Name: drosophila preparation and longitudinal imaging of heart function in vivo using optical coherence microscopy (ocm)
Uploaded: 2016-12-12T12:30:00
Description: Watch this Scientific Journal Video about Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy OCM at JoVE.com

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.

1. Preparation of OCM System for Optical Imaging of Drosophila16
<ol>
	<li>Select a spectrometer and a high-speed line scan camera that provides a frame rate of at least 80 frame/sec so the OCM system will be able to resolve the heartbeat of Drosophila.</li>
	<li>Use a broadband light source to ensure the axial resolution of 2 &#181;m to identify the heart structure of Drosophila.</li>
	<li>Use a 10X objective to obtain a high transverse resolution.</li>
	<li>Use a 45&#176; rod mir...

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 &#181;m), a high spatial resolution (better than 2 &#181;m) is required for resolving the heart tube structures. In this study, a high resolution and ultrahigh speed OCM system was developed, where a spectrometer wit...

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 &#181;m 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.

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			<td height="42" style="height:42px;width:168px;">Custom OCM imaging system</td>
			<td style="width:136px;">Developed in our lab</td>
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drosophila preparation and longitudinal imaging of heart function in vivo using optical coherence microscopy (ocm)

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.

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 Figu...

Watch this Scientific Journal Video about Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy OCM at JoVE.com

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy OCM

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.

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy (OCM)

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical ...

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