Here, the experimental protocols are described for preparing Drosophila at different developmental stages and performing longitudinal optical imaging of Drosophila heartbeats using a custom optical coherence microscopy (OCM) system. The cardiac morphological and dynamical changes can be quantitatively characterized by analyzing the heart structural and functional parameters from OCM images.
Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical coherence microscopy (OCM) has been demonstrated to be capable of imaging small animal hearts with high spatial resolution and ultrahigh imaging speed. The high image contrast and noninvasive properties make OCM ideal for performing longitudinal studies without requiring tissue dissections or staining. Drosophila has been widely used as a model organism in cardiac developmental studies due to its high number of orthologous human disease genes, its similarity of molecular mechanisms and genetic pathways with vertebrates, its short life cycle, and its low culture cost. Here, the experimental protocols are described for the preparation of Drosophila and optical imaging of the heartbeat with a custom OCM system throughout the life cycle of the specimen. By following the steps provided in this report, transverse M-mode and 3D OCM images can be acquired to conduct longitudinal studies of the Drosophila cardiac morphology and function. The en face and axial sectional OCM images and the heart rate (HR) and cardiac activity period (CAP) histograms, were also shown to analyze the heart structural changes and to quantify the heart dynamics during Drosophila metamorphosis, combined with the videos constructed with M-mode images to trace cardiac activity intuitively. Due to the genetic similarity between Drosophila and vertebrates, longitudinal study of heart morphology and dynamics in fruit flies could help reveal the origins of human heart diseases. The protocol here would provide an effective method to perform a wide range of studies to understand the mechanisms of cardiac diseases in humans.
Longitudinal study of the heart in small animals contributes to understanding a variety of human related cardiovascular diseases, such as gene related congenital heart defects1,2. In the past decades, various animal models, such as mouse3,4, Xenopus5,6, zebrafish7,8, avian9, and Drosophila10-16, have been used to conduct the human heart-development related research. The mouse model has been widely used to study normal and abnormal cardiac development and cardiac defect phenotypes due to its similarities with the human heart3,4. The Xenopus embryo is especially useful in the study of heart development due to its easy handling and partial transparency5,6. The transparency of the embryo and early larva of the zebrafish model allows for easy optical observation of cardiac development7,8. The avian model is a common subject of developmental heart studies because the heart can be easily accessed after removing the eggshells and the morphological similarity of avian hearts to humans9. The Drosophila model has some unique features which make it ideal for performing longitudinal studies of the heart. First, the heart tube of Drosophila is ~ 200 µm below the dorsal surface, which provides convenience for optical access and observation of the heart. Additionally, many molecular mechanisms and genetic pathways are conserved between Drosophila and vertebrates. The orthologs of over 75% of human disease genes were found in Drosophila, which have made it widely used in transgenic studies11,13. Furthermore, it has a short life cycle and low maintenance costs, and has been commonly used as a specimen model for developmental biology research14-16.
Previous reports described the protocols for monitoring Drosophila cardiac functions such as the heartbeat. However, dissection procedures were required17,18. Optical imaging provides an effective way to visualize cardiac development in animals due to its non-invasive nature. Different optical imaging modalities have been applied in performing animal cardiac study, such as two-photon microscopy19, confocal microscopy20,21, light sheet microscopy22, and optical coherence tomography (OCT)16,23-26. Comparatively, OCT is capable of providing great imaging depth in small animal hearts without using contrast agents, while keeping a high resolution and an ultrahigh imaging speed, which are important for imaging live animals. Additionally, the low cost of developing an OCT system has popularized this technique for optical imaging of specimens. OCT has been successfully used for the longitudinal study of Drosophila. Using OCT, cardiac morphological and functional imaging has been performed to study the heart structures, the functional roles of genes, and the mechanisms of cardiovascular defects in mutant models during cardiac development. For example, age-dependent cardiac function decline was confirmed with down-regulated angiotensin-converting enzyme-related (ACER) gene in Drosophila with OCT27. Phenotyping of gene related cardiomyopathy was demonstrated in Drosophila using OCT28-33. Research using OCT also revealed the functional role of the human SOX5 gene in the heart of Drosophila34. Compared with OCT, OCM uses an objective with a higher numerical aperture to provide better transverse resolution. In the past, the heart dysfunction caused by silencing an ortholog human circadian gene dCry/dClock has been studied using a custom OCM system15,16, as well as the effect of high-fat-diet on cardiomyopathies in Drosophila to understand obesity induced human cardiac diseases.15
Here, the experimental protocol is summarized for longitudinal study of the cardiac morphological and functional changes in Drosophila at second instar (L2), third instar (L3), pupa day 1 (PD1), pupa day 2 (PD2), pupa day 3 (PD3), pupa day 4 (PD4), pupa day 5 (PD5), and adult (Figure 1) using OCM to facilitate study of human-related congenital cardiac diseases. Cardiac functional parameters, such as HR and CAP were quantitatively analyzed at different developmental stages to reveal the cardiac development features.
1. Preparation of OCM System for Optical Imaging of Drosophila16
2. Drosophila Culture
3. Performing Optical Imaging with OCM
4. Imaging Analysis16
The longitudinal cardiac imaging was conducted using the fruit flies with the 24B-GAL4/+ strain at room temperature with OCM. Measurements were performed at L2, L3, and at 8 hr intervals from PD1 to PD4, and adult day 1 (AD1) to track the metamorphosis process (Table 1). Larva, early pupa, late pupa and adult flies were mounted on the glass slides as seen in Figure 1A. The segment features of the heart for larval and adult flies were shown in the schematic representations in Figure 1B.
In this developmental study, 4,096 frames were acquired in 32 sec with our custom OCM system to trace the heartbeat of a fruit fly. To improve measurement accuracy, five repeated measurements were taken for each specimen at each developmental stage. 3D data can also be obtained to observe the heart structure changes during metamorphosis.
Transverse M-mode and 3D images were created with custom Matlab programs and ImageJ. En face images and axial sections were also constructed from the acquired data to visualize the remodeling process of the heart during Drosophila metamorphosis (Figure 2). To quantify the cardiac function of fruit flies, the heart region was automatically segmented using a custom Matlab program from all 4096 frames. The fly heart rate (HR) can be quantified from the transverse M-mode OCM images (Figure 3a). During pupal stages, the Drosophila heart stops beating occasionally16. We introduced a new cardiac functional parameter, cardiac activity period (CAP) to quantify the ratio of the period with a heartbeat to the total imaging time (Figure 3b). EDD, ESD, EDA, ESA, and FS were also used to quantify the heart chamber changes in both axial and transverse dimensions during Drosophila development.16
At larval stages, the heart tube begins at the posterior abdominal region A8 with a broader lumen (A5 – A8 in Figure 1B) and ends at the anterior dorsal segment A1 with a narrower diameter (T3/A1 – A5 in Figure 1B). The heart chamber was located medially and dorsally and grew bigger during L2 (Figure 2 a, b) and L3 (Figure 2 c, d). After entering PD1, the heart tube was observed running axially over the top of a moving air bubble (Figure 2 e, f). Around 10 – 13 hr later, the bubble disappeared after puparium formation and the broad lumen became everted. Since the anterior heart tube was ventrally located, the whole heart tube was invisible except the posterior region in the OCM images ~12 hr after puparium formation. Later during PD2, the heart chamber gradually aligned along the dorsal abdomen, and the posterior part (A6 – A8) of the heart was eliminated (Figure 2 g, h)42,43. A conical chamber started to develop ~ A1 – A4 segment during PD2 and grew in size until the adult stage (Figure 2 i – m).
Besides observing structural changes, many functional changes were found as well during cardiac remodeling. The M-mode images shown in Figure 3 demonstrate that the heartbeat slowed down significantly from the larval stage to the pupal stage, and then increased substantially from pupa to adult. Significant HR changes were observed during the lifecycle (Figure 4a). Furthermore, the cardiac activity period (CAP) was analyzed for all the specimens measured from L2 to AD1 (Figure 4b). As shown in Figure 4, HR holds at ~ 277 beats per min (bpm) for L2 and L3. Upon entering the early pupal stages there is a marked decrease in HR and CAP. HR is reduced to 86 ± 11 bpm at the beginning of PD1, and continues to decrease to 26 ± 8 bpm by the end of PD1 finally coming to a complete stop early in PD2. One interesting discovery is the extended period of cardiac inactivity observed around PD2 stage (~ 24 hr – 48 hr after puparium formation), referred to as cardiac developmental diastalsis16. At the end of PD2, slow intermittent beating resumes (HR 17 bpm ± 6 with CAP 5 ± 2). Throughout PD3 and PD4, HR and CAP increase until reaching 392 ± 32 bpm and 95 ± 3 % at the first day of the adult stage (5 days after beginning the pupal stage).
Figure 1. Mounting of Drosophila at Different Stages and Schematic Representation of Heart Metamorphosis. (a) Mounting of larval, pupal, and adult WT (24B-GAL4/+) flies on glass slides. (b) Schematic representation of heart metamorphosis. Red arrows on larva and adult schematic denote the OCM M-mode imaging locations until PD1 24 hr and for subsequent time points, respectively. Please click here to view a larger version of this figure.
Figure 2. Drosophila Heart Morphological Changes. En face and axial sectional OCM images of a WT Drosophila obtained at (a, b) L2 (c, d) L3 (e, f) PD1 (g, h) PD2 (i, l) PD4 and (k, m) adult stages. M-mode images of the Drosophila heart were obtained from the A7 segment until PD1 and from A1 segment for later stages. The scale bars in en face and axial sectional images denote 200 µm and 500 µm, respectively. Please click here to view a larger version of this figure.
Figure 3. Drosophila Heart Functional Changes. (a) M-mode images at different developmental stages showing HR changes across lifecycle. (b) Examples demonstrating cardiac activity period (CAP) calculation. Please click here to view a larger version of this figure.
Figure 4. Quantitative Analysis of Functional Cardiac Parameters in WT Flies at Different Developmental Stages, including L2, L3, Pupal Stages at 8 hr intervals, and AD1. (a) HR. (b) CAP. The error bar of each group represents the standard deviation. Please click here to view a larger version of this figure.
Developmental Stage | |||||||||||||||||
L2 | L3 | PD1 | PD2 | PD3 | PD4 | AD1 | |||||||||||
8 hr | 16 hr | 24 hr | 32 hr | 40 hr | 48 hr | 56 hr | 64 hr | 72 hr | 80 hr | 88 hr | |||||||
Specimen Number | 21 | 17 | 13 | 19 | 19 | 19 | 19 | 19 | 19 | 18 | 17 | 18 | 9 | 25 |
Table 1. Number of WT Fruit Flies Measured at Various Developmental Stages in the Cardiac Developmental Study.
Video 1. Tracking of Heartbeat Along Temporal Dimension and Corresponding Heart Chamber Diameter Change along the z Direction (axial direction) in a WT fly at L2. The heart was beating relative fast at a steady rate. Please click here to view this video. (Right-click to download.)
Video 2. Tracking of Heartbeat along Temporal Dimension and Corresponding Heart Chamber Diameter Change along the z Direction (axial direction) in a WT fly at PD1. The HR started to decrease. Please click here to view this video. (Right-click to download.)
Video 3. Tracking of Heartbeat along Temporal Dimension and Corresponding Heart Chamber Diameter Change along the z Direction (Axial Direction) in a WT fly at PD2. The heart stopped beating completely during the time. The oscillation of plotted z-diameter was due to the imaging noise. Please click here to view this video. (Right-click to download.)
Video 4. Tracking of Heartbeat along Temporal Dimension and Corresponding Heart Chamber Diameter Change along the z Direction (Axial Direction) in a WT fly at PD4. After PD2, the HR and CAP started to increase. Please click here to view this video. (Right-click to download.)
Video 5. Tracking of Heartbeat along Temporal Dimension and Corresponding Heart Chamber Diameter Change along the z Direction (Axial Direction) in a WT Fly at AD1. The HR was the highest among all stages and CAP was almost 100%. Please click here to view this video. (Right-click to download.)
Video 6. 3D structural rendering of a larval fly. Please click here to view this video. (Right-click to download.)
The rapid heartbeat of Drosophila, with a maximum HR around 400 bpm at larval and adult stages, requires high imaging speed to resolve the heart diastoles and systoles (no less than 80 frames/sec based on experiences). Due to the small heart chamber size and micron scale heart wall thickness (5 – 10 µm), a high spatial resolution (better than 2 µm) is required for resolving the heart tube structures. In this study, a high resolution and ultrahigh speed OCM system was developed, where a spectrometer with 600 lines/mm transmission grating and a 2,048 pixel line-scan camera were used. An A-scan rate of 20 kHz is provided by the line-scan camera. The frame rate of 128 frames/sec is fast enough to capture the Drosophila heartbeat at multiple developmental stages, including L2, L3, PD1, PD2, PD3, PD4, PD5, and adult. The light source was a broad bandwidth supercontinuum light source with a central wavelength and bandwidth of ~ 800 nm and ~ 220 nm respectively and obtained an axial resolution of ~ 1.3 µm in tissue. A 10X objective was used in the sample arm to realize a transverse resolution of ~3.9 µm. Since the heart tube of Drosophila is around 200 µm below the dorsal surface, an imaging depth of hundreds of micron meters is required. A 45° rod mirror can be utilized to generate an annular sample beam and extend the depth of focus in the specimen44. The sensitivity and 3 dB roll-off were determined to be 96 dB and 600 µm, respectively with the sample arm power of ~ 9 mW. A custom computer program was used to control the OCM system and conduct the measurements. The cardiac structural images and functional parameters obtained demonstrate the feasibility of using OCM to quantitatively characterize the heart morphology and function of Drosophila throughout its whole lifecycle.
Currently, several other techniques are also used to image a small animal's heart structure or function, such as computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound. OCM provides higher spatial and temporal resolutions than these techniques, enabling visualization of fine structures and fast dynamics in animal hearts. Confocal microscopy is another widely used imaging technique, but its low imaging penetration and requisition of imaging contrast agents limit its applications in live animals. Comparatively, OCM enables high-speed and label-free imaging for visualizing fast cardiac dynamics non-invasively in small animals. However, there are still limitations of OCM. For example, the imaging depth provided by OCM is limited by light scattering from several hundred microns to about 1 mm in tissue while ultrasound has penetration depths of up to 10 cm. Compared to confocal microscopy, OCM has a higher speed and better imaging depth, but with lower resolution and poor molecular contrast. Furthermore, our current OCM system is based on spectral-domain detection systems. Higher imaging speed based on swept-source OCM45 may provide more distinct images of rapid dynamics like the heartbeat.
To perform longitudinal study of the heartbeat in Drosophila with OCM, there are several critical steps in the protocol. The flies must be handled very delicately at all stages of the experiment. Managing larva should be especially gentle since it is easy to damage the larva, which could affect heart structure and function in the following developmental stages. The flies must be positioned on the cover glass and the imaging stage very precisely. Poorly positioned flies will make it difficult to acquire quality images and may cause skewed structural and functional heart parameter values. Additionally, transferring adult flies from one tube to another and plugging the cotton ball should be very fast to prevent their escape from the tube.
Different studies on Drosophila heart development can be performed by modifying the protocol. The temperature at which the flies are cultured can be increased or decreased from 25 °C to alter the cardiac gene expression level and change the fly development period. By adding some ingredients such as coconut oil or ATR to the standard food, the heart development may be altered. Specific studies can be conducted in wild type or transgenic flies. When studying fruit fly heart development longitudinally, different time intervals can be used to perform the OCM measurements, for example, an 8 hr interval could be used during the pupal stages. Due to limited sensitivity of our OCM system, a lot of uniform speckle noise is found in the transverse M-mode images, which can make it difficult to correctly identify heart contraction signals with Matlab programs and decrease the efficiency of data analysis. Sensitivity can be increased by improving the alignment of the OCM system. Optimized filtering algorithms are recommended to remove a portion of the speckles.
The described protocol has been applied to study the silence of human circadian orthologs, dCry and dClock induced cardiac defects in Drosophila. Decreased HRs were observed at different developmental stages, including larva, pupa, and adult15,16. The role of circadian genes in heart development was revealed, which may explain the association between cardiovascular disorders and circadian rhythm related activity patterns. High-fat-diet (HFD) induced cardiac disorders were also studied by analyzing heart functional changes of fruit flies fed with HFD15. These studies demonstrated not only Drosophila as a powerful tool in the developmental study of heart structure and function, but also the significance of cardiac longitudinal study in understanding congenital and postnatal human diseases. The OCM platform will enable a wide range of future studies in gene related human cardiac disease.
The authors have nothing to disclose.
This work was supported by the Lehigh University Start-Up Fund, the NIH (R00EB010071 to C.Z., R15EB019704 to C.Z. and A.L., R03AR063271 to A.L., and R01AG014713 and R01MH060009 to R.E.T.), the NSF (1455613 to C.Z. and A.L.), the Cure Alzheimer’s Fund (to R.E.T.), and the Massachusetts General Hospital (Executive Committee on Research Award to A.L.). M.C. and Y.M. was supported by the National Key Basic Research Program of China (973 Program) under Grant No. 2014CB340404.
Custom OCM imaging system | Developed in our lab | ||
my Temp Mini Digital Incubator | Benchmark | H2200-HC | |
Cover glass | AmScope | 200PCS | |
Cotton Ball | RITE AID | ||
Instant Drosophila Formula | CAROLINA | formula 4-24 | |
Yeast | ActiveDry | ||
Microscope | SONY | WILD M420 | |
Brush | Loew-Cornell | 245B | being used to move specimens |
Labview software | National Instruments | ||
Image J | National Institutes of Health | ||
Matlab | Mathworks | ||
Tweezer | Wiha | AA SA | to fix the fruit fly wings |
FlyNap | Carolina Biological Supply Company | 4,224,898 | |
Scotch Permanent Double Sided Tape, 3M | Scotch | ||
Pipette | Fisherbrand | MU18837 | |
Organic Extra Coconut Oil | Spring Valley | 13183 | |
Microscope Slide | CapitolBrand | M3504-E | |
Drosophila Vials | SEOH | 8401SS | |
All-trans-retinal | Sigma-Aldrich Co. | R2500 |