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We report a rapid hyperspectral fluorescence lifetime imaging (FLIM) instrument that exploits high-speed FLIM technology in a line-scanning microscope. We demonstrate the acquisition of whole-field optically sectioned hyperspectral fluorescence lifetime image stacks (with 32 spectral bins) in less than 40 s and illustrate its application to unstained biological tissue. Microsc. Res. Tech., 2007. © 2007 Wiley-Liss, Inc.

Uptake of circulating macromolecules by the aortic wall is greater downstream than upstream of branch sites in immature rabbits, but the opposite pattern is seen at later ages. The mature pattern is nitric oxide dependent; we tested whether it is also flow dependent. Intercostal arteries of anesthetized rabbits were occluded, sham operated, or left alone. Uptake of rhodamine-labeled albumin was assessed by quantitative fluorescence microscopy of the sections through the aorta. In mature animals, uptake was higher upstream than downstream of the control and sham-operated branches, but the pattern was reversed at occluded branches. In young animals, uptake was not significantly different between regions upstream and downstream of control, sham-operated, or occluded branches. The absence of the normal immature pattern may reflect an influence of anesthesia and will assist in the elucidation of mechanisms underlying this pattern. The data for mature animals provide the first direct evidence that flow determines permeability near arterial branches and may account for the inverse spatial correlation between shear stress and disease prevalence at branches of adult human arteries.

Objective: The aim was to study the effects of altered heart rate and vasoactive drugs on the blood velocity patterns in the region of an arterial bifurcation. Methods: Blood velocity profiles were measured in an exposed iliofemoral bifurcation of paced dogs using a pulsed Doppler ultrasound velocimeter with high temporal and spatial resolution. Results: Decrease of the heart rate from 120 beats·min−1 (2 Hz) to 60 beats·min−1 (1 Hz) increased me peak forward velocity (30%), the peak reverse velocity (20%), and the duration of reverse flow (25%). Each drug caused qualitatively similar changes in velocity patterns at both heart rates. The systemic administration of angiotensin II reduced peak forward velocity (–26% at 2 Hz and –33% at 1 Hz) and forward flow duration (–15% at 1 Hz), the peak reverse velocity (–30% at 1 Hz), and reverse flow duration (–20% at 2 Hz and –28% at 1 Hz). Glyceryl trinitrate also reduced the peak forward velocity (–19% at both 2 and 1 Hz) but prolonged forward flow duration (28% at 2 Hz and 17% at 1 Hz) and that of reverse flow (45% at 2 Hz and 24% at 1 Hz), and also decreased the degree of oscillation (−16% at 2 Hz). Barnidipine hydrochloride (a calcium channel antagonist) also increased the duration of forward flow (48% at 1 Hz) and of reverse flow (31% at 2 Hz) but reduced the peak reverse velocity (−29% at 1 Hz) and flow oscillation (−22% at 2 Hz and 20% at 1 Hz). Conclusions: These dramatic changes in the pattern of blood flow, including alterations in the amplitudes and durations of the different phases of the flow cycle, are expected to have important consequences on the shear dependent responses of endothelial cells in the region of the bifurcation. Cardiovascular Research 1994; 28 :1686-1693

Publisher Summary This chapter describes the applications of multidimensional fluorescence imaging (MDFI) instrumentation. The chapter discusses fluorescence lifetime imaging (FLIM), with an emphasis on rapid wide-field time-gated imaging, including application to molecular biology, FLIM of tissue autofluorescence, and highspeed optically sectioned FLIM for live cell imaging. Optical sectioning is important to enhance image contrast and to minimize unwanted mixing of signals from axially separate fluorophores. The chapter discusses the extension to spectral FLIM, including (emission resolved) hyperspectral FLIM, implemented using line-scanning microscopy, and excitation-resolved imaging and FLIM utilizing supercontinuum generation to provide excitation throughout the visible spectrum. The combination of polarization-resolved and time resolved fluorescence imaging is described, mapping both lifetime and rotational correlation time as illustrated by an application to microfluidic devices. This chapter demonstrates that FLIM and MDFI can add significant value to microscopy, endoscopy, and assay technology. By resolving the fluorescence signal with respect to multiple dimensions including excitation and emission wavelength, lifetime, and polarization, it may be possible to enhance the fluorescence read-out for an experiment or assay, for example, in terms of improving the separation of multiple fluorophores or imaging variations in the local molecular environment.

Publisher Summary The body cannot survive without an intact cardiovascular system that pumps blood around the body and ensures that there is a blood supply to every organ to match its metabolic needs. Consequently, any pathology affecting either the heart or the blood vessels must be repaired if their normal function is inadequate. This chapter discusses various strategies that can be adopted to replace or reinforce different critical components of the system. It presents that every organ in the body needs a constant blood supply. Common causes for disruption of the cardiovascular system are heart failure due to age, heart valve disease, and vascular disease due to plaque build-up in the blood vessels, which restricts flow or causes aneurysms. Plaque rupture can release emboli or cause thrombosis, blocking the vessel locally or peripherally. Aged hearts can be assisted by pacemakers and diseased heart valves can be repaired or replaced, either with biological transplants or artificial mechanical prostheses. If none of these solutions is possible, pumps must be used, either extracorporeal or internal artificial hearts, although internal artificial hearts have had limited success. Constricted blood vessels can be widened by angioplasty (insertion of a balloon, which is inflated to stretch the narrowed vessel) and by the placement of a stent.

Publisher Summary This chapter discusses systems that deliver material to the body or that remove what is unwanted. Delivery systems include: implantable capsules for slow, sustained release of a drug, and implantable drug delivery pumps that can deliver material through a catheter. Exchange devices that are external to the body include: dialyzers, which remove excess fluid and waste products from the blood, aphaeresis devices that collect fractionated blood components, and artificial gas exchangers, which are commonly used in open-chest surgery and may be used to supplement pathologically ineffective lungs. For all these devices, there must be due consideration of the materials used and particularly for those that are in contact with tissues or body fluids. These factors will include: sterility, toxicity, resistance to corrosion, the mechanical properties of the components used, and the risk of the materials causing blood clotting. The latter is particularly important for dialyzers and gas exchangers as they are in direct contact with blood. Dialyzers and gas exchangers involve a direct contact between blood and foreign materials. In these cases, the material surfaces must be highly resistant to blood clotting. It is essential that devices do not expose the blood to flow conditions that might induce clotting or damage the cells. These include the avoidance of high shear and stagnation regions.

Publisher Summary This chapter describes some of the factors underlying the effective operation of implants and extracorporeal devices. It is concerned with the physical principles by which materials are transported, either in normal tissue, or in association with devices. Tissue-engineered constructs are a special case since the living cells within them require the establishment of normal exchange processes such as occur in all other tissues. Unicellular organisms and the simplest multi-cellular organisms are able to survive since diffusion can occur sufficiently rapidly for metabolites to enter cells at the rate at which they are being used and waste products to leave as soon as they are produced. Solutions of the diffusion equations to be presented indicate that diffusional transport of a very small molecule, such as oxygen in water will take roughly 16 hours to move 1 cm and about 8 minutes to move 1 mm. These rates would be totally inadequate for larger metabolic species. Consequently, all large organisms (even plants) have additional mechanisms for moving fluids to bring metabolites close to the cells. In this context, it is useful to differentiate between pressure-driven “convective” transport and “diffusive” transport, which results from intrinsic molecular motion. In strict thermodynamic terms, these mechanisms are equivalent, since both involve a minimization of free energy in a system.

Publisher Summary This chapter provides an overview of the cardiovascular system. Blood circulation in the body is essential for the delivery to the tissues of all the nutrients that they require, including oxygen, the removal of waste products, the control of fluid levels, and the control of multiple functions through the endocrine hormone system and for thermoregulation. Blood, blood vessels, and the heart comprise the cardiovascular system, which is essential to maintain life as the movement of blood cells and plasma throughout the body provides nutrition and removes waste products from cells. Blood is the fluid that is in continuous movement through all the tissues of the body, blood vessels are the conduits for the blood, and the heart is the muscular pump that is primarily responsible for driving the flow. Failure of any components of the cardiovascular system can lead to irreversible damage to cells and organs and is one of the leading causes of death in the developed world. Thus, repair and replacement of the components of the cardiovascular system is one of the great challenges of biomaterials and bioengineering.

We have applied fluorescence lifetime imaging (FLIM) to the autofluorescence of different kinds of biological tissue in vitro, including animal tissue sections and knee joints as well as human teeth, obtaining two-dimensional maps with functional contrast. We find that fluorescence decay profiles of biological tissue are well described by the stretched exponential function (StrEF), which can represent the complex nature of tissue. The StrEF yields a continuous distribution of fluorescence lifetimes, which can be extracted with an inverse Laplace transformation, and additional information is provided by the width of the distribution. Our experimental results from FLIM microscopy in combination with the StrEF analysis indicate that this technique is ready for clinical deployment, including portability that is through the use of a compact picosecond diode laser as the excitation source. The results obtained with our FLIM endoscope successfully demonstrated the viability of this modality, though they need further optimization. We expect a custom-designed endoscope with optimized illumination and detection efficiencies to provide significantly improved performance. © 2003 Optical Society of America.

Evans blue dye (EBD) conjugates with albumin in the circulation and is frequently used to measure vascular protein leakage. The fluorescence of the dye from tissue sections can be used to measure its uptake at very specific anatomical locations, but problems arise with dye quantification because tissue components also fluoresce; so-called autofluorescence. We have measured uptake of EBD by blood vessel walls at various points around the aorto-renal branch of rabbits. High resolution, digitised, fluorescence images of histological sections of artery wall allowed detailed microscopic analysis of EBD accumulation; and a ratiometric method was developed to enable autofluorescence to be separated from EBD fluorescence. When EBD-free tissue sections were illuminated with blue light, the ratio of red to green fluorescence was constant throughout the tissue (0.59 +/- 0.03, mean +/- S.D., n = 32). Therefore, at each individual pixel, the level of red autofluorescence could be determined by multiplying the green intensity at that pixel by the calculated red to green ratio. Since EBD fluorescence was detected only in the red region of the spectrum, intensity values of the dye alone were obtained from EBD-exposed tissue by subtracting the red autofluorescence estimated by this ratiometric method. In such cases the red to green fluorescence ratio was measured from adjacent sites known to be free of EDB (0.59 +/- 0.02, mean +/- S.D., n = 56). We were therefore able to increase the sensitivity of tracer quantification by complete elimination of background autofluorescence on a pixel-by-pixel basis. Use of EBD standards allowed calibration of corrected fluorescence intensities and calculation of mass transfer coefficients for albumin into the artery wall. Spatial variations in the permeability of the artery wall around the renal ostium were detected with the present high resolution technique, with an average mass transfer coefficient of (6.8 +/- 0.9) x 10(-8) cm s(-1) for all sites combined (n = 56). The present ratiometric method could potentially be applied to other quantitative fluorescence-based techniques.

In the microcirculation, the blood vessel wall is the major barrier which limits the transport of materials between the blood and tissues. Mass transport studies in the microcirculation are, therefore, primarily concerned with the mechanisms by which metabolites and catabolites are exchanged across the capillary wall. In contrast, in large vessels, there is more interest in the processes by which materials move into and out of the wall tissue itself. Somewhat paradoxically, the walls of large arteries are relatively avascular, having a slab of intimai and medial tissue which may be several hundred microns thick between the blood in the vessel lumen and that in the capillaries of the vasa vasorum. As thick walled vessels grow, angiogenesis occurs and capillaries penetrate the media from the adventitial side, but with increased thickening of the intima on ageing, the tissue may again become more avascular. For the tissue to undergo efficient metabolism, respiratory gases, nutrients and catabolites must be rapidly exchanged. In the field of large vessel mass transport though, the major interest has been in the movement of larger materials such as plasma proteins because of their putative role in atherosclerosis. Because this condition normally affects only arteries, transport processes in these vessels have been studied far more extensively than in veins or pulmonary vessels.

The blood vessels exhibit wide variations in structure from one site in the body to another. The marked differences in the composition, the ultrastructural organization of tissue components and the thickness of the vessel walls presumably reflect differences in the mechanical forces and chemical environment which prevailed during their growth. As the vessels age, differences also appear in their susceptibility to atherosclerosis. Veins are normally spared from the disease, except when they take on the features of arteries following the develoment of an arterio-venous fistula, or when they are used as arterial grafts. The pulmonary arteries also tend to be affected by atherosclerosis only if there is pulmonary hypertension. Even amongst the systemic arteries there is marked variability in susceptibility; vessels smaller than 1mm are rarely affected and the ascending portion of the aorta is normally affected to a lesser extent than the abdominal region.

During the last decade, the major emphasis in atherosclerosis research has been cell biological, with great advances being made in our understanding of the interactions of formed elements such as platelets with the vessel wall, and of the expression of a variety of substances by components of the vessel wall such as the endothelial cells. A major feature of the atherosclerotic lesion is the deposition of materials to a pathological degree within the interstitial tissue of the intima. Whether this excessive accumulation is a primary process which precipitates other changes, such as the migration of smooth muscle cells or monocytes into the intima, or whether the accumulation is a response to other factors remains unknown. Either way, the accumulation represents a failure of the normal homeostasis of the wall which presumably operates in healthy tissue to maintain a uniform composition of the interstitium. Because the major part of the material deposited consists of blood components such as lipoproteins, fibrinogen and lipids, many attempts have been made to investigate the rates of exchange of these substances between blood and vessel wall tissue. The studies have commonly used tracers to measure transient exchange processes and in doing so, perturbations to normal in vivo transport processes have been introduced, such as artificial concentration gradients. Only more recently has there been a recognition of the importance of the normal flux of most plasma components across the vessel wall, a process that occurs continuously in vivo (Walton 1975, Caro et al., 1980, Truskey et al., 1981, Fry 1987). Rather little attention has been given to the problems of the transport within wall tissue of small molecular weight metabolites or autacoids produced within the wall or at the blood-wall boundary.