2.1.

Bench-Top Optical Coherence Tomography and Optical Frequeny-Domain Imaging Systems

In time-domain OCT (TDOCT), axial ranging is performed by use of low-coherence reflectometry where the individual depth points are probed sequentially in time. A broad bandwidth [ $50\mathrm{nm}$ , full width at half maximum (FWHM)] source centered at $1.3\mu \mathrm{m}$ was used, providing an axial resolution of $\sim 10\mu \mathrm{m}$ in tissue. The frame rate was $20\text{per}\text{second}$ ( $2\mathrm{kHz}$ A-line rate, $100\times 500\text{pixels}$ ).

OFDI originates from a telecommunications ranging method termed optical frequency-domain reflectometry. The primary differences between OCT and OFDI are found in the system optics. With OFDI, the interferometer reference arm remains stationary and spectral interference is detected using a narrowband wavelength-tuning source. OFDI images, reconstructed by taking the Fourier transform of the spectral fringe pattern, are essentially identical to those obtained by OCT. OFDI provides a several-hundred-fold improvement in signal-to-noise ratio (SNR),^{16, 17} compared to OCT, which can be utilized to increase imaging speed to nearly $1000\text{frames}\text{per}\text{second}$ .

The wavelength tunable laser light source of our OFDI system was an extended cavity semiconductor laser employing an intracavity spectral filter.^{14, 17} The laser featured a sweep repetition rate of up to $54\mathrm{kHz}$ , a wide tuning range of $111\mathrm{nm}$ centered at $1320\mathrm{nm}$ , and a high average output power of $30\mathrm{mW}$ ( $7\mathrm{mW}$ on the tissue). The axial resolution was $10\mu \mathrm{m}$ in tissue. The frame rate was $900\text{per}\text{second}$ , resulting in a volumetric acquisition rate of $20\mathrm{Hz}$ ( $60\times 45\times 256\text{pixels}$ per volume). The system further comprised an acousto-optic frequency shifter $\left(25\mathrm{MHz}\right)$ to remove the depth degeneracy inherent in frequency-domain reflectometry.²¹

Polarization-diversity detection was implemented to eliminate polarization artifacts in the fiber-based OFDI system. Dual-balanced photoreceivers were used to improve imaging sensitivity through the reduction of laser intensity noise. The photoreceiver outputs were digitized with a two-channel analog-to-digital converter at a sampling rate of $100\mathrm{MHz}$ with $14\text{-}\text{bit}$ resolution. TDOCT and high-speed OFDI were incorporated into a dissecting light microscope. The scanning system was comprised of a collimating lens ( $5\text{-}\mathrm{mm}$ beam diameter), two synchronized galvanometric scanners for transverse scanning, a focusing lens ( $50\text{-}\mathrm{mm}$ focal length), and a small mirror that deflected the beam downward toward the sample. The transverse resolution was $15\mu \mathrm{m}$ with a confocal parameter of $280\mu \mathrm{m}$ .

Displacements associated with local cardiac motion (Figs. 5a, 5b, 5c, 5d) were estimated from the OFDI data by subtracting the heart surface locations at end diastole from those at end systole on a frame-by-frame basis. First, the heart was semiautomatically segmented using IPLab Spectrum (BD Biosciences, Franklin Lakes, New Jersey) in end diastole and end systole. Morphologic edge detection was then conducted on the segmented areas to determine the heart surfaces for end diastole and end systole. End diastole and end systole segmented areas were subtracted on a frame-by-frame basis. The subtracted image maintained its sign so that positive and negative displacements could be computed. For every point on the edge images, the thickness of the subtracted volumes was determined by summing the subtracted data within a local kernel centered at each edge point. The displacement value estimate for each surface position was then scaled according to the pixel dimensions and assigned a unique color that served as a color overlay on the 3-D volume rendering. Volumetric rendering and 3-D visualization was accomplished by using OsiriX software.²²

High-resolution OFDI was performed using a novel laser source with $200\text{-}\mathrm{nm}$ tuning range, centered at $1250\mathrm{nm}$ , in which two semiconductor optical amplifiers were utilized as the gain media.²³ An axial resolution of $4\mu \mathrm{m}$ in tissue was measured. The transverse resolution was $2\mu \mathrm{m}$ with $\mathrm{NA}=0.2$ objective lens. The imaging rate was $40\text{frames}\text{per}\text{second}$ with an A-line rate of $20\mathrm{kHz}$ (500 A-lines per frame, $666\text{pixels}$ per A-line).

Polarization-diversity and dual-balanced detection were performed and the photoreceiver outputs were digitized with a two-channel analog-to-digital converter at a sampling rate of $10\mathrm{MHz}$ with $12\text{-}\text{bit}$ resolution.

2.2.

Full-Field Optical Coherence Microscopy System

FFOCM is an interferometric technique that utilizes 2-D parallel detection to provide subcellular resolution images of reflected light within biological specimens.^{12, 24} The FFOCM system used spatially incoherent broadband light from a xenon arc lamp to illuminate the sample and the reference mirror of a Linnik interference microscope using two identical $\mathrm{NA}=0.3$ water-immersion microscope objective lenses. Interference images were captured with a complementary metal oxide semiconductor (CMOS) area scan camera with a spectral response centered at $650\mathrm{nm}$ . The transverse resolutions were $2\mu \mathrm{m}$ and the axial resolution was $1.1\mu \mathrm{m}$ . Maximum penetration depth was typically $1\text{to}2\mathrm{mm}$ . Acquisition time was $2\mathrm{s}$ per frame for a transverse field of view of approximately $700\times 700\mu \mathrm{m}$ ( $512\times 512\text{pixels}$ per frame, 800 frames per volume). Three-dimensional data was obtained by moving the sample through the focus at $1\text{-}\mu \mathrm{m}$ increments. Volumetric rendering and visualization was accomplished by using OsiriX software.

2.3.

Spectrally Encoded Confocal Microscopy System

SECM is a reflectance confocal microscopy technique that uses near-infrared light, allowing deeper penetration into tissue,²⁵ compared with confocal microscopes that utilize visible light. SECM differs from conventional laser scanning confocal microscopy in that it projects different wavelengths onto distinct locations on the sample.¹⁵ Rapid acquisition of spectra returned from the sample enables high-speed reconstruction of the image. In the SECM system,¹⁴ light from a rapid wavelength tuning source in the near-infrared (center $\text{wavelength}=1.32\mu \mathrm{m}$ , instantaneous line $\text{width}=0.1\mathrm{nm}$ , total $\text{bandwidth}=70\mathrm{nm}$ , repetition rate up to $15.7\mathrm{kHz}$ ) was collimated onto a diffraction grating (1100 lines per mm) and focused through a 1.2 NA, $60\times$ objective (Olympus UPlanApo/IR $60\times ∕1.20\mathrm{W}$ ). A multimode fiber was used for signal collection, resulting in $0.9\text{-}\mu \mathrm{m}$ transverse and $2.5\text{-}\mu \mathrm{m}$ axial resolutions. Images comprised of $500\times 500\text{pixels}$ were acquired at $10\text{frames}\text{per}\text{second}$ . The maximum imaging depth was limited to the $280\text{-}\mu \mathrm{m}$ working distance of the objective lens.

2.4.

Resolution Measurements

For all imaging modalities (TDOCT, OFDI, SECM, and FFOCM), transverse resolution was measured by imaging a resolution target and calculating the full width at half maximum (FWHM) of the derivative of the edge response. Axial resolution was measured by scanning a mirror along the optical axis and determining the FWHM.

2.5.

Specimen Preparation, Ethanol Treatment, and Histology

Xenopus laevis frogs were purchased from Nasco (Fort Atkinson, Wisconsin). Animal procedures were performed according to the approved protocols of Massachusetts General Hospital Subcommittee on Research Animal Care. Embryos were obtained by in vitro fertilization, incubated in $0.1\times$ Marc’s modified Ringer’s medium (MMR),²⁶ and staged according to Nieuwkoop and Faber tables.²⁷

Ethanol treatments were performed in $0.1\times$ MMR (vol/vol), from the mid-blastula transition (MBT, stage 8.5),²⁸ until the time of imaging, typically after $5\text{to}7\text{days}$ at room temperature. All embryos were imaged repeatedly during their development. Aligning the embryos was performed by observing them through the dissecting microscope and TDOCT cross sectional imaging of the heart area. Prior to in vivo imaging, to prevent the embryos from swimming, they were anesthetized using 0.02% 3-aminobenzoic acid ethyl ester (A-5040, Sigma). For TDOCT and OFDI imaging, embryos were positioned on a 1.5% agarose gel plate with their ventral side facing up, covered by the anesthesia working solution. For imaging with the SECM system, embryos were placed on a cover slip, lying on their ventral side in an anesthesia buffer, and imaged from below. FFOCM was conducted on fixed embryos. Fixation was done in MEMFA [ $0.1\text{-}\mathrm{M}$ MOPS (pH7.4), $2\text{-}\mathrm{mM}$ EGTA, $1\text{-}\mathrm{mM}$ $\mathrm{Mg}\mathrm{S}{\mathrm{O}}_4$ , and 3.7% formaldehyde] for more than one hour. Prior to imaging, the fixed embryos were transferred into a Petri dish with $1\times$ phosphate buffered saline (PBS) (8-gr NaCl, 0.2-gr KCl, 1.44-gr ${\mathrm{Na}}_2\mathrm{H}\mathrm{P}{\mathrm{O}}_4$ , and 0.24-gr $\mathrm{K}{\mathrm{H}}_2\mathrm{P}{\mathrm{O}}_4$ ), with their ventral side facing up, supported by clay. Developmental stages and phenotypes were determined by R. Yelin.

Plastic histology sections²⁹ were obtained after additional fixation in Karnovsky’s Fixative (KII) and embedding in tEpon-812 (Tousimis). Sections $1\mu \mathrm{m}$ thick were cut on a Reichert Ultracut Microtome and stained with methylene blue/toluidine blue in borate buffer (Tousimis). Paraffin sections ( $5\mu \mathrm{m}$ thick) were stained with hematoxylin and eosin.

3.1.

High-Resolution Three-Dimensional Imaging by Full-Field Optical Coherence Microscopy in Fixed Embryos

FFOCM offers the capability to image the microstructure of the embryonic heart with nearly isotropic cellular level resolution in a fixed embryo. Volumetric FFOCM images spanned a transverse field of view of $700\times 700$ and $1000\mu \mathrm{m}$ in depth. The transverse and axial resolutions were 2 and $1.1\mu \mathrm{m}$ , respectively. Acquisition time was $2\mathrm{s}$ for a single en face section, and $33\min$ for the entire volume. FFOCM sections of the heart in a fixed Xenopus tadpole (stage 49) allow visualization of the ventricular trabeculae [Figs. 1a and 1c ], the spiral valve [Figs. 1b and 1d, arrows], and the partial atrial septum [Fig. 1d, arrow head]. Partially transparent volumetric rendering of the heart [Figs. 1e, 1f, 1g, 1h] reveals the looping-compression structure with the angled TA [Fig. 1e], the aortic arches [Figs. 1f and 1g], and the thin wall of the atrium [Figs. 1g and 1h], in their 3D context. Cutaway views of Fig. 1e show fine 3-D internal structures, including the trabeculae [Figs. 1i and 1j] and the atrioventricular valve [Fig. 1k]. A magnified view of the atrioventricular valve, shown [Fig. 1l] next to a corresponding histology section of the same embryo [Fig. 1m], demonstrates its bicuspid morphology.

Fig. 1

3-D imaging of Xenopus heart (stage 49) using FFOCM in a fixed embryo. En face sections show trabeculae (a) within the ventricle and (b) the spiral valve (valve marked by arrow). Cross sections at different locations along the anterior-posterior axis show (c) the ventricular trabeculae and cavity, (d) the TA and spiral valve (marked by arrow), the thin atrium wall, and the partial atrial septum (arrowhead). Partially transparent volumetric rendering of the heart (e) through (h) is shown at different viewing angles, revealing (e) the characteristic looping-compression structure and the angled TA, (f) the TA splitting into the paired aortic arches, the carotid arches (ca), systemic arches (sa), the pulmocutaneous arches (pa), and (g) and (h) the thin walled atrium. (i), (j), and (k) Cut away views through the 3-D dataset. The atrioventricular valve is shown in a magnified view [(l), marked by star] next to the corresponding histological section [(m); resin-embedded, $1\text{-}\mu \mathrm{m}$ sections, methylene blue stain]. v, ventricle; t, truncus arteriosus; a, atrium. Scale bars correspond to $100\mu \mathrm{m}$ .

3.2.

Heart Abnormalities Due to Ethanol Exposure

Cardiovascular malformation can be caused by genetic³⁰ and teratogenic factors.³¹ Ethanol is a well-known teratogen; exposure of human embryo during pregnancy to alcohol (ethanol) is associated with fetal alcohol syndrome (FAS).^{32, 33} One estimate suggests that 54% of the children with FAS have heart defects.³⁴ To study the teratogenic effect of ethanol on Xenopus heart development, embryos were exposed to different concentrations of ethanol (0.5 to 2.5% vol/vol) from the mid-blastula transition (stage 8.5)²⁸ until they were imaged. Siblings developing under the same conditions, but not exposed to ethanol, were used as controls. During the developmental process, we screened the heart area of 121 live embryos in an attempt to identify and qualitatively evaluate the extent of the teratogenic effect. To image the large number of embryos repeatedly and reliably, we used a dedicated TDOCT system that was incorporated into a dissecting microscope, which allowed simultaneous imaging with the two systems, with easy access for embryo manipulation (accurate orientation alignment). What appears as a complete maturation with a substantial change in morphology compared to the controls was found in a minority (25%) of embryos that were exposed to 1% ethanol $(\mathrm{n}=28)$ , and in a majority (74%) of embryos that were exposed to 1.5% ethanol ( $\mathrm{n}=27$ , as in 1Video 1 ). Grossly abnormal rotation of the heart tube and/or incomplete maturation was found in all embryos in the 2.0 and 2.5% groups ( $\mathrm{n}=17$ and $\mathrm{n}=7$ , respectively, as in 2Video 2 ). We did not observe morphologic differences between the 0.5% ethanol treated group ( $\mathrm{n}=16$ , as in 3Video 3 ) and the control group ( $\mathrm{n}=42$ , as in 4Video 4 ).

Video 1

TDOCT movie of a Xenopus heart area from the group treated with 1.5% ethanol, acquired in vivo. Imaging rate was $20\text{frames}\text{per}\text{second}$ (QuickTime; $1\mathrm{MB}$ ). 1.

Video 2

TDOCT movie of a Xenopus heart area from the group treated with 2.0% ethanol, obtained in vivo. Imaging rate was $20\text{frames}\text{per}\text{second}$ (QuickTime; $1\mathrm{MB}$ ). 2.

Video 3

TDOCT movie of a Xenopus heart area from the group treated with 0.5% ethanol, obtained in vivo. Imaging rate was $20\text{frames}\text{per}\text{second}$ (QuickTime; $1\mathrm{MB}$ ). 3.

Video 4

TDOCT movie of a Xenopus (stage 48) heart area from the control group, obtained in vivo. Imaging rate was $20\text{frames}\text{per}\text{second}$ (QuickTime; $1\mathrm{MB}$ ). 4.

Video 5

SECM movie of the spiral valve in a stage 47 Xenopus heart, obtained in vivo. Imaging rate was $10\text{frames}\text{per}\text{second}$ (QuickTime; $3.1\mathrm{MB}$ ). 5.

Video 6

SECM movie of the aneurismal dilatation on the TA wall of a stage 47 tadpole, acquired in vivo. Imaging rate was $10\text{frames}\text{per}\text{second}$ (QuickTime; $2.9\mathrm{MB}$ ). 6.

Video 7

4-D imaging of stage 49 Xenopus heart, obtained in vivo using OFDI (QuickTime; $1.2\mathrm{MB}$ ). 7.

Video 8

Cross sectional imaging of stage 49 Xenopus heart acquired in vivo using high-resolution OFDI. Imaging rate was $40\text{frames}\text{per}\text{second}$ (QuickTime; $3\mathrm{MB}$ ). 8.

Cardiac motion was evident in all embryos, even those with the most severe malformations. Using TDOCT, we selected one tadpole (stage 48) from each of the control, 0.5, 1.5, and 2.0% ethanol treated groups to demonstrate typical phenotypes (1Videos 1 through 44). We determined that the four tadpoles’ hearts were in advanced developmental stages by identifying the existence of a partial atrial septum and an atrioventricular valve (histology data not shown).

The TDOCT images provided the first indication of damaged looping in the 1.5 and 2.0% groups (1Videos 1 and 22). We furthermore observed lower TDOCT signal from within the ventricle in those groups.

Photographs of the tadpoles, taken in vivo from the ventral aspect, are shown in Figs. 2a, 2b, 2c, 2d . The four embryos were then fixed and imaged with FFOCM to evaluate fine structural malformations. 3-D rendering of FFOCM data allowed evaluation of myocardial structure at high resolution, revealing the similarity between the control and the 0.5% tadpoles, and clearly showing defective heart tube looping in tadpoles from the 1.5 and 2.0% groups [Figs. 2e, 2f, 2g, 2h]. Based on sections from the FFOCM datasets, we measured the TA dimensions in each of the four representative embryos and found a decrease in maximum diameter of approximately $(20\pm 9)\%$ in the 1.5% embryo and $(66\pm 15)\%$ in the 2% embryo, when compared to the control and 0.5% groups. The smaller distorted TAs and spiral valves (marked by arrows) are shown in the 1.5% [Fig. 2k] and 2.0% embryos [Fig. 2l] compared with the control [Fig. 2i] and the 0.5% [Fig. 2j] embryos.

Fig. 2

Abnormal heart formation due to ethanol exposure. (a) through (d) Ventral-view photographs of the tadpoles, obtained in vivo. (e) through (h) 3-D renderings of the heart area using FFOCM in fixed embryos. (i) through (l) En face FFOCM sections through the volumetric datasets at the level of the spiral valve (marked by arrows). (m) through (p) Cross sections through the ventricle. (q) through (t) Corresponding histological sections (paraffin, H and E). v, ventricle; t, truncus arteriosus; a, atrium; tr, trabeculae; frame width in (a) through (d) is $2.5\mathrm{mm}$ . Scale bars correspond to $100\mu \mathrm{m}$ .

Pericardial edema was present in the 1.5 and 2.0% groups [Figs. 2k, 2l, 2o, 2p] compared with the control and 0.5% groups. Ethanol also affected the ventricle. The developed trabeculae in the control [Fig. 2m] and 0.5% [Fig. 2n] hearts contrast the less developed trabeculae in the 1.5% group [Fig. 2o] and the large ventricular cavity with sparse, stunted trabeculae in embryos exposed to 2.0% ethanol [Fig. 2p]. Corresponding histological sections confirmed some of our findings, including the less developed trabeculae [Figs. 2q, 2r, 2s, 2t] in embryos with the greater ethanol exposure.

3.3.

High-Resolution Imaging of the Embryonic Heart with Spectrally Encoded Confocal Microscopy In Vivo

To obtain high-resolution images within the Xenopus heart in vivo, we have used SECM that allows the imaging of both structure and dynamics with microscopic resolution. SECM provides a transverse resolution comparable to FFOCM, but at much higher frame rates, enabling microscopy of the heart in vivo. The Xenopus myocardium (stage 49) was imaged with SECM in vivo at a frame rate of $10∕\mathrm{s}$ , a transverse field of view of $220\times 220\mu \mathrm{m}$ , and transverse and axial resolutions of 0.9 and $2.5\mu \mathrm{m}$ , respectively. The maximum penetration depth was $280\mu \mathrm{m}$ . SECM images show the thin cusps of the atrioventricular valve [Fig. 3a ], approximately $280\mu \mathrm{m}$ below the ventral surface, and parts of the ventricle and TA [Fig. 3c], containing individual blood cells within the trabecular spaces. SECM images correlated well with corresponding histology sections [Figs. 3b and 3d]. A series of frames from a different tadpole (stage 47) demonstrates the spiral valve as it closes [Fig. 3e] and opens [Figs. 3f and 3g], regulating blood flow, seen at the single-cell level, from the TA to the aortic bifurcation (5Video 5 ). Blood cells are also apparent between the trabeculae [Fig. 3h], and smaller, bright features that may represent nuclei and organelles can be observed.

Fig. 3

High-resolution confocal imaging in vivo using SECM. En face SECM images showing the atrioventricular valve [(a), marked by star], the ventricular trabeculae, and the TA (c) are compared with plastic histological sections [(b) and (d); resin-embedded, $1\text{-}\mu \mathrm{m}$ sections, methylene blue stain]. The regions of interest in (b) and (d), marked by the dotted rectangles, correspond to the approximate fields of view of the SECM images in (a) and (c), respectively. (e), (f), and (g) A series of three SECM images demonstrate the opening of the spiral valve, allowing the flow of blood cells to the aortic bifurcation. Arrows mark the flow direction. (h) Trabecular microstructure within the ventricle. Stars mark valve locations. v, ventricle; t, truncus arteriosus; s, spiral valve; b, blood cells. Scale bars correspond to $50\mu \mathrm{m}$ .

3.4.

Aneurismal Dilatation in the Xenopus Embryo

In one of the embryos (stage 47), we noticed a protrusion emanating from the TA wall. SECM sections obtained in vivo at two different depths [Figs. 4a and 4b ], taken from 6Video 6 reveal its saccular shape, its location with respect to the spiral valve, as well the flow of individual blood cells through the defect. This abnormality was also observed in vivo with TDOCT [Fig. 4a, inset]. The embryo was then fixed and imaged with FFOCM. An FFOCM section [Fig. 4c] and a 3-D volumetric rendering of the dataset [Fig. 4d] shows the dilatation in the context of the entire heart. Difficult to see under conventional brightfield microscopy [Fig. 4e], but clearly visualized with TDOCT, FFOCM, and SECM, this protrusion may represent a saccular aneurismal dilatation of the TA in a heart that otherwise appeared to have a normal phenotype.

Fig. 4

Aneurismal dilatation in the Xenopus heart (stage 47). (a) SECM image, obtained in vivo, shows the presence of blood cells within the protrusion. (b) A deeper SECM section reveals the location of the protrusion relative to the spiral valve. TDOCT was used to confirm the abnormality in vivo [(a), inset, arrow]. (c) FFOCM en face section, obtained in fixed embryo, and (d) 3-D rendering of the FFOCM data, show additional views of the aneurysm. (e) Brightfield photo showing ventral view of the tadpole (frame $\text{width}=2\mathrm{mm}$ ). The aneurismal defect is marked by arrows. v, ventricle; t, truncus arteriosus; a, atrium; s, spiral valve. Scale bars correspond to $100\mu \mathrm{m}$ .

3.5.

Optical Frequency Domain Imaging Allows Real-Time Four-Dimensional Imaging of the Xenopus Tadpole Heart

Direct imaging of heart dynamics in the whole mount embryo, in situ, including valve activity and myocardial motion, can help to elucidate heart function and the etiology of failure. While FFOCM and SECM are too slow, in their current implementation, to acquire the 3-D volume of the heart in real time, OFDI allows 4-D imaging without using gating or synchronization techniques.^{19, 20} OFDI images of the Xenopus heart (stage 49) were acquired in vivo at a rate of $900\text{frames}\text{per}\text{second}$ (fps), while the OFDI image plane was scanned through the heart at a rate of $20\mathrm{Hz}$ . Volumetric OFDI images were therefore obtained at a rate of 20 3-D datasets per second [Figs. 5a, 5b, 5c, 5d ]. The heart beat rate was measured to be approximately $2\mathrm{Hz}$ , resulting in ten volumetric datasets captured during a single cardiac cycle. A 4-D rendering of the myocardial dynamics is shown in 7Video 7 . At end systole, OFDI demonstrated that the ventricle was at its smallest volume; the volumes of the atrium and truncus arteriosus (TA) were conversely at their maxima [Figs. 5a and 5c]. At end diastole, the ventricle was dilated to its greatest volume, whereas the volumes of the atrium and TA were at their minima [Figs. 5b and 5d].

3.6.

High-Resolution Optical Frequency Domain Imaging of the Embryonic Heart In Vivo

To increase OFDI resolution, a novel broadband $\left(200\mathrm{nm}\right)$ wavelength-swept source²³ was used to obtain cross sections of a stage 49 Xenopus heart, in vivo. Compared to the $15\text{-}\mu \mathrm{m}$ transverse and $10\text{-}\mu \mathrm{m}$ axial resolutions of the previously described OFDI, the transverse and axial resolutions of high-resolution OFDI were 2 and $4\mu \mathrm{m}$ , respectively. Imaging speed was $40\mathrm{fps}$ .

Details within the three-chamber Xenopus heart can be clearly resolved in the high-resolution OFDI movie (8Video 8 ), including atrioventricular valve dynamics [Figs. 5e, 5f, 5g]. Individual blood cells can also be seen flowing from the atrium to the ventricle through the atrioventricular valve [Fig. 5g]. The ventricular trabeculae [Fig. 5h] and the spiral valve [Fig. 5j] structure and dynamics are clearly resolved with the high-resolution OFDI. For comparison, we present FFOCM sections [Figs. 5i and 5k] of corresponding areas from embryos at similar developmental stages.

Fig. 5

Imaging of Xenopus laevis hearts (stage 49) in vivo using OFDI. (a) to (d) High-speed OFDI 4-D imaging is presented through displacement color maps, revealing myocardial motion within a single cardiac cycle. The $900\text{-}\mathrm{fps}$ imaging rate allows in vivo real-time 3-D imaging at a rate of $20\text{volumes}\text{per}\text{second}$ (vps). (e), (f), and (g) High-resolution OFDI shows a series of frames of the atrioventricular valve (e) as it closes and [(g), valve marked by arrows] opens. Different anatomic structures within the heart can be clearly resolved, including (h) the ventricle with the trabeculae and [(j), spiral valve marked by arrow] the spiral valve within the TA. FFOCM images of the areas of the ventricle and the spiral valve are shown in (i) and [(k), spiral valve marked by arrow], respectively. v, ventricle; t, truncus arteriosus; a, atrium. Scale bars correspond to $100\mu \mathrm{m}$ .