We propose ultra-high resolution optical coherence tomography to study the morphological development of internal organs in medaka fish in the post-embryonic stages at micrometer resolution. Different stages of Japanese medaka were imaged after hatching in vivo with an axial resolution of 2.8 µm in tissue. Various morphological structures and organs identified in the OCT images were then compared with the histology. Due to the medaka’s close resemblance to vertebrates, including humans, these morphological features play an important role in morphogenesis and can be used to study diseases that also occur in humans.
© 2015 Optical Society of America
Medaka, Oryzias lapties is a small fresh water fish inhibiting the rice fields and ponds throughout Japan. Along with the zebrafish, the medaka has become a popular model organism, for the study of early developments and in understanding the elements involved in organogenesis . Due to its close resemblance to vertebrates in genetics and organogenesis, the medaka has been used in various studies on diseases that also occur in humans. Their short generation time of 2-3 months and the transparency of its eggs make it easy to follow their early development and they are easily grown in a laboratory environment. Most of the studies follow the growth of different organs of the medaka in the embryo . Stereo and confocal microscopy are commonly used tools to study the early developmental stages, utilizing the transparency of the embryo. Changes in internal organs continue after hatching in the form of post-embryonic developments and metamorphosis . Moreover, the influence of environmental factors also affects the morphology and growth of certain organs in the juvenile and adult stages [4–6]. Morphological features of internal organs in medaka post hatching that have been reported earlier are presented below.
Cubbage et al.  and Renn et al.  studied the skeletal morphogenesis in medaka to understand the ossification (which is the process of transforming the cartilage tissues to bone tissue), which could give better insights into the ossification of human fetal bones. The stages of ossification of neurocranium and the viscerocranium were also studied and reported . Langille et al. studied the development of the skeletal bones at each stage of medaka in detail  and showed the effect of removing the neural crest (24 hours post fertilization) on the morphological distortions in fish at later stages .
Ishikawa et al. have shown that brain morphology is different for different species of medaka  with a specific strain having a larger optic tectum and smaller telencephalon compared to other strains. Morphogenesis of optic tectum  and cerebellum  has been investigated in detail. Finn et al. studied the effect of chemicals on heart rate and heart development . Barrionuevo et al. have monitored the heart rate of zebrafish and the consumption of oxygen as a function of temperature from embryo to adult stage . Iwamatsu et al.  has reported about the growth of the dorsal aorta, dorsal vein and ventral vein that run along the vertebral column and also about the shifts in position of these blood vessels as the medaka grows.
Germ cells in medaka continue to develop after hatching and the development has been studied with fluorescent microscopy using green fluorescent protein (GFP) markers . There is also much interest in the genetic control of sex determination as medaka was the first vertebrate in which cross over between X and Y chromosomes was demonstrated [1,15]. The genes responsible for the possession of both oral and pharyngeal teeth in medaka have been studied in detail to understand evolution of feeding and interspecies comparison .
These studies demonstrate that there is a need for monitoring the morphological features of internal organs from post-hatching until the adult stage. However, the medaka loses most of its transparency when it grows. The tools that aided the developmental study in the embryonic stage might not perform well in the post-embryonic stage due to a lack in transparency. The most popular method for studying post-embryonic development in medaka is by dissecting, fixing, and staining the fish and then studying it using electron microscopy or confocal microscopy. Fluorescent microscopy is also used to image the post-embryonic stages but only on GFP mutated medaka strain. Techniques such as magnetic resonance microscopy  do not have a resolution sufficiently high enough to resolve features in internal organs of the fish. Therefore another imaging tool is necessary to study the morphological changes and development of internal organs in vivo in the post-embryonic stages.
Optical coherence tomography (OCT) has proven itself to be successful in in vivo three-dimensional (3D) imaging of biological samples, and as a non-invasive imaging modality it could therefore complement existing imaging modalities to study the fish in its post-embryonic developmental stage in vivo and without mutation. The very small coherence gate in ultra-broad band spectral-domain OCT (UHR-SD-OCT) systems allows for resolving structures up to a few micrometers in the axial direction [18,19] and usage of microscopic objectives provides micrometer resolution in the lateral direction. These properties have been exploited for the study of small animals like drosophila and zebrafish for full animal imaging. Iftimia et al. have reported imaging of heart, notochord, mouth, retina and blood flow in zebrafish using a dual beam OCT with a bandwidth of 62 nm, which gave an axial resolution of 5.4 µm in air . Rao et al. could successfully resolve anatomic features such as telencephalon, tectum opticum, eminentia granularis and cerebellum in the zebrafish brain . Degeneration and regeneration of photoreceptors in the adult zebrafish retina have been studied by Weber et al. at an axial resolution of 3.2 µm . Vishal et al. has imaged the cornea of an Indian mourala fish eye in vitro with a wide field white light OCT system . Similar studies on adult mouse retina, embryonic avian heart and drosophila heart have been reported [24–29]. An OCT image of the medaka embryo has been published by Davis et al , and to the best of our knowledge no such studies have been reported for medaka in the post-hatching stages.
In the following sections we will show how UHR-SD-OCT can be used to perform the above mentioned studies in vivo.
2. Material and methods
2.1 Sample preparation
The medaka strain, T5 (MT827) was supplied by the National BioResource Project (Medaka) (http://www.shigen.nig.ac.jp/medaka/). The fish were kept separately according to their stages in plastic aquariums at 26 - 28 °C and maintained on a 14 hour light and a 10 hour dark time schedule. Medaka were fed fish food twice a day. Before imaging with our OCT system fish were paralyzed by placing them on ice for 7-10 seconds in case of young fish i.e. 30 days post hatching (dph) or younger and 20 - 25 seconds in case of older fish (60 dph or 90 dph). Then the paralyzed fish was placed on a small petri dish filled with water and imaged. After imaging, fish were returned back to the aquarium where they became active again. We also performed histology on the fish for comparison with our OCT images. For histology the fish were fixed in Bouin’s solution overnight, dehydrated in a series of ethanol solutions and embedded in paraffin. Cross, sagittal, or coronal sections of the body were cut serially at 5 µm thickness and stained with heamatoxylin and eosin. Sections were observed carefully under a Leica MZ16F stereo microscope with transmitted light and were captured with an Olympus DP73 digital camera. All the histology and microscopic images presented in this paper are produced originally.
We developed an UHR-SD-OCT system with a center wavelength of 840 nm and bandwidth of 140 nm. Since light with a center wavelength of 1.3 µm is strongly attenuated by water, a central wavelength of 840 nm is a better choice for our purpose as the fish has to be imaged under water. The light source was a super luminescent diode from Superlum (BLMD-D-840-B-1-10) and for UHR-SD-OCT, the shorter central wavelength of this light source combined with its large bandwidth make this light source a better choice for our purposes than a standard SLD at 840 nm or a light source at 1.3 µm. The light was split into sample and reference arms at a ratio of 50:50 using a fiber coupler (AC photonics) as shown in Fig. 1. The sample arm consisted of a dual axis galvanometer scanner (Thorlabs) and an objective lens mount housed on an X translation stage with rotating platform, offering flexibility in illuminating the sample from various angles and also for changing the focus. Three types of objective lenses i) 60 mm (achromatic), ii) 36 mm (telecentric objective) and iii) 10x microscope objective were used for the experiments. The reference arm consisted of a lens and mirror assembly mounted on a translation stage. The custom-built high-speed Cobra spectrometer (Wasatch photonics) contained a collimating lens (f-115 mm) for collimation and a 1045 line/mm grating at an incident angle of 26.75 deg for dispersing the light. An f-150 mm multi-element focusing lens was used to focus the dispersed light onto the central 2048 pixels of a line scan camera (Basler SPL-4096-140k).
The axial resolution of the system was measured on a mirror in air and is equal to 3.9 µm. In tissue (n = 1.38) the coherence length is found to be 2.8 µm. The sensitivity roll-off was 3.85 dB/mm over the first 500 µm and 9.45 dB/mm over the first mm. The depth range of the system in air is 2.04 mm, as shown in Fig. 2. We used a beam size of 2.8 mm in the sample arm, which produced 1/e2 spot sizes of 20 µm, 12 µm and 6 µm when using the f-60 mm, f-36 mm and a 10x microscopic lens, respectively. The scan ranges were varied according to the area of interest by adjusting the voltage supplied to the galvanometer scanner. The maximum scan range for both f-60 mm and f-36 mm objective lenses was 12 mm. For the 10x microscopic objective lens, the maximum scan range was 5 mm. The integration time of the camera was fixed at 33 µs and the camera was operated at 27,000 A-scan/s. Each B-scan consists of 1000 A-scans and the galvanometer scanner and camera were synchronized to acquire 27 B-scans /s. The incident beam power at the sample arm was 5 mW and the sensitivity was 100.69 dB.
The acquisition and analysis program was developed in LabVIEW and was used to control the experiment. The acquisition program synchronized the galvanometer scanner with the camera through a data acquisition board and achieved a maximum line rate of 70 kHz. The analysis program performed background subtraction, spectral calibration, dispersion compensation, windowing, fast Fourier transform (FFT) and logarithmic scaling . All the above mentioned operations were done in real time. Each B-scan (cross sectional image) was saved as an image for visualization or further processing
3. Results and discussion
The images were taken at three different post-embryonic development stages, i) 30 dph, ii) 60 dph and iii) 90 dph. These stages were chosen in order to divide the life span of medaka from post-hatching to adult at equal intervals. The B-scans were made either vertically from head to tail or horizontally from fin to fin according to the area of interest. The images that were taken close to the heart were motion-artifact corrected using the ‘rigid body correction’ option present in the ‘Turboreg plugin’ for ImageJ software . Data was also rendered in 3D with Amira, FEI visualization sciences group.
3.1 Results – brain
Figure 3(A) shows the histology of 90 dph medaka where the brain features can be identified. Figures 3(B), 3(C) and 3(D) show the raw OCT B-scans of 30 dph, 60 dph and 90 dph medaka acquired using the 10x microscope objective and f-60 mm lens objective. Comparison of Fig. 3(A) with Figs. 3(B), 3(C) and 3(D) shows that the same features can be identified from the OCT images. The distinct areas of the brain like telencephalon, optic tectum, torus longitudinalis, torus semicircularis and cerebellum could be easily recognized. Media 1(Fig. 3(C)) shows the raw B-scans from a 60 dph medaka compared with a single horizontally sliced image of the same fish played at 15 frames per second (fps). Figure 3(E) shows the en face scan of all B-scans of a 30 dph medaka and its corresponding 3D rendered volume is shown in Media 2, which is found to be in agreement with the microscopic image of the dissected brain shown in Fig. 3(F).
3.2 Discussion – brain
It has been reported that the morphology of the brain is different for different genotypes of medaka , but the studies were done by first anaesthetizing the fish and then fixing the brain in a fixative solution such as para-formaldehyde and observing it through a stereomicroscope. Morphogenesis of optic tectum and cerebellum were studied separately using histology, where various stages and patterns in the development process were identified [12,33]. OCT could provide the freedom of doing those studies in vivo for different stages on the same fish. Moreover, the boundaries of the brain regions are visible in the OCT image, which are generally associated with proliferation zones and are considered as an important element in neurogenesis [34,35]. These zones are usually less than 10 µm in thickness and require ultra-high resolution imaging. OCT could therefore play an important role in the developmental and morphological studies of medaka brain.
3.3 Results – gonads
The gonads of a medaka can be identified from the histological slice shown in Fig. 4(A). A single B-scan in the OCT image of a 30 dph female medaka reveals the same features as seen from Fig. 4(B). From the comparison of Figs. 4(A) and 4(B) regions such as i) spinal cord along with the vacuolated tissue, ii) the abdomen with the digestive tract and iii) the gonads on one side can be identified. Figures 4(C) and 4(D) compare the histology of an adult female medaka at 90 dph with the corresponding OCT image (which is an average of 10 B-scans). Both mature and immature eggs in the ovary of the adult female fish can be seen. Immature eggs were also identified in the 60 dph medaka. Figures 4(E) and 4(F) compare the histology and OCT images of the testis of the male medaka at 90 dph. An average of 20 B-scans is required to identify the testis. Only a part of the testis (Fig. 4(F)) and a part of the matured egg (Fig. 4(D)) are visible in the OCT image due to the limited penetration depth of the light in the fully grown adult.
3.4 Discussion – gonads
Sex is determined in medaka only at a later stage (20-40 dph) and until then it has a bi-potential gonad which has the ability to either become male or female. The medaka also has the special ability of sex reversal (transition from male to female or vice versa) . These transitions are strongly influenced and triggered by genetic factors  and environmental factors  as the medaka grows. To understand these factors, monitoring the fish throughout its life cycle from juvenile to adult stage under the influence of the triggering factors is required. Moreover, GFP markers are extensively used to study the gonadal development using fluorescent microscope, but it is also reported that the GFP markers create a bias in the development of gonads in medaka . This issue can be circumvented by using OCT as an imaging tool. The obvious advantage is that the same fish can be followed over time, without the need for staining and dissection, while a GFP marker is not required. Moreover, identification of individual germ cells (which are a few microns in diameter) within the gonads could help in early sex determination . However, those microstructures could not be distinguished in our images due to poor contrast between the cells.
3.5 Results – vertebral column and blood vessels
Figure 5(A) shows the anatomy of a 30 dph medaka vertebral column imaged with a binocular microscope. Figure 5(B) shows a single horizontal sliced image from the 3D data and Fig. 5(C) is the en face image of the same data. The OCT image could resolve various developmental characteristics of vertebra such as neural arch, hemal arch, vacuolated tissue and centrum as can be seen in Figs. 5(B) and 5(C). These OCT images are in good agreement with the microscopic images from Fig. 5(A). Figure 5(D) is a single B-scan which shows the position of dorsal artery and ventral vein. From the media associated with the same figure (Media 3) we can see the shift in position of the artery and vein along the length of the vertebral column, which is played at 15 fps.
3.6 Discussion – vertebral column and blood vessels
The development of the vertebral column continues into the post-embryonic stage and this has been extensively studied both in zebrafish and in medaka. Such studies can be performed in vivo using OCT as it can resolve most of the anatomical features of the vertebral column. As shown in Figs. 5(B) and 5(C), both the inside of the vertebra and the outside structures can be seen in the OCT images. Deformations that occur in the vertebra of medaka  can be imaged post hatching with the OCT system. This was earlier demonstrated using a zebrafish embryo with standard OCT . The vacuolated tissue structures determine the development of notochord in the early stages of post-embryonic development . These structures, visible in Fig. 5(B) measure 5-10 µm and demand ultra-high resolution imaging. For a comparison with published work on notochord imaging with OCT, reconstruction with smaller bandwidths of 50 nm and 30 nm was done in a post processing analysis. Figure 5(E) shows an UHR-OCT image with 140 nm bandwidth where the vacuolated tissues can be easily identified as seen from Fig. 5(F). Figures 5(G) and 5(H) show the same image simulated with 50 nm and 30 nm bandwidths respectively, which demonstrates the necessity for ultra-high resolution imaging. An important feature that allows us to follow the development of circulatory system in medaka post-hatching is the shift in position of the ventral vein with respect to the dorsal aorta. A relationship between the total length of the fish and the distance of separation between the ventral vein and dorsal aorta has been reported by . Such developmental characteristics can be studied in vivo using OCT, where the shift in position of the blood vessels is visible as can be seen in Media 3. The position of dorsal aorta stays parallel to the vertebrae while the position of the ventral vein shifts. The flow of blood inside the blood vessels can also be seen.
3.7 Results – teeth and ear
Figure 6(A) shows a binocular microscope image of the oral jaw of a stained adult (90 dph) medaka with protruding canine-like teeth. Figure 6(B) shows a single B-scan of a 30 dph medaka, where the same canine-like teeth can be identified in the oral jaw. Figure 6(C) shows the dentary bone where small teeth are arranged in a single row along the middle part of the bone in another B-scan of a 30 dph medaka.
In Fig. 7(A) histology of a 30 dph fish is shown with pharyngeal bones (gills) and pharyngeal teeth. The same area has been imaged using OCT where the same features can be identified as shown in Fig. 7(B) and in the associated Media 4. OCT images close to the eye also reveal the morphological features of the ear which agrees with the histological image of the same area, as can be seen in Fig. 8.
3.8 Discussion - teeth and ear
Medaka has the peculiar feature of possessing both oral and pharyngeal teeth and can be compared with the pharyngeal teeth lacking species (mouse) or with the oral teeth lacking species (zebrafish) . This helps in understanding the responsible gene expressions i.e. the set of genes acting on oral or pharyngeal cavities during odontogenesis. This makes medaka a promising model for understanding the evolution of feeding and interspecies comparison as its genome is fully sequenced . But such studies are typically performed using histology.
3.9 Result - heart
Figure 9(A) shows the histology of a horizontal slice of a 30 dph medaka where the heart along with liver, gills and digestive tract can be identified. The same organs can be identified in the horizontal depth slice of the OCT B-scans as shown in Fig. 9(B). Motion artifacts created by the pumping heart obscure the heart and gills. Figure 9(C) shows the OCT image of the same heart from a single horizontal slice without motion artifacts. Figure 9(D) shows a single B-scan of a 30 dph medaka showing both the atrium and the ventricle along with the bulbus arteriosus. In Media 5, a video of the pumping action of the same heart (played at 12 fps) can be seen, where contraction and relaxation of the two chambers of the heart can be distinctively identified. By counting the cardiac cycles against the number of B-scans (27 scans/s) the heart rate of the medaka was measured to be 120 beats/minute.
3.10 Discussion - heart
External factors such as chemicals and temperature affect the development of the heart and also the heart rate in medaka. Exposure to propranolol (a beta-adrenergic antagonist chemical which inhibits the beta-adrenergic receptors in the heart that are responsible for contractility and heart rate) causes decreased heart rate and impairment in cardiac development in medaka . These studies could help in the development of better drugs to treat blood pressure related diseases in humans. It can also help to understand the genetic and external factors hindering normal heart development. However the reported studies have been done with medaka in the embryonic stages using microscopy. These studies can be extended to post-hatching and adult stages using OCT, where the medaka loses its transparency. Furthermore, the ability to image the pumping action of the heart offers a great benefit over other imaging modalities, which are often limited to ex vivo studies.
In our OCT images we successfully identified the morphological features of the brain such as telencephalon, optic tectum, cerebellum and other parts in all the three developmental stages (30, 60 and 90 dph). Moreover, the bi-potential gonad (30 dph) and the female gonad in both 60 and 90 dph and male gonad at 90 dph were identified as well. Regarding the vertebra and the blood vessels that run along it: both can been seen and even the shift of the blood vessels as the medaka grows can be tracked. Other organs such as the internal ear, both the oral and the pharyngeal teeth and the pumping heart were imaged as well. The imaging of vacuolated tissue structures in vertebrae, proliferation zones in brain and germ cells in the gonads demand ultra-high resolution for which the UHR-SD-OCT system was used.
The study of development and morphological features of internal organs of medaka using UHR-SD-OCT reveals vital information that cannot be provided with other imaging techniques that often require fixing and staining. Medaka resembles vertebrates, including humans, and can therefore be considered as a model organism for studies on diseases that affect humans. While some existing imaging tools perform well in in vivo developmental studies during the embryo stage, they often fail during the post-hatching stage, as the medaka loses its transparency. UHR-SD-OCT can therefore be used complementary to other available imaging techniques for developmental studies of Medaka fish.
The authors would like to thank Mr. Kawashima, Mr. Kamiyama, Mr. Takahashi and Mr. Nakayama from the mechanical engineering department for machining of parts for this project. This research is part of the Project for Bio-imaging and sensing at Utsunomiya University which is funded by the Ministry of Education, Culture, Sports, Science and Technology (MEXT). This work was also partly supported by MEXT KAKENHI Grant Number 23360026.
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