We used high speed, multidimensional optical microscopy to dynamically assess metrics of vascular function (blood flow and vascular reactivity) in real time in unanaesthetized zebrafish embryos. Until recently, the high speed detectors required to make these measurements (video-rate and CCDs devices) were only fast enough to measure either slower peripheral blood flow, or flow after treatment to slow heartrate (i.e. anesthesia, cooling). Erythrocyte tracking methods such as digital motion analysis 
and digital particle image velocimetry (DPIV) 
have been used extensively to obtain gross measurements of blood flow and approximations of vessel diameters in developing zebrafish. In fact, an early example of applying DPIV to study early cardiac function in the developing heart tube of zebrafish embryos yielded data that contradicted the accepted model of peristalsis as the main pumping mechanism in the embryonic vertebrate heart 
. However, as with traditional erythrocyte tracking methodologies, the utility of DPIV is limited by the temporal resolution of the camera and the ability of the system to resolve the particles within the flow field 
. In contrast, the CMOS detector employed in the present studies enabled us to image a very large (1 mm) segment of the region of interest (in this case, the dorsal aorta of the zebrafish embryo) over many cardiac cycles, with sufficient speed and sensitivity that the trajectories of individual red blood cells could be resolved in real time. We feel that the system used here provides significant advantages over existing techniques, certainly from the point of view of the camera technology employed. But also, the use of a relatively high numeric aperture (NA) low magnification objective not only allows large areas to be imaged, it does so with a high photon recovery, and with a relatively large depth of field. Certainly higher NA objectives will provide a higher resolution, and perhaps brighter image, but the decreased field of view, and reduced axial focal volume makes these objectives much less attractive for this type of imaging, as erythrocytes moving axially within the vessel will move out of the focal plane. This approach allows a much higher event count at any time point. Using these methods, we were able to obtain flow velocity profiles over multiple cardiac cycles in 3 and 5 dpf zebrafish embryos, and assess dynamic changes in response to a TXA2
agonist (U-46619) which has well-characterized effects on vascular tone in other vertebrate models 
. We propose that the described methods represent a simple, inexpensive, and reproducible approach to evaluate vascular performance in animal models of human disease and may be extended to an array of microscopic measurements that are currently limited by sensitivity and time resolution.
Zebrafish form fully lumenized vessels with intact barrier function in the dorsal aorta, cardinal vein and intersegmental vessels, as early as 54 hours post fertilization 
. Endothelial permeability has been previously assessed using both wide-field and confocal methods to compare extravasation of fluorescent dextrans or microspheres from the vascular space at fixed time points, between treatment groups 
. Here we use high resolution, time-lapse imaging to show real-time changes in permeability in response to a vasoactive agonist (thrombin), and further demonstrate that it is possible using high NA optics to identify regional changes in endothelial continuity. In future studies, these measurements can be combined with measurements of calcium flux, and pharmacological or genetic inhibition of key signaling pathways to provide mechanistic information about the pathways regulating endothelial dysfunction in models of human disease.
Beyond the technical advances described above, our observation that 5 dpf embryos regulate aortic blood flow in response to a vasoconstricting agent (U46619), at a point in development when there is only limited expression of vascular smooth muscle 
, suggests that endothelial constriction contributes to the regulation of vessel tone. While the heart and circulation of the zebrafish embryo are functional from 1 dpf, expression of embryonic smooth muscle cell markers is relatively weak and discontinuous throughout the length of the dorsal aorta even at 5 dpf 
, as we confirmed in our model using transmission electron microscopy (TEM, Figure S2
). Vascular endothelium contains all the molecular machinery required to generate contractile force via the actomyosin motor, and has been shown to play a role in capillary constriction in a number of vascular beds in other vertebrate models 
. Furthermore, endothelial cells in early stage zebrafish embryos have been shown to produce vasoconstrictive agents including prostacyclin 
; and nitric oxide (NO) has been shown to modulate vascular tone early in development when both nervous control and smooth muscle cells are limited or lacking 
The actions of TXA2 are mediated by a G-protein-coupled receptor (TxA2 receptor) triggering activation of phospholipase Cβ via Gq/11, and ultimately inducing increases in intracellular Ca2+ and cell contraction 
. While untested in this study, the observed age related differences in responsiveness to U-46619 could reflect developmental changes in the expression patterns of TXA receptors, or other proteins involved in the prostanoid/thromboxane signaling pathway. For example, the TXA synthase transcript does not become apparent until 3 dpf 
. Relative changes in protein expression between the hatching period (3 dpf) and larval stage (5 dpf) have been described for a number of canonical signaling proteins, including those related to calcium handling/signaling 
It is well recognized that the endothelium modulates vascular tone via production of vasoactive mediators including endothelin and NO, while also acting as a semi-permeable barrier between the vascular lumen and surrounding tissue. Disruption of the endothelial barrier has been shown to be mediated via Ca2+
/calmodulin-dependent assembly of actin stress fibers, cell contraction, disruption of cell–cell contacts, and intercellular gap formation 
. The cellular effects of thrombin are mediated by protease-activated receptors (PARs), a subfamily of related G-coupled protein receptors (GCPRs) that are activated by cleavage of part of their extracellular domain. To date, four zebrafish par
genes have been cloned and identified as homologs of mammalian PARs1–3
. While evidence for direct acute action of thrombin to increase permeability in intact vascular beds is limited 
, it has been previously shown that human thrombin can stimulate zebrafish Par1 to induce a Ca2+
response in Xenopus
. In these studies we used high speed slit scanning confocal microscopy to confirm that thrombin can induce time- dependent changes in endothelial barrier function in situ
in 5 dpf zebrafish larvae.
Our data suggest that the scientific opportunities presented using an integrated suite of high speed imaging techniques are extensive, and are penetrant in attempting to answer temporally important questions in vascular physiology. A limitation of the approach used here is that it will work best on embryonic trunk vessels, because of the restriction in the depth of penetration of wide field, non-multiphoton microscopy. While the working distance of the 10X objective used in these studies would work on older embryos, the chrna1 deficient transgenic model we have employed (which is paralyzed) will not live once nutrients from the yolk are exhausted. However, if measurements are made on anaesthetized fish, then the age of imaging can be extended out further, such that flow velocities in animals with varying degrees of vascular smooth muscle coverage can be compared.