Velocimetry of red blood cells in microvessels by the dual-slit method: Effect of velocity gradients

The dual-slit is a photometric technique used for the measurement of red blood cell (RBC) velocity in microvessels. Two photometric windows (slits) are positioned along the vessel. Because the light is modulated by the RBCs flowing through the microvessel, a time dependent signal is captured for each window. A time delay between the two signals is obtained by temporal cross correlation, and is used to deduce a velocity, knowing the distance between the two slits. Despite its wide use in the field of microvascular research, the velocity actually measured by this technique has not yet been unambiguously related to a relevant velocity scale of the flow (e.g. mean or maximal velocity) or to the blood flow rate. This is due to a lack of fundamental understanding of the measurement and also because such a relationship is crucially dependent on the non-uniform velocity distribution of RBCs in the direction parallel to the light beam, which is generally unknown. The aim of the present work is to clarify the physical significance of the velocity measured by the dual-slit technique. For that purpose, dual-slit measurements were performed on computer-generated image sequences of RBCs flowing in microvessels, which allowed all the parameters related to this technique to be precisely controlled. A parametric study determined the range of optimal parameters for the implementation of the dual-slit technique. In this range, it was shown that, whatever the parameters governing the flow, the measured velocity was the maximal RBC velocity found in the direction parallel to the light beam. This finding was then verified by working with image sequences of flowing RBCs acquired in PDMS micro-systems in vitro. Besides confirming the results and physical understanding gained from the study with computer generated images, this in vitro study showed that the profile of RBC maximal velocity across the channel was blunter than a parabolic profile, and exhibited a non-zero sliding velocity at the channel walls. Overall, the present work demonstrates the robustness and high accuracy of the optimized dual-slit technique in various flow conditions, especially at high hematocrit, and discusses its potential for applications in vivo

Roles of nitric oxide and shear stress in the regulation of microvessel permeability in intact rat mesenteric venules

Mechanical forces have been indicated to play important roles in the regulation of inflammatory cell interaction with endothelium resulting in localized leakage formation and contributing to many disease-associated microvascular dysfunctions. However, most of the mechanical force related studies were conducted in vitro. The underlying mechanisms are still controversial. There is a need to investigate how shear stress regulates the endothelial cell (EC) signaling and related vascular barrier function using intact microvessels with experimental conditions closely replicating in vivo situations. The overall aim of my dissertation is to understand the molecular and cellular mechanisms of how shear stress and nitric oxide (NO) regulate microvessel function under physiological and pathological conditions. Studies were conducted on individually perfused intact rat mesenteric venules.;It is well known that shear stress is one of most important regulators in stimulating endothelial cells to produce NO. NO, in addition to being a potent vasodilator, has also been considered a double edged sword -mediator in inflammation. Under basal conditions, it prevents leukocyte and platelet adhesion, whereas under inflammatory conditions, the inflammatory mediator-induced excessive NO production contributes to permeability increases. In Chapter 2, we investigated the roles of endothelial basal NO production in leukocyte adhesion and adhesion-induced changes in microvessel permeability. The results indicated that the application of the eNOS specific inhibitor, caveolin-1 scaffolding peptide (CAV), caused reduction of basal NO and promoted ICAM-1-mediated leukocyte adhesion through Src activation-mediated ICAM-1 phosphorylation. Also, CAV-induced leukocyte adhesion was uncoupled from leukocyte oxidative burst and microvessel barrier function, unless in the presence of a secondary stimulation.;In Chapter 3, we investigated the roles of shear stress (SS) in the regulation of microvessel permeability and its related EC signaling involving blood cells in individually perfused intact microvessels. Our results demonstrated that in response to a sudden change of SS, transient shear magnitude-dependent increases in EC [Ca2+]i occurred only in vessels perfused with whole blood or perfusate containing RBCs, which was correlated with EC gap formation illustrated by fluorescent microsphere accumulation. Carbenoxolone, a Pannexin 1 inhibitor, significantly reduced shear magnitude-dependent ATP release from RBCs and also abolished SS-induced increases in EC [Ca 2+]i and EC gap formation. Meanwhile, both plasma and whole blood perfusion induced shear magnitude-dependent NO production and eNOS-Ser 1177 phosphorylation.;It is unknown how EC sense SS, but the Glycocalyx (GCX), a layer of proteoglycans covering the endothelium, has been implicated as a mechanical sensor for changes in SS in vitro. The objective of chapter 4 is to identify the changes in GCX in microvessels of streptozotocin-induced diabetic rats and evaluate the associated changes in sensing SS and SS-induced NO production in individually perfused venules of diabetic rats. Our results indicated that the impaired GCX in diabetic microvessels enhances EC response to mechanical force and potentiates NO production and EC responses to ATP, resulting in enhanced endothelial gap formation.;Advances in micromanufacturing and microfluidic technologies have enabled a variety of insights into biomedical sciences while curtailing the high experimental costs and complexities associated with animals and in vivo studies. In Chapter 5, we presented and discussed our research work in creating engineered microvessels using a microfluidic platform and demonstrated the formation of the microvascular network in vitro and validated the key features that have been observed in microvessels in vivo. In our future studies, this may provide us a novel platform for studying spatial and temporal change of shear stress in the regulation of microvessel function in a close in vivo situation.;In conclusion, we revealed the role of shear stress and NO in the regulation of endothelial cell signaling and microvessel permeability in vivo, involving blood and non-blood components. The results also suggest the potential in using a microfluidic device in studying the physiological microvessel function

Theranostic Window to the Brain for Multispectral Light Delivery and Microcirculation Imaging

Interest in using optical methods in the development of noninvasive clinicaldiagnostic and therapeutic techniques for brain diseases has widely increased due to theirsimplicity, safety, and affordability. The main limitations of light-based techniques usedfor brain theranostics are the strong light scattering in the scalp and skull, which causedecrease of spatial resolution, low contrast, and small penetration depth. To address thischallenge, our group has previously introduced a transparent nanocrystallineyttria-stabilized-zirconia (nc-YSZ) cranial implant material which implant possesses themechanical strength and biocompatibility that are prerequisites for a clinically-viablepermanent cranial implant for patients.This implant possesses the mechanical strength and biocompatibility that areprerequisites for a clinically-viable permanent cranial implant for patients. A potentialbenefit of this optical window is an improvement of light-based therapeutic techniquesthat rely on sufficient light penetration to a target embedded in tissue such asphotobiomodulation, photodynamic therapy, and optogenetics. Another application of thisoptical window is noninvasive visualization of brain blood vessels, hematomas, and smallpathologic structures (including cancerous growth) with high resolution. This isimportant for diagnosis and treatment of many diseases such as tumors of the brain,vascular pathologies, and so forth. In this dissertation, I investigated (1) characteristicsand durability of transparent nc-YSZ implants; (2) feasibility of chronic brain imagingthrough the implant; (3) multimodal imaging across the implant to generate anarteriovenous vascular map; (4) through-scalp VIS-NIR light delivery and microvascularimaging

A Novel Microbial Source Tracking DNA Microarray Used for Pathogen Detection in Environmental Systems

Pathogen detection and the identification of fecal contamination sources can be challenging in environmental and engineered treatment systems. Factors including pathogen diversity and ubiquity of fecal indicator bacteria hamper risk assessment and remediation of contamination sources. Therefore, a quick method that can detect and identify waterborne pathogens in environmental systems is needed. In this work, a custom microarray targeting pathogens (viruses, bacteria, protozoa), microbial source tracking (MST) markers, mitochondria DNA (mtDNA) and antibiotic resistance genes was used to detect over 430 selected gene targets in whole genome amplification (WGA) DNA and complementary DNA (cDNA) isolated from sewage and animal (avian, cattle, poultry and swine) feces, freshwater and marine water samples, sewage spiked surface water samples, treated wastewater and sewage contaminated produce.;A combination of perfect match and mismatch probes on the microarray reduced the likelihood of false positive detections, thus increasing the specificity of the microarray for various gene targets. A linear decrease in fluorescence of positive probes over a 1:10 dilution series demonstrated a semi-quantitative relationship between gene concentrations in a sample and microarray fluorescence. Various pathogens, including norovirus, Campylobacter fetus, Helicobacter pylori, Salmonella enterica, and Giardia lamblia were detected in sewage via the microarray, as well as MST markers and resistance genes to aminoglycosides, beta-lactams, and tetracycline. Sensitivity (percentage true positives) of MST results in sewage and animal waste samples (21--33%) was lower than specificity (83--90%, percentage of true negatives). Next generation sequencing (NGS) of DNA from the fecal samples revealed two dominant bacterial families that were common to all sample types: Ruminococcaceae and Lachnospiraceae. Five dominant phyla and 15 dominant families comprised 97% and 74%, respectively, of sequences from all fecal sources.;Waterborne pathogens were also detectable via the microarray in freshwater, marine water and sewage spiked surface water samples as well as treated wastewater. Ultrafiltration was used to concentrate microorganisms (bacteria, viruses, protozoa and parasites) from several liters of environmental and treated water samples. Dead-end ultrafiltration (DEUF) was shown to have a 61.4 +/- 47.8 % recovery efficiency and 46-fold concentration increasing ability. Then WGA was utilized to increase gene copies and lower the microarray detection limit. Viruses, including adenovirus, bocavirus, Hepatitis A virus, and polyomavirus were detected in human associated water samples as well as pathogens like Legionella pneumophila, Shigella flexneri, C. fetus and genes coding for resistance to aminoglycosides, beta-lactams, tetracycline. Microbial source tracking results indicate that sewage spiked freshwater and marine samples clustered separately from other fecal sources including wild and domestic animals via non-metric dimensional scaling. A linear relationship between qPCR and microarray fluorescence was found, indicating the semi-quantitative nature of the MST microarray.;Multiple displacement amplification (MDA), which is an important type of WGA, is a widely used tool to amplify genomic nucleic acids. The strong amplification efficiency of MDA and low initial template requirement make MDA an attractive method for environmental molecular and NGS studies. However, like other nucleic acid amplification techniques, various factors may influence MDA efficiency including template concentration (e.g. rare species swamping out), GC amplification bias and genome length favoring amplification of longer genomes. It was found that MDA increased nucleic acids in mixed environmental samples approximately 4.24 +/- 1.40 (log, average +/- standard deviation) for 16S rRNA gene of Enterococcus faecalis, 1.90 +/- 1.70 for RNA polymerase gene of human norovirus, 8.83 +/- 2.88 for T antigen gene of human polyomavirus, 3.83 +/- 0.93 for uidA gene of Escherichia coli, 4.96 +/- 0.32 for invA gene of S. enterica and 8.77 +/- 2.85 for 16S rRNA gene of human Bacteroidales. The template length, concentration and GC content were found to influence MDA efficiency. The results mainly show that the MDA will be more efficient the longer the template length, the greater the initial concentration of nucleic acids and the lower the GC content of the template.;Overall, the results of this work show that 1) the microarray and sample handling technique is suitable for pathogen detection from feces and sewage; 2) when combined with ultrafiltration techniques, the microarray can also be used as a pathogen detection tool in environmental waters; 3) template length, and initial concentration increase MDA efficiency, but higher GC content template negatively effects MDA efficiency. The proposed microarray can be used for pathogen detection in feces, wastewater treatment plant sewage, treated wastewater and environmental waters. Further the proposed method is potentially applicable to pathogen/microorganism detections on vegetables, seafood, in hospital settings, industrial wastewater, and aquaculture settings

Review of methodological developments in laser Doppler flowmetry

Laser Doppler flowmetry is a non-invasive method of measuring microcirculatory blood flow in tissue. In this review the technique is discussed in detail. The theoretical and experimental developments to improve the technique are reviewed. The limitations of the method are elaborated upon, and the research done so far to overcome these limitations is critically assessed

Laser doppler perfusion imaging of the normal and diseased vulva.

Vulval lichen sclerosus (LS) and high-grade intraepithelial neoplasia (VIN 3) are two common and distressing diseases. Significant morbidity is caused by symptoms of persistent pruritus and surgical treatment of skin areas suspicious of malignancy. The risk of developing cancer in a background of LS and VIN 3 is poorly defined. The methods currently available for clinical assessment of the vulva are limited. There is abundant research on the application of the LASER Doppler technique - laser Doppler Flowmetry (LDF) - showing changes in perfusion within the small blood vessels of the skin as a useful parameter for more accurate disease classification. There is also research on immunohistochemical microvessel density (MVD) studies showing increases in blood supply in tissues prone to develop cancer or as a prognostic marker of cancer outcome. The Laser Doppler perfusion imager (LDPI) provides a rapid, real time, non-invasive and non-contact method to measure skin blood flow in an area as opposed to a single point by the LDF, making the LDPI more suitable for application to the vulva. This thesis reports for the first time, the application of the LDPI to the vulva. Initially the LDPI was applied to the clinically normal vulva to study perfusion variance related to menstrual cycle, age and local skin temperature provocation. The application was then extended to vulval disease, LS and VIN 3, and validated against morphological differences in MVD. The LDPI and MVD studies suggest that in VIN 3 there is an actual increase in skin perfusion. In LS the situation is more complex and suggests that the LDPI measured perfusion at a greater depth than the MVD. Studies on base line perfusion variance of vulval LS to topical therapy show that there is no overall difference in baseline perfusion in spite of symptom improvement. Temperature provocation studies suggest differences in skin blood flow response in diseased compared to the normal vulva

Evaluation of the Role of Microvascular Pathology on Peripheral Artery Disease

Background: Peripheral Artery Disease (PAD) begins with atherosclerotic narrowing of arteries, including those that supply the legs. Individuals with PAD experience pain during walking, which becomes increasingly limiting. Studies from our group and others have shown that a myopathy is present in the skeletal muscle of PAD patients, and is characterized by myofiber degeneration, fibrosis, and remodeling of vessels ranging from 50 – 150 mm in diameter. However, microvascular pathology, particularly of the smallest microvessels (5 – 15 mm in diameter) remains poorly characterized. Furthermore, little is known about the relationships between microvascular architecture, microperfusion, and patient walking performance. We hypothesize that microvascular pathology is present in the terminal microvasculature of PAD muscle compared to control and worsens with PAD severity. Additionally, we hypothesize that microvascular architecture is associated with deficits in micro- and macro- perfusion and walking performance in PAD patients with intermittent claudication (IC). Methods: Gastrocnemius biopsy specimens were collected from control, PAD patients with IC, and PAD patients with critical limb ischemia. Microvascular architecture, microvascular fibrosis, total collagen, and the abundance and phenotype of pericytes were quantified. Microvascular perfusion was assessed by Contrast Enhanced Ultrasonography (CEU). Gardner walking protocols were used to assess claudication onset time (COT) and peak walking time (PWT). Patients also completed the Walking Impairment Questionnaire (WIQ). Results: Microvascular pathology increased with advancing PAD severity and included progressive increases in basement membrane thickening, abundance of aSMA+ pericytes, and microvessel density. In advanced PAD muscle, increases were observed in total fibrotic burden and peri-microvascular Collagen I and IV deposition. aSMA+ pericytes expressed TGF-b1. Relationships were observed between microvascular architecture and microperfusion both at rest and after ischemic stress. Microvascular architecture was associated with macrovascular hemodynamic restrictions. Microvascular architecture was associated with COT, PWT, and patient self-reports of walking speed, walking distance, and stair climbing ability. Conclusions: Microvascular pathology worsens with PAD severity in association with fibrosis. Alteration of microvascular architecture contributes to microperfusion deficits and walking limitations in PAD

Optical-Resolution Photoacoustic Microscopy

Optical microscopy, providing valuable biomedical insights at the cellular and organelle levels, has been widely recognized as an enabling technology. Mainstream optical microscopy technologies, including single-/multi-photon fluorescence microscopy and OCT, have demonstrated extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been fully assessable. The emergence of biomedical photoacoustics has led to a new branch of optical microscopy--OR-PAM. As a valuable complement to existing optical microscopy technologies, OR-PAM detects optical absorption contrasts with exquisite sensitivity: i.e., 100%). Combining OR-PAM with fluorescence microscopy or optical-scattering-based OCT: or both) provides comprehensive optical properties of biological tissues. Moreover, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and thus provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences: i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra and imperfect extinction of the filter. Unique for high-resolution imaging of optical absorption, OR-PAM has demonstrated broad biomedical applications in fields such as neurology, ophthalmology, vascular biology, and dermatology. My doctoral research focuses on developments and biomedical applications of OR-PAM. The first part of my dissertation discusses the development of three generations of OR-PAM towards high-resolution, high-sensitivity, high-speed, and wide FOV in vivo imaging. In this section, I provide a comprehensive description of OR-PAM, including the principle, system design, system configuration, experimental procedures, laser safety, functional imaging scheme, and example biomedical applications at a variety of in vivo anatomical sites: i.e., skins, eyes and brains). The second part of my dissertation focuses on the application of OR-PAM in vascular biology, with an emphasis on neovascularization. In this section, I demonstrate longitudinal OR-PAM monitoring of the morphological: i.e., vessel diameter, length, tortuosity and volume) and functional: i.e., sO2) changes of angiogenic microenvironment at the capillary level, in both a non-disease TetON-HIF-1 transgenic mouse model and a cancer xenograft model in mouse ear. The last part of my dissertation focuses on the application of OR-PAM in neurology, with an emphasis on cortical stimulation, Alzheimer\u27s disease, and ischemic stroke. In this section, I use label-free OR-PAM for both acute monitoring of microvascular responses to direct electrical stimulations of the mouse somatosensory cortex through a cranial opening and longitudinal monitoring of the morphological and functional changes of cortical vasculature in a transient middle cerebral artery occlusion mouse model. I also explore the potential of OR-PAM for transcranial monitoring of amyloid plaque growth in an AD mouse model

Noninvasive imaging of flowing blood cells using label-free spectrally encoded flow cytometry

Optical microscopy of blood cells in vivo provides a unique opportunity for clinicians and researchers to visualize the morphology and dynamics of circulating cells, but is usually limited by the imaging speed and by the need for exogenous labeling of the cells. Here we present a label-free approach for in vivo flow cytometry of blood using a compact imaging probe that could be adapted for bedside real-time imaging of patients in clinical settings, and demonstrate subcellular resolution imaging of red and white blood cells flowing in the oral mucosa of a human volunteer. By analyzing the large data sets obtained by the system, valuable blood parameters could be extracted and used for direct, reliable assessment of patient physiology