials with SPE have not been demonstrated. We sought to develop a method for TPE of di-4-ANEPPS and test whether the method yields voltage-dependent fluorescence in cardiac tissue. We modified our SPE and ratio-metric fluorescence recording system to use a femtosecond pulsed near-infrared laser. Modifications were made to enhance fluorescence collection efficiency and to block infrared laser light from entering the fluorescence collection system. Fluorescence was collected simultaneously in green (510-570 nm) and red (590-700 nm) wavelength bands. Action potentials were observed in the ratio of the green signal to the red signal, but were not observed above the noise level in either of the individual signals. Incorporation of a common-mode noise subtraction method revealed action potentials in green and red signals. We also found that the di-4-ANEPPS fluorescence emission spectrum for TPE at 930 nm was similar to the emission spectrum for SPE at 488 nm. The multiphoton method may be beneficial for highly localized cardiac optical measurements.
Subject(s)
Action Potentials/physiology , Diagnostic Imaging , Fluorescence , Fluorescent Dyes/pharmacology , Heart Conduction System/physiology , Pyridinium Compounds/pharmacology , Animals , Diagnostic Imaging/instrumentation , Diagnostic Imaging/methods , Organ Culture Techniques , RabbitsABSTRACT
Major effects of stimulation on cardiac transmembrane potentials (Vm) are thought to occur under the electrode, however these have not been optically mapped due to blockage of light by electrodes. Here we optically mapped under translucent indium tin oxide (ITO) electrodes in hearts stained with transmembrane voltage sensitive fluorescent dye, di-4-ANEPPS excited at 488 nm. Emissions in wavelength bands 510-570 nm and >590 nm were similarly affected by changes in ITO transmittance due to electrochemical effects of current at the electrode interface. Dual-wavelength ratiometric mapping with the two emission bands revealed Vm under the electrode during plateau-phase stimulation (220 mA). Changes in Vm were heterogeneous under the electrode, and were anisotropic with larger values along the fiber axis. These results explain early excitation sites for sufficiently strong diastolic stimulation, and agree with theoretical predictions based on summation of anisotropic effects of point stimulation and a linear 3-d cardiac bidomain computer model. The bidomain model and experiments disagree under the edge of the electrode, where modeled Vm is much larger. Thus, changes in Vm under an electrode are anisotropic with greater Vm in the direction parallel to fibers. Nonlinear effects of stimulation in hearts may limit changes in Vm under the electrode edge.
Subject(s)
Body Surface Potential Mapping/methods , Electric Countershock/methods , Electric Stimulation/methods , Electrodes , Microscopy, Confocal/methods , Microscopy, Fluorescence/methods , Models, Cardiovascular , Animals , Body Surface Potential Mapping/instrumentation , Computer Simulation , Electric Countershock/instrumentation , Electric Stimulation/instrumentation , Heart Conduction System/physiopathology , In Vitro Techniques , Membrane Potentials/physiology , Pyridinium Compounds , RabbitsABSTRACT
The adhesion of platelets to the subendothelium of blood vessels at sites of vascular injury under high shear conditions is mediated by a direct interaction between the platelet receptor glycoprotein Ibalpha (GpIbalpha) and the A1 domain of the von Willebrand factor (VWF). Here we report the 2.6-A crystal structure of a complex comprised of the extracellular domain of GpIbalpha and the wild-type A1 domain of VWF. A direct comparison of this structure to a GpIbalpha-A1 complex containing "gain-of-function" mutations, A1-R543Q and GpIbalpha-M239V, reveals specific structural differences between these complexes at sites near the two GpIbalpha-A1 binding interfaces. At the smaller interface, differences in interaction show that the alpha1-beta2 loop of A1 serves as a conformational switch, alternating between an open alpha1-beta2 isomer that allows faster dissociation of GpIbalpha-A1, as observed in the wild-type complex, and an extended isomer that favors tight association as seen in the complex containing A1 with a type 2B von Willebrand Disease (VWD) mutation associated with spontaneous binding to GpIbalpha. At the larger interface, differences in interaction associated with the GpIbalpha-M239V platelet-type VWD mutation are minor and localized but feature discrete gamma-turn conformers at the loop end of the beta-hairpin structure. The beta-hairpin, stabilized by a strong classic gamma-turn as seen in the mutant complex, relates to the increased affinity of A1 binding, and the beta-hairpin with a weak inverse gamma-turn observed in the wild-type complex corresponds to the lower affinity state of GpIbalpha. These findings provide important details that add to our understanding of how both type 2B and platelet-type VWD mutations affect GpIbalpha-A1 binding affinity.
Subject(s)
Platelet Glycoprotein GPIb-IX Complex/chemistry , von Willebrand Factor/chemistry , Binding Sites/genetics , Humans , Models, Molecular , Mutation , Protein Binding , Protein Conformation , Protein Structure, Tertiary , von Willebrand Diseases/genetics , von Willebrand Factor/geneticsABSTRACT
The aim of this study is to develop a system for the first simultaneous measurements using 31P NMR and optical transmembrane potential-sensitive fluorescence. 31P NMR is used to evaluate 5 metabolic markers (pH, sugar phosphates, phosphocreatine, phospholipid intermediates and ATP). Action potential duration is measured with a transmembrane potential-sensitive fluorescent dye, di-4ANEPPS. The system is intended to correlate the action potentials with the metabolic markers and their changes during the time course of cardiac ischemia. The requirements of this system include fabrication of a NMR probe large enough for rabbit heart experiments, incorporation of light guides for excitation of dye and collection of fluorescence in hearts located within a NMR magnet and with minimal disturbance of the magnetic field. The quality factor (Q) of the probe's coil was measured. Control NMR spectra were then acquired with phosphorus test solution. Further spectra were obtained after addition of the optical elements. Results show the ability to use our new probe to acquire a spectrum in the presence of the optical elements within the magnet, suggesting the possibility that 31P NMR spectroscopy and optical transmembrane potential measurements can be performed simultaneously in hearts.
ABSTRACT
Direct interaction between platelet receptor glycoprotein Ibalpha (GpIbalpha) and thrombin is required for platelet aggregation and activation at sites of vascular injury. Abnormal GpIbalpha-thrombin binding is associated with many pathological conditions,including occlusive arterial thrombosis and bleeding disorders. The crystal structure of the GpIbalpha-thrombin complex at 2.6 angstrom resolution reveals simultaneous interactions of GpIbalpha with exosite I of one thrombin molecule,and with exosite II of a second thrombin molecule. In the crystal lattice,the periodic arrangement of GpIbalpha-thrombin complexes mirrors a scaffold that could serve as a driving force for tight platelet adhesion. The details of these interactions reconcile GpIbalpha-thrombin binding modes that are presently controversial,highlighting two distinct interfaces that are potential targets for development of novel antithrombotic drugs.
Subject(s)
Platelet Aggregation , Platelet Glycoprotein GPIb-IX Complex/chemistry , Platelet Glycoprotein GPIb-IX Complex/metabolism , Thrombin/chemistry , Thrombin/metabolism , Binding Sites , Blood Platelets/chemistry , Blood Platelets/physiology , Crystallization , Crystallography, X-Ray , Humans , Hydrogen Bonding , Hydrophobic and Hydrophilic Interactions , Models, Molecular , Platelet Adhesiveness , Protein Binding , Protein Conformation , Protein Structure, Secondary , Protein Structure, TertiaryABSTRACT
Early endosome antigen 1 (EEA1) is a 170-kDa polypeptide required for endosome fusion in mammalian cells. The COOH terminus of EEA1 contains a FYVE domain that interacts specifically with phosphatidylinositol 3-phosphate (PtdIns-3-P) and a Rab5 GTPase binding region adjacent to the FYVE domain. The dual interaction of EEA1 with both PtdIns-3-P and Rab5 has been hypothesized to provide the specificity required to target EEA1 to early endosomes. To test this hypothesis, we generated truncated (amino acids 1277--1411) and full-length EEA1 constructs containing point mutations in the COOH terminus that impair Rab5 but not PtdIns-3-P binding. These constructs localized to endosomes in intact cells as efficiently as their wild-type counterparts. Furthermore, overexpression of the truncated constructs, both wild-type and mutated, impaired the function of endogenous EEA1 resulting in the accumulation of small, untethered endosomes. These results suggest that association with Rab5 is not necessary for the initial binding and tethering functions of EEA1. A role for Rab5 binding was revealed, however, upon comparison of endosomes in cells expressing full-length wild-type or mutated EEA1. The mutant full-length EEA1 caused the accumulation of endosome clusters and suppressed the enlargement of endosomes caused by a persistently active form of Rab5 (Rab5Q79L). In contrast, expression of wild-type EEA1 with Rab5Q79L enhanced this enlargement. Thus, endosome tethering depends on the interaction of EEA1 with PtdIns-3-P, and its interaction with Rab5 appears to regulate subsequent fusion.