Multi-photon Microscopy in the Evaluation of Human Saphenous Vein (original) (raw)
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The Annals of Thoracic Surgery, 2003
Background. Injury to endothelium can compromise the patency of bypass grafts harvested during coronary artery bypass graft (CABG) surgery. Maintaining structural and functional viability of endothelium in grafts may lead to improved long-term patency. The information gained from the application of multi-photon microscopy in transmission and epifluorescence mode was used to assess the structural and functional integrity of human saphenous vein segments stored in multiple preservation solutions, and to design a superior storage solution.
Journal of Vascular Research
Introduction Imaging and visualization of Nitric Oxide (NO), an important messenger that regulates a multitude of function within the vascular system, is essential to better define its role in (patho)physiology. We study a novel NO-sensitive copper-fluorescein complex (CuFL) with two-photon laser scanning microscopy (TPLSM). Methods To study cellular NO production both in vitro and ex vivo CuFL fluorescence was determined using TPLSM. CuFL was characterized and compared with DAF-2-DA. In endothelial cell (EC) cultures, hydrogen peroxide (H2O2 ), acetylcholine and shear stress were used as stimulant. Isolated murine carotid arteries were incubated ex vivo with H2O2 and acetylcholine to stimulate NO production. Isolated arteries were mounted in a home-built perfusion chamber and transmural pressure was applied to mimic physiological condition. Endothelium functional capacity of artery, that depends on the amount of NO produced and the vasodilatation effect was evaluated by measuring l...
PLoS ONE, 2013
To study the role and (sub) cellular nitric oxide (NO) constitution in various disease processes, its direct and specific detection in living cells and tissues is a major requirement. Several methods are available to measure the oxidation products of NO, but the detection of NO itself has proved challenging. We visualized NO production using a NOsensitive copper-based fluorescent probe (Cu 2 FL2E) and two-photon laser scanning microscopy (TPLSM). Cu 2 FL2E demonstrated high sensitivity and specificity for NO synthesis, combined with low cytotoxicity. Furthermore, Cu 2 FL2E showed superior sensitivity over the conventionally used Griess assay. NO specificity of Cu 2 FL2E was confirmed in vitro in human coronary arterial endothelial cells and porcine aortic endothelial cells using various triggers for NO production. Using TPLSM on ex vivo mounted murine carotid artery and aorta, the applicability of the probe to image NO production in both endothelial cells and smooth muscle cells was shown. NO-production and time course was detected for multiple stimuli such as flow, acetylcholine and hydrogen peroxide and its correlation with vasodilation was demonstrated. NO-specific fluorescence and vasodilation was abrogated in the presence of NOsynthesis blocker L-NAME. Finally, the influence of carotid precontraction and vasorelaxation validated the functional properties of vessels. Specific visualization of NO production in vessels with Cu 2 FL2E-TPLSM provides a valid method for studying spatial-temporal synthesis of NO in vascular biology at an unprecedented level. This approach enables investigation of the pathways involved in the complex interplay between NO and vascular (dys) function. Citation: Ghosh M, van den Akker NMS, Wijnands KAP, Poeze M, Weber C, et al. (2013) Specific Visualization of Nitric Oxide in the Vasculature with Two-Photon Microscopy Using a Copper Based Fluorescent Probe. PLoS ONE 8(9): e75331.
Intravital imaging of tumour vascular networks using multi-photon fluorescence microscopy
Advanced Drug Delivery Reviews, 2005
The blood supply of solid tumours affects the outcome of treatment via its influence on the microenvironment of tumour cells and drug delivery. In addition, tumour blood vessels are an important target for cancer therapy. Intravital microscopy of tumours growing in dwindow chambersT in animal models provides a means of directly investigating tumour angiogenesis and vascular response to treatment, in terms of both the morphology of blood vessel networks and the function of individual vessels. These techniques allow repeated measurements of the same tumour. Recently, multi-photon fluorescence microscopy techniques have been applied to these model systems to obtain 3D images of the tumour vasculature, whilst simultaneously avoiding some of the problems associated with the use of conventional fluorescence microscopy in living tissues. Here, we review the current status of this work and provide some examples of its use for studying the dynamics of tumour angiogenesis and vascular function. D
Multimodal imaging of vascular network and blood microcirculation by optical diagnostic techniques
Quantum Electronics, 2011
We present a multimodal optical diagnostic approach for simultaneous non-invasive in vivo imaging of blood and lymphatic microvessels, utilising a combined use of êuorescence intravital microscopy and a method of dynamic light scattering. This approach makes it possible to renounce the use of êuorescent markers for visualisation of blood vessels and, therefore, signiécantly (tenfold) reduce the toxicity of the technique and minimise side effects caused by the use of contrast êuorescent markers. We demonstrate that along with the ability to obtain images of lymph and blood microvessels with a high spatial resolution, current multimodal approach allows one to observe in real time permeability of blood vessels. This technique appears to be promising in physiology studies of blood vessels, and especially in the study of peripheral cardiovascular system in vivo.
2002
Recent interest in vascular targeting and anti-angiogenic drug treatments for cancer has stimulated fundamental research regarding the modes of action of these drugs as well as studies of the development and re-modelling of the vascular network following treatment. Multiphoton fluorescence microscopy is employed for in vivo mapping of threedimensional blood vessel distribution in tumours grown in rodent dorsal skin-flap window chamber preparations. Accurate visualisation of the vasculature in three-dimensions allows us to perform dynamic experiments in thick biological specimens in vivo. Examples of in vivo imaging of tumour vasculature are given and compared to normal tissue vasculature. The dynamic responses of blood vessels to treatment with the vascular targeting drug combretastatin A4-P are presented and discussed. The implementation of time-domain imaging by reversed stop-start time-correlated single photon counting (RSS-TCSPC) is discussed as a method for feature extraction in the presence of exogenous and endogenous fluorophores. In particular, the segmentation of the vascular network is demonstrated. Additional contrast, indicative of probe environmental factors, may also be realised. We present examples of in vivo lifetime imaging as a method to elucidate the physiological processes of the tumour microenvironment.
Proceedings of SPIE, 2002
Recent interest in vascular targeting and anti-angiogenic drug treatments for cancer has stimulated fundamental research regarding the modes of action of these drugs as well as studies of the development and remodelling of the vascular network following treatment. Multiphoton fluorescence microscopy is employed for in vivo mapping of threedimensional blood vessel distribution in tumours grown in rodent dorsal skin-flap window chamber preparations. Accurate visualisation of the vasculature in three-dimensions allows us to perform dynamic experiments in thick biological specimens in vivo. Examples of in vivo imaging of tumour vasculature are given and compared to normal tissue vasculature. The dynamic responses of blood vessels to treatment with the vascular targeting drug combretastatin A4-P are presented and discussed. The implementation of time-domain imaging by reversed stop-start time-correlated single photon counting (RSS-TCSPC) is discussed as a method for feature extraction in the presence of exogenous and endogenous fluorophores. In particular, the segmentation of the vascular network is demonstrated. Additional contrast, indicative of probe environmental factors, may also be realised. We present examples of in vivo lifetime imaging as a method to elucidate the physiological processes of the tumour microenvironment.
Advances in renal (patho)physiology using multiphoton microscopy
Kidney International, 2007
Multi-photon excitation fluorescence microscopy is a state-of-the-art confocal imaging technique ideal for deep optical sectioning of living tissues. It is capable of performing ultra-sensitive, quantitative imaging of organ functions in health and disease with high spatial and temporal resolution that other imaging modalities can not achieve. For more than a decade, multi-photon microscopy has been successfully used with various in vitro and in vivo experimental approaches to study many functions of organs, including the kidney. This mini-review focuses on recent advances in our knowledge of renal (patho)physiological processes made possible by the use of this imaging technology. Visualization of cellular variables like cytosolic calcium, pH, cell-to-cell communication and signal propagation, interstitial fluid flow in the juxtaglomerular apparatus (JGA), real-time imaging of tubuloglomerular feedback (TGF) and renin release mechanisms are reviewed. Brief summary is provided how one can perform quantitative imaging of kidney functions in vivo including glomerular filtration and permeability, concentration, dilution, and activity of the intra-renal reninangiotensin system using this minimally invasive approach. New visual data challenge a number of existing paradigms in renal (patho)physiology. Also, quantitative imaging of kidney function with multi-photon microscopy has excellent potential to eventually provide novel non-invasive diagnostic and therapeutic tools for future applications in clinical nephrology.