This work has received funding from the Carlsberg Foundation (CF17-0778; CF18-0658), the Lundbeck Foundation (R324-2019-1470; R346-2020-1210), the Velux Foundations (00022458), The A.P. M&#248;ller Foundation for the Advancement of Medical Science, the European Union&#39;s Horizon 2020 research and innovation program under the Marie Sk&#322;odowska-Curie grant agreement (No. 754513), and The Aarhus University Research Foundation.

The protocol below was performed with permission from the Danish Inspectorate for Animal Experimentation within the Danish Ministry of Food, Agriculture, and Fisheries, Danish Veterinary and Food Administration (Permit number 2016-15-0201-00835).
1. Anesthesia and ultrasound medium
<ol>
	<li>Anesthetize the research animal. 
	NOTE: Type and dose of appropriate anesthesia are highly species-dependent. In general, immersion-based anesthetics such as MS-222 (ethyl 3-aminobenzoate methanesulfonic acid), benzocaine (ethyl 4-aminobenzoate), and propofol (2,6-diisopropyl phenol) are useful in fish and amphibians which readily absorbs the anesthetic over gills or skin (e.g., 0.05 mg&#183;L-1 benzocaine in rainbow trout). A range of dissolved compounds that can be administered intravenously, intramuscularly, intraperitoneally is available for amniotes, as are gas-based anesthetics. Alfaxalon administered intramuscularly is useful in reptiles (e.g., 30 mg&#183;kg-1 in lizards), and isoflurane administered as gas is useful in birds (e.g., 2% in air for pigeons). Refer to the published literature30,31,32 for a full overview of available anesthetics across species.</li>
	<li>Test reflexes in the animal to confirm an optimal level of anesthesia. Ensure that the animal is completely motionless during the procedure as the flow-enhanced ultrasound procedure is sensitive to motion noise.
	<ol>
		<li>Too deep anesthesia can alter blood flow patterns, so conduct a dose titration in the start-up phase of an experiment.</li>
		<li>Increase the anesthesia dosage in steps and observe blood flow in the eye aided by simple brightness mode (B-mode) ultrasound. 
		NOTE: An optimal level of anesthesia is obtained when the animal is motionless (except respiration) with visible ocular blood flow.</li>
	</ol>
	</li>
	<li>If the type/dose of anesthetic is not permissive for respiratory movements, then ensure adequate ventilation of the animal, e.g., using an air pump to oxygenate the water for aquatic species or a ventilator for air-breathing species.</li>
	<li>Position the animal in a posture that allows direct access from above to the eye. 
	NOTE: Depending on species, this can be in either a supine or lateral position. It can be useful to construct a simple holding device using a small piece of non-reactive metal (e.g., stainless steel) and loose rubber bands (see Figure 1).</li>
	<li>Place appropriate ultrasound medium on the eye of the animal. If scaled eyelids (ultrasound impermeable) cover the eye, then displace these gently with a cotton swab. 
	&#8203;NOTE: For aquatic species, the best ultrasound medium is clean tank water in which the animal usually lives. For terrestrial species, a generous amount of ultrasound gel ensures free movements and imaging of the ultrasound transducer (i.e., linear array probe) across the entire surface of the eye. Vet ointment on the contralateral eye is required for terrestrial species.</li>
</ol>
2. 2D and 3D ocular ultrasound image acquisition
<ol>
	<li>Position the ultrasound transducer medial to the eye in either a dorsal/ventral or rostral/caudal orientation depending on desired image orientation.</li>
	<li>In B-mode, with a maximum depth of field, image the medial and deepest portion of the eye and make sure that all structures of interest are visible in the image field. 
	NOTE: In some species, the crystalline lens takes up a comparatively large proportion of the vitreous humor, which may absorb the ultrasound, especially at higher frequencies.</li>
	<li>Slowly translate the transducer to each side while inspecting the real-time images. Make sure all structures of interest are visible in the image field; if not, switch to a transducer with a lower frequency and larger depth of field. 
	NOTE: The following center frequencies allow for the following maximum depth of field: 21 MHz: 3 cm, 40 MHz: 1.5 cm, 50 MHz: 1 cm (see Table 1). However, these maximum depth of field values can be markedly lower if the eye contains calcified or other ultrasound impermeable structures.</li>
	<li>Adjust image depth, depth offset (distance from the top of the image to the structure of interest), image width, as well as number and position of focal zones to cover the desired region of interest in all three spatial dimensions (e.g., 1 cm image depth, 2 mm depth offset, 1 cm image width, one focal zone). 
	NOTE: Although specific naming of buttons that adjust these parameters may vary between ultrasound systems, most systems will have buttons with logical names for these adjustments. These image parameter settings usually affect the range of possible temporal resolutions of the ultrasound acquisition.</li>
	<li>Set frame rate in the range of 50-120 frames&#183;s-1. 
	NOTE: The temporal resolution (i.e., the time interval between successive B-scans) must be adequate to display large pixel intensity variability in imaged blood vessels, i.e., the temporal resolution must not be too high. On the other hand, to complete a full 3D recording of the eye in a reasonable time, temporal resolution cannot be too low. A temporal resolution ranging from 50-120 frames&#183;s-1 is usually adequate for the flow-enhanced procedure in most species. On some ultrasound systems, this desired temporal resolution can be obtained by switching between the &#34;general imaging&#34; (high spatial/low temporal resolution) and &#34;cardiology&#34; (low spatial/high temporal resolution) modes.</li>
	<li>Adjust 2D gain to a level (~5 dB), so anatomical structures are only just visible in the B-mode acquisition to increase the signal-to-noise ratio in the subsequent flow-enhanced reconstruction.</li>
	<li>To acquire a 2D flow-enhanced image at a single slice position, translate the transducer to this position and continue at step 3.1.</li>
	<li>To acquire a 3D recording of an entire region of interest, e.g., the retina, translate the transducer to one extreme of the region of interest.
	<ol>
		<li>To determine the exact position of the extreme end of the region of interest, increase the 2D gain briefly.</li>
		<li>After correct transducer placement is complete, lower the 2D gain before recording to ensure maximal signal-to-noise ratio in the subsequent flow-enhanced reconstruction.</li>
	</ol>
	</li>
	<li>For each step (slice) in the 3D recording, acquire &#8805;100 frames (optimally &#8805;1000 frames).</li>
	<li>Using a micromanipulator or build-in transducer motor, translate the transducer across the entire region of interest in steps of, e.g., 25 &#181;m or 50 &#181;m (remember to note the step size) and repeat the &#8805;100 frames acquisition for each step.</li>
	<li>Euthanize the research animal according to the animal care guidelines of the institution.</li>
</ol>
3. Flow-enhanced image reconstruction
<ol>
	<li>Export the recordings into digital imaging and communications in medicine (DICOM) file format (little-endian).</li>
	<li>To produce a single flow-enhanced image based on a &#8805;100 frames (T) cine recording, calculate the standard deviation on pixel level (STD(x,y)) using the formula: 
	<img alt="Equation 1" src="/files/ftp_upload/62986/62986eq01.jpg" /> 
	Where It(x,y) is the intensity of the pixel at the (x,y) pixel coordinate at time t, and &#298;t(x,y) is the arithmetic mean value of I over time.</li>
	<li>Repeat step 3.2 for each slice in the 3D recording.</li>
	<li>To automate the STD-calculation and image reconstruction process for multiple slices in a 3D recording, conduct this operation in batch mode using, e.g., ImageJ and the supplementary macro script (Supplementary File 1).</li>
	<li>Combine all reconstructed slices into one image stack (Images to Stack command in ImageJ).</li>
	<li>Specify slice thickness from the step size used during acquisition (Properties command in ImageJ).</li>
	<li>Save the image stack as a 3D TIF file. 
	NOTE: Flow-weighted three-dimensional recordings of ocular blood vessels can subsequently be used to create volume renderings and build digital and physical anatomical models of vascular structures of the eye. These image processing options are outside the scope of this protocol; refer to the previously published articles for more details33,34,35.</li>
</ol>

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The authors declare that no completing interests exists.

Vascular imaging using flow-enhanced ultrasound provides a new method for non-invasive imaging of the vasculature of the eye that offers several advantages over present techniques but has its intrinsic limitations. The primary advantage of flow-enhanced ultrasound is the ability to generate ocular angiographies with a depth of field that exceeds the retinal pigment epithelium, which limits the depth of field in optical techniques. In ultrasound imaging, spatial resolution and depth of field are ultimately determined by the ultrasound transducer frequency, where higher frequencies increase the spatial resolution, but at the expense of a shallower depth of field, thus the choice of transducer frequency introduces a tradeoff between image depth and spatial resolution. In our experience, optimal retinal ultrasound imaging is achieved using high-frequency ultrasound transducers (&#8805;50 MHz) in small eyes with image depths of &lt;1 cm and lower frequency transducers (20-40 MHz) in larger eyes with image depths of 1.5-3.0 cm. For a 3D ultrasound scan, the resolution of the additional slice-dimension is set by the step size between scans in the stack of 2D ultrasound scans. In our experience, it is difficult to conduct a 3D scan with a step size smaller than 20 &#181;m. 
Flow-enhanced 2D ultrasound has a high temporal resolution. Ideally, &#8805;1000 frames per image are required for flow-enhanced vascular imaging, so at least 8 s are required per image scan. The temporal resolution is significantly reduced when performing 3D flow-enhanced ultrasounds, where the scanning time increases with the number of images in the 3D stack of scans. Given the high temporal resolution, the flow-enhanced 2D ultrasound workflow shows strong potential as a method for identifying temporal changes in relative blood flow velocities and blood flow distribution during experimental manipulation. Thus, future studies can use the workflow to identify how altered environmental conditions (e.g., temperature, pO2, pCO2) or pharmacological administration affect blood flow in the eye and other organs. 
The ultrasound workflow relies on the positive contrast of nucleated red blood cells from most non-mammalian vertebrates. Thus, the enucleated red blood cells of adult mammals and some salamander species37 provide too little contrast to effectively enhance blood flow using the present workflow (Figure 3E,F). In traditional ultrasound workflows, vascular injection of microbubbles provides high enough contrast to identify the vasculature in mammals38, which has been used to generate vascular angiographies of the retrobulbar vessels within the rat eye39. However, the microbubbles burst within minutes, so the generation of 3D angiographies requires successive microbubble injections. 
Flow-enhanced ultrasound depends on sequential recordings in the same position of the eye, so the technique is not possible in awake animals, where minor random movements may offset the image and undermine flow-enhancement calculations. Thus, the present method must be performed under proper anesthesia for immobilization to enhance image quality by reducing random movements. However, regular movements of the eye that occur during regular respiratory movements can be offset by prospectively or retrospectively gating to the ventilation pattern of the animal, so only scan recording from the same time interval within the ventilation cycle is used in the data analysis. While the retrospective gating approach to offset ventilatory movements of the image significantly improves the image stability, it pronouncedly reduces the number of frames included in calculating the standard deviation of signal intensity leading to a decrease in signal-to-noise ratio (compare Figure 4E to Figure 2C and Figure 2I). This effect is alleviated using prospective gating at the ultrasound scanner, in which image data is only acquired when the animal is in the desired phase of respiration. However, this causes a marked increase in acquisition time if the desired number of frames &#8805;1000 must be acquired.
We see multiple applications in zoological and veterinarian research for the flow-enhanced ultrasound workflow to map the physiology and anatomy of the eye's vasculature. The vasculature of ray-finned fishes, mammals, and birds are relatively well-described1,3,4,8,9,12,15,40, but this is not the case for non-bony fishes (jaw-less vertebrates and chondrichthyans), amphibians, and reptiles, that represent their respective earlier diverging sister groups. Implementing flow-enhanced ultrasound on these poorly understood animal groups and integrating these data with knowledge on the more well-studies groups will provide fundamental insight into the evolution of the vasculature of the vertebrate eye. Because the eye's vasculature is similar in closely related species4, such detailed information on the ocular vasculature in a broad range of species will provide a point-of-reference for veterinarians to identify malformations in the eye's vasculature due to developmental defects, diseases, or physical injuries. Furthermore, the ability to acquire 2D blood flow information with a high spatiotemporal resolution provides the means for quantifying pharmacokinetic effects on blood flow distribution in deep vascular beds, with vast applications in drug development and testing. Future studies on this technique should focus on identifying injectable compounds that enhance the contrast of blood in species with enucleated red blood cells, which will expand the applicability of this technique to mammals with vast applications in biomedical research and clinical diagnostics of vascular dysfunction in the eye and other deep vascular beds.

The high metabolism on the vertebrate retina imposes an intrinsic tradeoff between two contrasting needs; high blood flow rates and a light path devoid of blood vessels. To avoid visual disturbance of perfusing red blood cells, the retina of all vertebrates receives oxygen and nutrients via a sheet of capillaries behind the photoreceptors, the choriocapillaris1,2,3. However, this single source of nutrients and oxygen imposes a diffusion limitation to the thickness of the retina4,5, so many visually active species possess a variety of elaborate vascular networks to provide additional blood supply to this metabolically active organ6. These vascular beds include blood vessels perfusing the internal retinal layers in mammals and some fishes4,7,8,9,10, blood vessels on the inner (light-facing) side of the retina found in many fishes, reptiles, and birds4,11,12,13, and countercurrent vascular arrangements of the fish choroid, the choroid rete mirabile, that allows for the generation of super-atmospheric oxygen partial pressures14,15,16,17,18,19,20. Despite that these additional non-choroidal paths for retinal nutrient supply play an essential role in fueling the metabolic requirements of superior vision4, the three-dimensional anatomy of these vascular structures is poorly understood, limiting our understanding of the morphological evolution of the vertebrate eye.
Traditionally, retinal blood supply has been studied using optical techniques, such as fundus ophthalmoscopy. This category of techniques provides high-throughput non-destructive information on non-choroidal blood vessel anatomy in high-resolution21 and is therefore readily used in clinical diagnosis of abnormalities in retinal vessel structure22. However, the retinal pigment epithelium absorbs the transmitted light and limits the depth of view in these optical techniques, providing reduced information on choroidal structure and function without the use of contrast agent21. Similar depth limitations are experienced in optical coherence tomography (OCT). This technique can generate high-resolution fundus angiographies using light waves at the technical expense of depth penetration23, while the enhanced depth imaging OCT can visualize the choroid at the expense of retinal imaging quality24. Magnetic resonance imaging overcomes the optical limitations of ophthalmoscopy and OCT and can map vascular layers in the retina, albeit at a low resolution25. Histology and microcomputed tomography (&#181;CT) maintain the high-resolution of the optical techniques and provide information on whole-eye vascular morphology4, but both techniques require ocular sampling and are therefore not possible in the clinic or rare or endangered species. To overcome some of the limitations of these established retinal imaging techniques, the study here presents an ultrasound protocol on anesthetized animals, where blood movement is mapped in silico on a series of equally-spaced two-dimensional ultrasound scans spanning a whole eye by applying a comparable technique as described previously for embryonic and cardiovascular imaging26,27,28 and in OCT angiography29. This approach allows for the generation of non-invasive three-dimensional deep ocular angiographies without using a contrast agent and opens up new avenues for mapping blood flow distribution within the eye across species.

<table><tbody><tr><td>MS-222</td><td>Sigma</td><td>E10521-50G</td><td></td></tr><tr><td>Benzocaine</td><td>Sigma</td><td>E-1501</td><td></td></tr><tr><td>Propofol</td><td>B Braun</td><td>&lt;br/&gt; 12260470_0320</td><td></td></tr><tr><td>Alfaxalon</td><td>Jurox</td><td>NA</td><td></td></tr><tr><td>Isoflurane</td><td>Zoetis</td><td>50019100</td><td></td></tr><tr><td>Ultrasound scanner</td><td>VisualSonics</td><td>Vevo 2100</td><td></td></tr></tbody></table>

deep vascular imaging in the eye with flow-enhanced ultrasound

The retina within the eye is one of the most energy-demanding tissues in the body and thus requires high rates of oxygen delivery from a rich blood supply. The capillary lamina of the choroid lines the outer surface of the retina and is the dominating source of oxygen in most vertebrate retinas. However, this vascular bed is challenging to image with traditional optical techniques due to its position behind the highly light-absorbing retina. Here we describe a high-frequency ultrasound technique with subsequent flow-enhancement to image deep vascular beds (0.5-3 cm) of the eye with a high spatiotemporal resolution. This non-invasive method works well in species with nucleated red blood cells (non-mammalian and fetal animal models). It allows for the generation of non-invasive three-dimensional angiographies without the use of contrast agents, and it is independent of blood flow angles with a higher sensitivity than Doppler-based ultrasound imaging techniques.

The flow-enhanced ultrasound technique to image vascular beds of the eye can be applied in a range of species and has currently been used in 46 different vertebrate species (Figure 1, Table 1). The presence of nucleated red blood cells in non-adult-mammalian vertebrates provides positive contrast of flowing blood compared to static tissue in cine recordings (Supplementary File 2). However, when analyzed on a frame-by-frame basis, the clear distinction between blood and surrounding tissue is less obvious (Figure 2A). The blood flow enhancement procedure described in this protocol essentially compiles a multi-time point recording in 2D space (a slice made of T frames) into a single image in which the inherent signal value fluctuations in pixels positioned in flowing blood scores a higher standard deviation than surrounding static tissue, hence producing positive contrast (Figure 2B). To perceivably enhance the blood vessel contrast, Look Up Tables can be used to produce pseudocolor images (Figure 2C). In 3D acquisitions, multiple parallel slices with known spacing can be combined into 3D image data (Supplementary File 3 and Supplementary File 4) that can be used for three-dimensional volume rendering (Figure 2D) and anatomical modeling (Figure 2E and Supplementary File 5). Doppler-based ultrasound imaging also provides the option to specifically image blood flow, however with less sensitivity than the described method (compare Figure 2G with Figure 2H and Figure 2I), and importantly not if blood flow orientation is directly or close to perpendicular to the direction of the sound wave. The flow-enhanced procedure described in this protocol is independent of the orientation of blood flow both in-plane and out-of-plane.
The flow-enhanced ultrasound procedure allows for blood flow imaging in a range of species with nucleated red blood cells (Figure 3A-D). Deep ocular vascular beds such as the choroid rete mirabile in some fish can be imaged if present in the species (yellow arrowhead in Figure 2, Figure 3B, Figure 4). The method is limited by the absence of nucleated red blood cells in adult mammals in which the flow enhancement procedure produces some degree of blood flow contrast but is not as distinct as in species with nucleated red blood cells (Figure 3E,F).
Flow-enhanced ultrasound is sensitive to motion noise, and, e.g., respiratory movements can cause image blurring and artifacts such as tissue border enhancement (Figure 4A-C, Supplementary File 6). Prospective or retrospective gating can be used to adjust for motion noise (Figure 4D,E).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62986/62986fig01.jpg" /> 
Figure 1: Examples of the variety of species suitable for flow-enhanced ultrasound imaging of ocular vasculature. (A) Goldfish (Carassius auratus). (B) Siberian sturgeon (Acipenser baerii). (C) European seabass (Dicentrarchus labrax). (D) Clown featherback (Chitala ornata). (E) Crucian carp (Carassius carassius). (F) Embryonic domestic chicken (Gallus gallus domesticus). It can be useful to construct a simple holding device using a non-reactive metal weight and loose rubber bands (A,C,D). Both large, immobile lab-based ultrasound imaging systems can be used for the procedure (A-D,F) as well as small field operative systems (E). When imaging small and highly temperature-sensitive species that cannot be retained in a temperature-controlled water bath like embryonic birds, imaging can be performed while the sample is inside the incubator (F).&#160;<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62986/62986fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62986/62986fig2v5.jpg" /> 
Figure 2: Effect of flow enhancement on ocular ultrasound scans. (A) Examples of raw B-mode ultrasonographic images of the eye of a goldfish in a 1000 frame cine recording. Whereas blood flow can be observed in the cine recording (Supplementary File 2) it is difficult to see in static frames. (B) Flow-enhanced grayscale image (same slice as in A). Both retinal and post-retinal vascular beds are enhanced. (C) Pseudo-colored version of the image in B with ImageJ Fire Look Up Table. (D) Volume-rendered display of blood flow in the eye of the same goldfish as in A-C, based on 3D acquisition. (E) Two-segment (retinal and post-retinal vessels) anatomical model of eye in A-D (for interactive model see supplementary material 5). (F-I) Raw B-mode ultrasonographic image of the eye of another goldfish (F) comparing color Doppler based flow imaging (G) to the flow-enhanced methods described in this protocol (H-I, note I is an overlay of H on F). Green arrows indicate retinal vessels, yellow arrowheads indicate the choroid rete mirabile.&#160;<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62986/62986fig2v5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62986/62986fig3v5.jpg" /> 
Figure 3: Representative examples of flow-enhanced ocular ultrasound images in a variety of vertebrate species. (A) Senegal bichir (Polypterus senegalus). (B) Red-bellied piranha (Pygocentrus nattereri). (C) Green iguana (Iguana iguana). (D) Embryonic (day 18) domestic chicken (Gallus gallus domesticus). (E) House mouse (Mus musculus). (F) Brown rat (Rattus norvegicus). In species with nucleated red blood cells, the flow-enhancement procedure yields useful images of ocular blood flow (A-D), whereas in adult mammals (enucleated red blood cells), it produces only limited contrast between flowing blood and surrounding tissue (E-F). Green arrows indicate retinal vessels; blue arrowheads indicate post-retinal vessels such as the choriocapillaris; yellow arrowheads indicate choroid rete mirabile. In the late embryonic domestic chicken, blood flow in the pecten oculi can be observed (lower green arrow in F).&#160;<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62986/62986fig3v5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62986/62986fig4v5.jpg" /> 
Figure 4: Respiratory movements induce motion noise that can be alleviated by retrospective gating. (A-B) Example of respiratory movements in the eye of a European plaice (Pleuronectes platessa). The red dot is at the same image coordinate in A (slice 54/410)&#160;and B (slice 92/410), but it can be observed that the eye has shifted position (see also cine recording in supplementary material 6). (C) Attempt to perform the flow-enhancement operation on the full 410 frames recording fails due to motion noise. Tissue borders are artificially enhanced due to movements. (D) retrospective gating operation based on normalized signal intensity (SI) at the red dot in A-B. Only frames with normalized SI &#62; 50 (in total 38 frames), i.e., indicating that the eye is at the same position as in B, are included for the flow-enhancement procedure. (E) Resulting image of retrospectively gated flow-enhancement procedure. Compare with C. In the gated image, artificial border enhancement is avoided, and blood flow in the choroid rete mirabile (yellow arrowhead) can be observed. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62986/62986fig4v5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: List of species on which the flow-enhanced ultrasound technique to image ocular blood flow has been used. The applicability of the method is based on the ability to produce a contrast-rich representation of vascular beds compared to the static background.&#160;<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62986/Table_1.xlsx" target="_blank">Please click here to download this Table.</a>
Supplementary File 1: Macro script to automate flow-enhancement calculations. The script is written in IJ1 Macro language and can be executed both using the ImageJ macro function (for single slice recording) or the ImageJ Batch Process (for multiple slice 3D recording). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62986/Supplementary file_1.txt" target="_blank">Please click here to download this File.</a>
Supplementary File 2: Raw B-mode cine recording on the eye of a goldfish (Carassius auratus). Blood flow can be observed as the video is playing, but not on a single frame as in Figure 2A. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62986/Supplementary file_2.mp4" target="_blank">Please click here to download this File.</a>
Supplementary File 3: Slice video through the eye of a goldfish (Carassius auratus) of blood flow-enhanced sections. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62986/Supplementary file_3.mp4" target="_blank">Please click here to download this File.</a>
Supplementary File 4: Three-dimensional TIF file of the flow-enhanced eye of goldfish (Carassius auratus). Images have been binned by 3 x 3 x 3 to minimize file size (27-fold reduction in spatial resolution and file size).<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62986/Supplementary file_4.tif" target="_blank">Please click here to download this File.</a>
Supplementary File 5: Interactive 3D model of pre- and post-retinal vessels in the eye of a goldfish (Carassius auratus).&#160;<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62986/Supplementary file_5.pdf" target="_blank">Please click here to download this File.</a>
Supplementary File 6: Raw B-mode cine recording on the eye of a European plaice (Pleuronectes platessa). Note respiratory movements. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62986/Supplementary file_6.mp4" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Deep Vascular Imaging in the Eye with Flow-Enhanced Ultrasound at JoVE.com

Deep Vascular Imaging in the Eye with Flow-Enhanced Ultrasound

We present a non-invasive ultrasound technique for generating three-dimensional angiographies in the eye without the use of contrast agents.

The retina within the eye is one of the most energy-demanding tissues in the body and thus requires high rates of oxygen delivery from a rich blood ...

deep-vascular-imaging-in-the-eye-with-flow-enhanced-ultrasound

Universidad San Sebastian

Research

JoVE Journal

Biology

2.3K Views.  Aarhus University. We present a non-invasive ultrasound technique for generating three-dimensional angiographies in the eye without the use of contrast agents. 

Video: Deep Vascular Imaging in the Eye with Flow-Enhanced Ultrasound

Doppler Optical Coherence Tomography of Retinal Circulation

Noncontact retinal blood flow measurements are performed with a Fourier domain optical coherence tomography (OCT) system using a circumpapillary double circular scan (CDCS) that scans around the optic nerve head at 3.40 mm and 3.75 mm diameters. The double concentric circles are performed 6 times consecutively over 2 sec. The CDCS scan is saved with Doppler shift information from which flow can be calculated. The standard clinical protocol calls for 3 CDCS scans made with the OCT beam passing through the superonasal edge of the pupil and 3 CDCS scan through the inferonal pupil. This double-angle protocol ensures that acceptable Doppler angle is obtained on each retinal branch vessel in at least 1 scan. The CDCS scan data, a 3-dimensional volumetric OCT scan of the optic disc scan, and a color photograph of the optic disc are used together to obtain retinal blood flow measurement on an eye. We have developed a blood flow measurement software called "Doppler optical coherence tomography of retinal circulation" (DOCTORC). This semi-automated software is used to measure total retinal blood flow, vessel cross section area, and average blood velocity. The flow of each vessel is calculated from the Doppler shift in the vessel cross-sectional area and the Doppler angle between the vessel and the OCT beam. Total retinal blood flow measurement is summed from the veins around the optic disc. The results obtained at our Doppler OCT reading center showed good reproducibility between graders and methods (&lt;10%). Total retinal blood flow could be useful in the management of glaucoma, other retinal diseases, and retinal diseases. In glaucoma patients, OCT retinal blood flow measurement was highly correlated with visual field loss (R2&gt;0.57 with visual field pattern deviation). Doppler OCT is a new method to perform rapid, noncontact, and repeatable measurement of total retinal blood flow using widely available Fourier-domain OCT instrumentation. This new technology may improve the practicality of making these measurements in clinical studies and routine clinical practice.

Total retinal blood flow is measured by Doppler optical coherence tomography and semi-automated grading software.

Noncontact retinal blood flow measurements are performed with a Fourier domain optical coherence tomography (OCT) system using a circumpapillary double ...

Medicine

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite fluorescent in situ elements through the optical fibers, and then record some of the emitted photons, via the same optical fibers. The light source is a laser that sends the exciting light through an element within the fiber bundle and as it scans over the sample, recreates an image pixel by pixel. As this scan is very fast, by combining it with dedicated image processing software, images in real time with a frequency of 12 frames/sec can be obtained.
We developed a technique to quantitatively characterize capillary morphology and function, using a confocal fluorescence videomicroscopy device. The first step in our experiment was to record 5 sec movies in the four quadrants of the tumor to visualize the capillary network. All movies were processed using software (ImageCell, Mauna Kea Technology, Paris France) that performs an automated segmentation of vessels around a chosen diameter (10 &mu;m in our case). Thus, we could quantify the 'functional capillary density', which is the ratio between the total vessel area and the total area of the image. This parameter was a surrogate marker for microvascular density, usually measured using pathology tools.
The second step was to record movies of the tumor over 20 min to quantify leakage of the macromolecular contrast agent through the capillary wall into the interstitium. By measuring the ratio of signal intensity in the interstitium over that in the vessels, an 'index leakage' was obtained, acting as a surrogate marker for capillary permeability.

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

In this paper, we present a method to analyze tumor microvessels in vivo using dynamic contrast-enhanced fluorescence videomicroscopy. Two quantitative parameters were acquired: functional capillary density reflecting the vascularity of the tumor, and index leakage reflecting the leakiness of the endothelial wall.

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite ...

Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered perfusion can aid research into the mechanisms of retinal pathologies. Intravital video microscopy of fluorescent tracers can be used to measure vascular diameters and bloodstream velocities of the retinal vasculature, specifically the arterioles branching from the central retinal artery and of the venules leading into the central retinal vein. Blood flow rates can be calculated from the diameters and velocities, with the summation of arteriolar flow, and separately venular flow, providing values of total retinal blood flow. This paper and associated video describe the methods for applying this technique to mice, which includes 1) the preparation of the eye for intravital microscopy of the anesthetized animal, 2) the intravenous infusion of fluorescent microspheres to measure bloodstream velocity, 3) the intravenous infusion of a high molecular weight fluorescent dextran, to aid the microscopic visualization of the retinal microvasculature, 4) the use of a digital microscope camera to obtain videos of the perfused retina, and 5) the use of image processing software to analyze the video. The same techniques can be used for measuring retinal blood flow rates in rats.

Intravital microscopy can be used in animals to visualize and measure retinal vascular diameters, bloodstream velocities, and total retinal blood flow.

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered ...

Ultrasound Assessment of Endothelial-Dependent Flow-Mediated Vasodilation of the Brachial Artery in Clinical Research

The vascular endothelium is a monolayer of cells that cover the interior of blood vessels and provide both structural and functional roles. The endothelium acts as a barrier, preventing leukocyte adhesion and aggregation, as well as controlling permeability to plasma components. Functionally, the endothelium affects vessel tone.
Endothelial dysfunction is an imbalance between the chemical species which regulate vessel tone, thombroresistance, cellular proliferation and mitosis. It is the first step in atherosclerosis and is associated with coronary artery disease, peripheral artery disease, heart failure, hypertension, and hyperlipidemia.
The first demonstration of endothelial dysfunction involved direct infusion of acetylcholine and quantitative coronary angiography. Acetylcholine binds to muscarinic receptors on the endothelial cell surface, leading to an increase of intracellular calcium and increased nitric oxide (NO) production. In subjects with an intact endothelium, vasodilation was observed while subjects with endothelial damage experienced paradoxical vasoconstriction.
There exists a non-invasive, in vivo method for measuring endothelial function in peripheral arteries using high-resolution B-mode ultrasound. The endothelial function of peripheral arteries is closely related to coronary artery function. This technique measures the percent diameter change in the brachial artery during a period of reactive hyperemia following limb ischemia.
This technique, known as endothelium-dependent, flow-mediated vasodilation (FMD) has value in clinical research settings. However, a number of physiological and technical issues can affect the accuracy of the results and appropriate guidelines for the technique have been published. Despite the guidelines, FMD remains heavily operator dependent and presents a steep learning curve. This article presents a standardized method for measuring FMD in the brachial artery on the upper arm and offers suggestions to reduce intra-operator variability.

Endothelial dysfunction is associated with numerous disease states and is predictive of adverse cardiovascular events in humans. Flow-mediated vasodilation (FMD) is a non-invasive ultrasound method of evaluating endothelial function. Methodological choices and operator experience may affect results. A systematic approach to FMD in human studies is discussed here.

The vascular endothelium is a monolayer of cells that cover the interior of blood vessels and provide both structural and functional roles. The ...

Non-invasive Parenchymal, Vascular and Metabolic High-frequency Ultrasound and Photoacoustic Rat Deep Brain Imaging

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central nervous system of small animals. However, transdermal and transcranial neuroimaging is frequently affected by low sensitivity, image aberrations and loss of space resolution, requiring scalp or even skull removal before imaging. To overcome this challenge, a new protocol is presented to gain significant insights in brain hemodynamics by photoacoustic and high-frequency ultrasounds imaging with the animal skin and skull intact. The procedure relies on the passage of ultrasound (US) waves and laser directly through the fissures that are naturally present on the animal cranium. By juxtaposing the imaging transducer device exactly in correspondence to these selected areas where the skull has a reduced thickness or is totally absent, one can acquire high quality deep images and explore internal brain regions that are usually difficult to anatomically or functionally describe without an invasive approach. By applying this experimental procedure, significant data can be collected in both sonic and optoacoustic modalities, enabling to image the parenchymal and the vascular anatomy far below the head surface. Deep brain features such as parenchymal convolutions and fissures separating the lobes were clearly visible. Moreover, the configuration of large and small blood vessels was imaged at several millimeters of depth, and precise information were collected about blood fluxes, vascular stream velocities and the hemoglobin chemical state. This repertoire of data could be crucial in several research contests, ranging from brain vascular disease studies to experimental techniques involving the systemic administration of exogenous chemicals or other objects endowed with imaging contrast enhancement properties. In conclusion, thanks to the presented protocol, the US and PA techniques become an attractive noninvasive performance-competitive means for cortical and internal brain imaging, retaining a significant potential in many neurologic fields.

The present work describes a new protocol to perform non-invasive high-frequency ultrasound and photoacoustic based imaging on rat brain, to efficiently visualize deep subcortical regions and their vascular patterns by directing signals on skull foramina naturally present on animal cranium.

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central ...

Neuroscience

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy (oSLO) and Optical Coherence Tomography (OCT)

While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big challenge with the state-of-the-art retinal imaging modalities because they would require z-stacking to compile a volumetric dataset. Newer optical coherence tomography (OCT) and OCT angiography (OCTA) systems overcome these restrictions to provide three-dimensional (3D) anatomical and vascular images, but the dye-free nature of OCT cannot visualize leakage indicative of vascular dysfunction. This protocol describes a novel oblique scanning laser ophthalmoscopy (oSLO) technique that provides 3D volumetric fluorescence retinal imaging. The setup of the imaging system generates the oblique scanning by a dove tail slider and aligns the final imaging system at an angle to detect fluorescent cross-sectional images. The system uses the laser scanning method, and therefore, allows an easy incorporation of OCT as a complementary volumetric structural imaging modality. In vivo imaging on rat retina is demonstrated here. Fluorescein solution is intravenously injected to produce volumetric fluorescein angiography (vFA).

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy oSLO and Optical Coherence Tomography OCT

Here, we present a protocol to get a large field of view (FOV) three-dimensional (3D) fluorescence and OCT retinal image by using a novel imaging multimodal platform. We will introduce the system setup, the method of alignment, and the operational protocols. In vivo imaging will be demonstrated, and representative results will be provided.

While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big ...

Bioengineering

Fluorescent Dye Labeling of Erythrocytes and Leukocytes for Studying the Flow Dynamics in Mouse Retinal Circulation

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, diabetic retinopathy, age-related macular degeneration (AMD) and other ocular inflammatory conditions. It may also help to monitor the therapeutic responses in the eye. The proper labeling of the blood cells, coupled with live-cell imaging of the labeled cells, allows for the investigation of the flow dynamics in the retinal and choroidal circulation. Here, we describe the standardized protocols of 1.5% indocyanine green (ICG) and 1% sodium fluorescein labeling of mice erythrocytes and leukocytes, respectively. Scanning laser ophthalmoscopy (SLO) was applied to visualize the labeled cells in the retinal circulation of C57BL/6J mice (wild type). Both methods demonstrated distinct fluorescently labeled cells in the mouse retinal circulation. These labeling methods can have wider applications in various ocular disease models.

Live-cell imaging of the labeled blood cells in ocular circulation can provide information about inflammation and ischemia in diabetic retinopathy and age-related macular degeneration. A protocol to label blood cells and image the labeled cells in the retinal circulation is described.

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, ...

Blood Flow Imaging with Ultrafast Doppler

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler modes, it has several limits. Firstly, a finely tuned signal filtering operation is needed to distinguish blood flows from surrounding moving tissues. Secondly, the operator must choose between localizing the blood flows or quantifying them. In the last two decades, ultrasound imaging has undergone a paradigm shift with the emergence of ultrafast ultrasound using unfocused waves. In addition to a hundredfold increase in framerate (up to 10000 Hz), this new technique also breaks the conventional quantification/localization trade-off, offering a complete blood flow mapping of the field of view and a simultaneous access to fine velocities measurements at the single-pixel level (down to 50 &#181;m). This data continuity in both spatial and temporal dimensions strongly improves the tissue/blood filtering process, which results in an increase sensitivity to small blood flow velocities (down to 1 mm/s). In this method paper, we aim to introduce the concept of ultrafast Doppler as well as its main parameters. Firstly, we summarize the physical principles of unfocused wave imaging. Then, we present the Doppler signal processing main steps. Particularly, we explain the practical implementation of the critical tissue/blood flow separation algorithms and on the extraction of velocities from these filtered data. This theoretical description is supplemented by in vitro experiences. A tissue phantom embedding a canal with flowing blood-mimicking fluid is imaged with a research programmable ultrasound system. A blood flow image is obtained and the flow characteristics are displayed for several pixels in the canal. Finally, a review of in vivo applications is proposed, showing examples in several organs such as carotids, kidney, thyroid, brain and heart.

This protocol shows how to apply ultrafast ultrasound Doppler imaging to quantify blood flows. After a 1 s long acquisition, the experimenter has access to a movie of the full field of view with axial velocity values for each pixel every &#8776;0.3 ms (depending on the ultrasound time of flight).

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler ...

The Preparation of Chicken Ex Ovo Embryos and Chorioallantoic Membrane Vessels as In Vivo Model for Contrast-Enhanced Ultrasound Imaging and Microbubble-Mediated Drug Delivery Studies

The chicken embryo and the blood-vessel rich chorioallantoic membrane (CAM) is a valuable in vivo model to investigate biomedical processes, new ultrasound pulsing schemes, or novel transducers for contrast-enhanced ultrasound imaging and microbubble-mediated drug delivery. The reasons for this are the accessibility of the embryo and vessel network of the CAM as well as the low costs of the model. An important step to get access to the embryo and CAM vessels is to take the egg content out of the eggshell. In this protocol, three methods for taking the content out of the eggshell between day 5 and&#160;8 of incubation are described thus allowing the embryos to develop inside the eggshell up to these days. The described methods only require simple tools and equipment and yield a higher survival success rate of 90% for 5-day, 75% for 6-day, 50% for 7-day, and 60% for 8-day old incubated eggs in comparison to ex ovo cultured embryos (~50%). The protocol also describes how to inject cavitation nuclei, such as microbubbles, into the CAM vascular system, how to separate the membrane containing the embryo and CAM from the rest of the egg content for optically transparent studies, and how to use the chicken embryo and CAM in a variety of short-term ultrasound experiments. The in vivo&#160;chicken embryo and CAM model is extremely relevant to investigate novel imaging protocols, ultrasound contrast agents, and ultrasound pulsing schemes for contrast-enhanced ultrasound imaging, and to unravel the mechanisms of ultrasound-mediated drug delivery.

This protocol describes three methods on how to obtain and use 5 to 8-day old chicken embryos and their chorioallantoic membrane (CAM) as an in vivo&#160;model to study contrast-enhanced ultrasound imaging and microbubble-mediated drug delivery.

The chicken embryo and the blood-vessel rich chorioallantoic membrane (CAM) is a valuable in vivo model to investigate biomedical processes, new ...

Real-Time Intravital Multiphoton Microscopy to Visualize Focused Ultrasound and Microbubble Treatments to Increase Blood-Brain Barrier Permeability

The blood-brain barrier (BBB) is a key challenge for the successful delivery of drugs to the brain. Ultrasound exposure in the presence of microbubbles has emerged as an effective method to transiently and locally increase the permeability of the BBB, facilitating para- and transcellular transport of drugs across the BBB. Imaging the vasculature during ultrasound-microbubble treatment will provide valuable and novel insights on the mechanisms and dynamics of ultrasound-microbubble treatments in the brain.
Here, we present an experimental procedure for intravital multiphoton microscopy using a cranial window aligned with a ring transducer and a 20x objective lens. This set-up enables high spatial and temporal resolution imaging of the brain during ultrasound-microbubble treatments. Optical access to the brain is obtained via an open-skull cranial window. Briefly, a 3-4 mm diameter piece of the skull is removed, and the exposed area of the brain is sealed with a glass coverslip. A 0.82 MHz ring transducer, which is attached to a second glass coverslip, is mounted on top. Agarose (1% w/v) is used between the coverslip of the transducer and the coverslip covering the cranial window to prevent air bubbles, which impede ultrasound propagation. When sterile surgery procedures and anti-inflammatory measures are taken, ultrasound-microbubble treatments and imaging sessions can be performed repeatedly over several weeks. Fluorescent dextran conjugates are injected intravenously to visualize the vasculature and quantify ultrasound-microbubble induced effects (e.g., leakage kinetics, vascular changes). This paper describes the cranial window placement, ring transducer placement, imaging procedure, common troubleshooting steps, as well as advantages and limitations of the method.

This protocol describes the surgical and technical procedures that enable real-time in vivo multiphoton fluorescence imaging of the rodent brain during focused ultrasound and microbubble treatments to increase blood-brain barrier permeability.

The blood-brain barrier (BBB) is a key challenge for the successful delivery of drugs to the brain. Ultrasound exposure in the presence of microbubbles ...

Deep Vascular Imaging in the Eye with Flow-Enhanced Ultrasound

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The Preparation of Chicken Ex Ovo Embryos and Chorioallantoic Membrane Vessels as In Vivo Model for Contrast-Enhanced Ultrasound Imaging and Microbubble-Mediated Drug Delivery Studies

Real-Time Intravital Multiphoton Microscopy to Visualize Focused Ultrasound and Microbubble Treatments to Increase Blood-Brain Barrier Permeability