2.1.

Imaging Setup and Characterization

The schematic diagram of the SD-OCM system is shown in Fig. 1.³⁷ A broadband superluminescent diode (SLD); T-850-HP, Superlum with a bandwidth of 170 nm and a center wavelength of 845 nm was used as the light source. The actual system bandwidth was reduced to $\sim 90\mathrm{nm}$ (780 to 870 nm, $\text{center wavelength}=\sim 825\mathrm{nm}$) due to the imperfect optics and limited spectral coverage of optical components in the system. In the sample arm, a pair of $X-Y$ galvanometer mirrors (8315 K, Cambridge Technology) performed two-dimensional lateral scanning. An objective lens with $10\times$ magnification (LSM02-BB, Thorlabs) focused the light onto the sample. The back aperture of the objective was underfilled, with an effective numerical aperture of 0.11. In the reference arm, a dispersion compensation block (LSM02DC, Thorlabs) matched the dispersion of the $10\times$ objective lens. Polarization controllers in the SLD arm, sample arm, and reference arm were used to optimize the sensitivity of the system. A custom LabVIEW (National Instruments) program controlled the galvanometer scanners and data acquisition from the spectrometer.

Fig. 1

Schematic diagram of the SD-OCM system for small animal imaging. SLD, superluminescent diode; ISO, isolator; DC, dispersion component.

Backscattered light from the sample arm and reference arm generated interference spectra that were detected by a custom-built spectrometer. The spectrometer data were transferred to the computer through a frame grabber (PCIe-1433, National Instruments). The custom-built spectrometer consisted of a collimating lens ($f=75\mathrm{mm}$), a $1200\text{lines}/\mathrm{mm}$ volume phase holographic transmission grating (1002-2, Wasatch Photonics), a focusing lens ($f=150\mathrm{mm}$), and a line scan complementary metal-oxide-semiconductor (CMOS) camera (spL4096-140km, Basler). It was calibrated to determine the wavelength distribution on CMOS pixels. A calibration approach was developed, which combined methods of using spectral phase difference from single-layer reflections^38–40 and the spectrum of the light source provided by the manufacturer. This calibration approach was cost-effective, as it did not require extra instruments, such as fiber Bragg gratings and narrowband light sources.

The lateral resolution of the system was measured by imaging a United States Air Force (USAF) resolution target (R1DS1P, Thorlabs) in water. In Fig. 2(a), line intensity profiles of elements in group 7 show that the smallest full width at half maximum (FWHM) that was resolved by the SD-OCM system was in element 4 (blue dashed box), which has a line width of $2.8\mu \mathrm{m}$. This is the measured lateral resolution in agreement with the theoretical focal spot size of $\sim 2.8\mu \mathrm{m}$.

Fig. 2

Characterization of the SD-OCM system. (a) Measurement of the lateral resolution by imaging a USAF resolution target in water. Line intensity profiles over the red dashed line area show that the smallest lines clearly resolvable are in element 4 of group 7, which have the widths of $2.8\mu \mathrm{m}$. Scale bars: $50\mu \mathrm{m}$. (b) Measurement of the axial resolution by determining the width of the axial PSF in water. The FWHM of the axial PSF at a depth of $40\mu \mathrm{m}$ is $2.8\mu \mathrm{m}$. (c) Axial PSFs at various depths show a sensitivity roll-off of $-3.5\mathrm{dB}$ across $\sim 380\text{-}\mu \mathrm{m}$ depth.

The axial resolution of the system (also referred to as the axial point spread function; axial PSF) was measured by imaging the reflectivity profile of a mirror reflection in water (index of refraction $n=1.33$). Figure 2(b) shows the axial PSF (axial resolution) at a depth of $40\mu \mathrm{m}$ has an FWHM of $2.8\mu \mathrm{m}$, which was close to the theoretical value of $\sim 2.5\mu \mathrm{m}$ calculated from the effective system bandwidth.⁴¹ Due to finite spectral resolution and finite pixel size in the spectrometer, the sensitivity of the system decreased as the depth increased. Figure 2(c) shows that the sensitivity roll-off over $\sim 380\text{-}\mu \mathrm{m}$ depth is only $-3.5\mathrm{dB}$, indicating that the spectrometer is suitable for imaging zebrafish embryos that are typically $<400\text{-}\mu \mathrm{m}$ thick.

2.2.

Sample Preparation

Live zebrafish embryos were collected for in vivo imaging. Adult zebrafish and embryos were raised and maintained as described in the guidance book⁴² and in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals by the University of Southern California, where the protocol was approved by the Institutional Animal Care and Use Committee (permit number: 12007 USC). The transgenic zebrafish line used in our experiments expressed GFP in its vascular endothelial cells: Tg(kdrl:eGFP). Zebrafish embryos were raised in egg water ($60\mu \mathrm{g}/\mathrm{mL}$ of instant ocean and $75\mu \mathrm{g}/\mathrm{mL}$ of CaSO₄ in milli-Q water) at 28.5°C in an incubator. Most embryos were imaged at developmental stages of 2 to 5 days postfertilization (dpf), because their vasculature is maturing and undergoing remodeling during these stages.^7,8,14 Zebrafish embryos were anesthetized with tricaine (0.02%) so that they stayed stationary during imaging experiments. The area of interest was the fish trunk near the tail because it had rich anatomy and a predictable vasculature for both structural and functional imaging with the SD-OCM system [Fig. 5(a)].

For in vivo imaging with confocal laser scanning microscopy, typically low melting point agarose is used to stabilize zebrafish embryos in place inside glass bottom dishes. To avoid image distortion and accommodate short working distances of objective lenses, thin glass bottom dishes are used (thickness $\sim 170\mu \mathrm{m}$), and the sample is mounted very near to the glass bottom, as shown in Fig. 3(a). For OCM, mounting of the sample must be adjusted for the best imaging:

Fig. 3

Mount of the animal sample in a glass bottom dish for in vivo imaging. (a) Conventional way of mounting the sample in confocal laser scanning microscopy. (b) Mounting method was adjusted to optimize SD-OCM imaging.

2.3.

Technique of Phase Variance OCM

The phase variance technique was integrated into our SD-OCM system for imaging the microvasculature in zebrafish embryos. In pvOCM, multiple B-scans taken at the same location over time (BM-scan) were used to calculate the phase variance. Regions of blood flow were distinguished from stationary tissues by larger phase variance (motion contrast). The phase variance is a statistical measure and defined as^26,32

In our pvOCM imaging experiments, four B-scans were taken for the same position as a BM-scan. The B-scan frame rate was $\sim 56\mathrm{Hz}$, resulting in a BM-scan rate of $\sim 14\mathrm{Hz}$. In offline data processing, B-scan images of the intensity and phase were generated following standard SD-OCT processing procedures, including background removal,⁴⁵ spectra interpolation, numerical dispersion compensation,⁴⁶ spectral reshaping, zero padding,⁴⁵ and Fourier transforms. B-scan images of phase variance values were then calculated after the bulk motion was removed. The bulk motion was estimated using weighted mean phase changes between aligned successive B-scans at depths within the sample region,^21,47 defined as

With bulk motion removed, binary masks based on the structural intensity and median filters (3 pixels laterally and 1 pixel axially) were applied. 3-D en face images of blood flow at multiple depths were created for later visualization. General processing procedures of the pvOCM technique are shown in the flowchart of Fig. 4.

Fig. 4

Flowchart of pvOCM data processing procedures. In experiments, more B-scans per BM-scan would improve the statistics of phase variance, but this would slow the imaging. One BM-scan was chosen to be four B-scans to balance the quality of pvOCM images and imaging speed.

3.1.

Structural and Vascular Imaging

In SD-OCM imaging experiments, the system acquired a 3-D volume of B-scan images, or cross-sectional images, as shown in Fig. 5(d). The scan direction within each B-scan was parallel to the zebrafish trunk; each B-scan image contains 800 A-scans spanning a distance of 1.20 mm. En face intensity images were created at multiple depths via average intensity projection of $5\text{-}\mu \mathrm{m}$ depths from the volumetric set of imaging data to provide a lateral view of the zebrafish structure, as shown in Fig. 5(e). En face images clearly identified the structure of the somites, notochord, spinal cord, yolk, and gut. The aorta and cardinal vein were also resolved. Images of zebrafish embryos were taken with a bright-field microscope (MVX10, Olympus), with a representative image shown in Fig. 5(b). Such bright-field microscopic images served as references for OCM structural images.

Fig. 5

Structural and vascular imaging of zebrafish embryos. (a) Composite wide-field microscopic image of a 3--dpf zebrafish embryo: Tg(kdrl:eGFP). The red dashed box marks the trunk area of interest for SD-OCM imaging. (b) Bright-field microscopic image of the structure of a 3-dpf zebrafish trunk. (c) Fluorescence microscopic image of the GFP-labeled vascular endothelium in the trunk. (d) OCM B-scan intensity images at roughly the same area. The yellow dashed line represents one example plane for creating an en face image. (e) En face OCM intensity images at various depth layers. (f) En face pvOCM images of the vasculature at various depth layers. In both (e) and (f), each en face image was created from average intensity projection of $5\text{-}\mu \mathrm{m}$ depth and spanned an area of $1.20{\times 0.45\mathrm{mm}}^2$. The depth $z$ was relative to the top surface of the zebrafish trunk. Scale bars: $100\mu \mathrm{m}$.

Imaging of the vasculature in zebrafish embryos was implemented by the pvOCM technique, and en face pvOCM images were created at multiple depths, by averaging the intensity from $5\text{-}\mu \mathrm{m}$ slabs within the volumetric data set, as shown in Fig. 5(f). The intersegmental vessels (Se), dorsal aorta (DA), caudal artery (CA), posterior cardinal vein (PCV), caudal vein (CV), and dorsal longitudinal anastomotic vessel (DLAV) were clearly visualized in these datasets. Fluorescence images of the GFP-labeled vasculature were taken with a wide-field fluorescence microscope (MVX10, Olympus) for comparison, with a representative image shown in Fig. 5(c).

3.2.

Visualization of the Vascular Network

To visualize the vasculature at all depths of the sample in a single image, en face projection pvOCM image was created via average intensity projection over depths ranging from 30 to $170\mu \mathrm{m}$ below the top surface of the zebrafish trunk, as shown in Fig. 6(a). Features present in the projection pvOCM image corresponded well to the vasculature image obtained by fluorescence microscopy shown in Fig. 5(c). Color coding of the rendering, based on depth, reveals the 3-D organization of the microvascular network, as shown in Fig. 6(b). The vasculature on the ipsilateral half of the embryo (closer to the objective lens in the experiment, depths 30 to $100\mu \mathrm{m}$) was coded as green, and the vasculature on the contralateral half of the embryo (further away from the objective lens in the experiment, depths 100 to $170\mu \mathrm{m}$) was coded as red. This is especially helpful for distinguishing the intersegmental vessels (Se) on two sides of the embryo. Interestingly, in some segments (white arrows), Se blood flow was detected by pvOCM only on one side of the embryo. Such differences in the flow did not follow a fixed pattern, reminiscent of the randomness of the arterial–venous identity of the Se along the trunk of zebrafish embryos.⁷ These differences in blood flow may result from the Se remodeling process because some Se had not yet formed functional connections with the CA and CV.¹⁴ If an Se had no blood flow, it would not be visualized in pvOCM images.

Fig. 6

Visualization of the microvascular network by pvOCM. (a) Projection pvOCM image, created by average intensity projection over the depth range of 30 to $170\mu \mathrm{m}$ below the top surface of the zebrafish trunk. (b) Depth color-coded projection pvOCM image, with green denoting the vasculature on the ipsilateral half and red denoting the vasculature on the contralateral half of the embryo. (b’) Sagittal views of pvOCM images are shown for two white dashed box areas in (b). White arrows mark projection artifacts below strong flow regions of the aorta and vein. Scale bars: $100\mu \mathrm{m}$.

In Fig. 6(b), sagittal views of pvOCM images at selected white dashed box areas display high-phase variance contrast that corresponds to major vessels, the DA, PCV, CA, and CV. Note that there are inevitable projection artifacts, marked by white arrows (depths $>100\mu \mathrm{m}$). These artifacts resulted from the large phase variance of overlying regions of strong flow in the aorta and veins.^21,32,33 Artifacts in regions outside of sample areas were removed by applying binary masks according to the procedure shown in Fig. 4. In Fig. 6(b), projection artifacts made the depth color-coded blood flow of the aorta and vein (DA, CA, and PCV) have a mix of green and red colors that appears to be yellow. These vessels appear as if they covered the entire contralateral half of the embryo.

3.3.

Imaging the Developing Vascular System

Images of the developing vascular system were taken using the pvOCM technique at a range of developmental stages (2 to 5 dpf; Fig. 7). The pvOCM images were compared with corresponding fluorescence microscopic images of the GFP-labeled vascular endothelial cells. These projection pvOCM images provide both anatomical and functional information on vasculature, which is important in the study of vascular development.

Fig. 7

pvOCM images of the developing vascular system. Projection pvOCM images of zebrafish embryos at developmental stages of (a) 2 dpf, (b) 3 dpf, (c) 4 dpf, and (d) 5 dpf. All images were created by average intensity projection over entire depths across samples. Corresponding fluorescence microscopic images are shown on the right column for comparison. Yellow dashed boxes represent areas for analysis in Fig. 8. Scale bars: $100\mu \mathrm{m}$.

Regional differences in the phase variance contrast values suggest that not all vessels present in embryos had the same magnitude of flow. Enlarged views in Fig. 8 highlight some bright regions in fluorescence images of the vasculature with little or no pvOCM contrast (red arrows). The absence of phase variance signals in these regions suggests that there were regions of the vasculature with little or no blood flow, offering valuable functional information that is unavailable in fluorescence images of labeled endothelial cells. In addition, in some small regions, the absence of phase variance signal was an artifact of the pulsatile nature of blood flow. Given that the BM-scan rate ($\sim 14\mathrm{Hz}$) was much faster than the heart rate ($\sim 2\mathrm{Hz}$) of a zebrafish embryo, some BM-scans should be recorded during the phase of peak blood flow (resulting in high-phase variance), whereas the others were recorded at the phase of slowest blood flow (resulting in low or no phase variance). This is responsible for the flow that appears to be discontinuous in some Se (yellow asterisks, Fig. 7), in which there are alternating regions of high and low/no phase variance contrast along the same vessel. Furthermore, in the smallest vessels, a discontinuous pvOCM signal could correspond to locations of individual blood cells since the majority of reflections that contributed to the phase variance contrast came from the highly reflective blood cells and not the plasma.

Fig. 8

Enlarged views of pvOCM images of developing zebrafish embryos, corresponding to yellow dashed box areas in (a–d) of Fig. 7. Red arrows highlight some parts of the vasculature that appear in fluorescent images but are absent in pvOCM images. The absence of phase variance signals is related to the pulsatile nature of blood flow. Blue arrows highlight veins that had no blood flow detected after vascular junctions that are denoted by yellow arrows. Scale bars: $50\mu \mathrm{m}$.

Remodeling processes were observed by pvOCM as shown in Figs. 8(b)–8(d). At some vascular junctions (yellow arrows), one path of the bifurcation had blood flow, whereas the other path (blue arrows) did not. This suggests that at these developmental stages remodeling processes were occurring in CV areas, where one of the veins (blue arrows) was not functional for blood circulation. In these areas, the blood flow circulated only through the more ventral veins that would eventually develop into CV.