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

Summary

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.

Abstract

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.

Introduction

Longitudinal study of the heart in small animals contributes to understanding a variety of human related cardiovascular diseases, such as gene related congenital heart defects^1,2. In the past decades, various animal models, such as mouse^3,4, Xenopus^5,6, zebrafish^7,8, avian⁹, and Drosophila^10-16, 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 heart^3,4. The Xenopus embryo is especially useful in the study of heart development due to its easy handling and partial transparency^5,6. The transparency of the embryo and early larva of the zebrafish model allows for easy optical observation of cardiac development^7,8. 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 studies^11,13. Furthermore, it has a short life cycle and low maintenance costs, and has been commonly used as a specimen model for developmental biology research^14-16.

Previous reports described the protocols for monitoring Drosophila cardiac functions such as the heartbeat. However, dissection procedures were required^17,18. 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 microscopy^20,21, 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 OCT^28-33. 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 system^15,16, 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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Protocol

1. Preparation of OCM System for Optical Imaging of Drosophila¹⁶

1. 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.
2. Use a broadband light source to ensure the axial resolution of 2 µm to identify the heart structure of Drosophila.
3. Use a 10X objective to obtain a high transverse resolution.
4. Use a 45° rod mirror to reflect the reference arm light beam and to generate an annular sample arm light beam to extend the depth of focus in the specimens.
5. Develop a custom computer program to control the OCM system and perform measurements.

2. Drosophila Culture

1.  Standard Fly Food Preparation
   1. Put ~ 5 ml instant Drosophila formula into a polystyrene vial tube with the assistance of a paper chute.
   2. Pour ~ 8 ml water into the formula to properly saturate the food.
   3. Add different supplements to the standard fly food for different experiments. When preparing for all-trans-retinal (ATR) food for the optogenetic pacing experiment³⁵, use a pipette to extract 100 mM ATR and dissolve into ~ 8 ml water to get the ATR concentration of 1 mM in food. After mixing the solution uniformly, pour the solution into the formula and stir it sufficiently.
   4. To prepare a high-fat-diet for studying obesity related heart dysfunctions in Drosophila,^10,15 mix ~10 ml formula with 15 ml water in a cup and heat for 30 sec in a microwave oven. Put some organic extra virgin coconut oil in another cup and heat it for 90 sec in the microwave oven.
   5. Extract 7.5 ml coconut oil and mix with the prepared formula sufficiently to make the weight/volume ratio of coconut oil to food ~ 30/100, and then extract ~ 2 ml mixed food and put it to the bottom of a tube.
   6. Wait for 1 min until the medium is thoroughly saturated. Compact the food carefully with a flat surface to optimize the living conditions for the Drosophila. Add 6 - 8 grains of yeast to the prepared formula, and plug the tube with a cluster of cotton.
2. Fruit Fly Crosses and Culture
   1. Take a tube with prepared standard fly food, and remove the plugged cotton. Carefully transfer the adult flies (male and female) to the tube, and plug the tube with cotton immediately. Check the cotton to make sure that there is no gap between the cotton and the tube wall to prevent flies from escaping from the tube.
   2. Keep the fruit flies in the incubator at 25 °C for cross breeding. Most of the genes are active and the cellular proteins are synthesized at 25 °C^36-39.
   3. Take the tube out from the incubator after 8 hand transfer the adult flies out of the tube to obtain the eggs at similar age for experimental control.
   4. Continue culturing the eggs in the incubator at 25 °C, which is the standard temperature for Drosophila development with the development period of 8.5 days^40,41.
      NOTE: Temperature influences the developmental period (egg to adult) and expression level of various genes.

3. Performing Optical Imaging with OCM

1. Mount Fly Larva for Optical Imaging
   NOTE: The egg of Drosophila hatches in 22 - 24 hr at 25 °C to the first instar larva (L1). The second instar larva emerges after another 24 hr. The largest larval form is the third instar larva, which molts after about 24 hr. Structural characteristics in larva can be used to distinguish their different developmental stages. The size of the mouthparts between the first instar and the second instar is different. The mouth hooks of the first instar larva are very small and look like two pairs of tiny black spots, while the mouth hooks of the second instar larva are larger and the structure is clearer. The spiracles are usually used to identify the second instar and the third instar. The second instar larva has clubbed anterior spiracles, while, for the third instar, the anterior spiracles are branched. A dark orange ring will begin to appear at the tip of the posterior spiracles in the third instar larva.
   1. Apply a piece of double sided tape to a clean microscope glass slide. Expel the air bubbles under the tape to avoid the reflections caused by air bubbles during imaging.
   2. Take one of the tubes with the cultured flies out from the incubator at the larval stage.
   3. Identify the larva in the media, remove it from the media with a soft brush and place on a clean tissue. Remove any food stuck to the larva with a wet soft brush and dry it on the tissue.
   4. Move the cleaned fly to a tissue under the objective lens of a wide field microscope.
   5. Adjust the focus of the microscope to find a clear view of the fly. Identify the right developmental stage of the larva by its structural characteristics with the microscope.
   6. Position the fly using the soft brush. Ensure the body is straight with the dorsal side facing upward to prepare for mounting on the glass slide by the dorsal side. Perform this step under the microscope.
   7. Ensure the larva is completely dry before mounting on the tape. Otherwise, the larva will not adhere to the tape.
   8. Stick the dorsal side of the positioned fly to the double side tape on the glass slide with moderate pressure. Note that too much pressure may kill the fly and too little force will lead to fly movement during imaging.
2. Optical Imaging of Drosophila at Larval Stages (L2 and L3) with OCM
   NOTE: A broad lumen of the heart tube can be found located in the segments between A5 to A8 at the larval stages (Figure 1). The transverse OCM M-mode images (2D + time) were acquired at the A7 segment of the heart tube for each larva to facilitate the systolic and diastolic analysis.
   1. Place the mounted larva on the adjustable sample stage of the OCM system along the y-transverse direction with the dorsal side facing upward below the objective lens. A small hole in the sample stage is necessary for placing the larva to avoid its contact with the stage plane.
   2. Adjust the sample stage to move the heart tube of the fruit fly to the focal plane of the imaging beam. To easily find the A7 segment, find the posterior region of the heart tube with the real time cross sectional OCM images in the image acquisition software. Then move the stage forward until the A7 segment is visible.
   3. Set parameters of the image acquisition software to 100 A-scans per B-scan (frame), 100 B-scans, and the scanner voltage to cover ~ 0.28 mm in the x-transverse direction, and 0 V in the y-transverse direction. Click the "start" button in the software to acquire the background noise data for background subtraction by blocking the sample beam path with a dark cloth.
      NOTE: 3 of the 100 frames can be used for the background subtraction.
   4. Set parameters of the data acquisition software to 128 A-scans per B-scan, 4096 B-scans, and the scanner voltage to cover ~ 0.28 mm in the x-transverse direction, and 0 V in the y-transverse direction. Click the "start" button in the software to acquire the transverse M-mode images across the A7 segment of the fly heart tube over a region covering 0.28 x 0.57 mm² for about 30 sec.
   5. Block the imaging beam using a dark cloth during the data saving process to avoid lengthy exposure of the fly heart to the imaging light.
   6. Repeat the measurement for 5 times to get reliable measurement of the heart function.
   7. Set parameters of the image acquisition software to 400 A-scans per B-scan, 800 B-scans, and the scanner voltage to cover ~ 1.7 mm in the x-transverse direction, and ~ 4 mm in the y-transverse direction. Move the stage in both directions to ensure the entire fruit fly can be imaged. Click the "start" button in the software to acquire one dataset to obtain images of the fruit fly in 3 dimensions. Note: The 3D fly structure can be rendered using Amira 3D software
   8. Use a wet soft brush to moisten the measured fly and gently remove it from the glass slide. Move it into a separate tube for continuous development. Label the tube for longitudinal study through the next developmental stages.
3. Image Drosophila at Pupal Stages
   NOTE: All the fruit flies were taken out for imaging from PD1 to PD5. As shown in the larva schematic in Figure 1b, a broad lumen remains in A5 to A8 segments of the heart tube until PD1. From PD2, a conical chamber starts to develop between A1 to A4 segments. To acquire consistent images and facilitate heart analysis, transverse M-mode images were obtained from the A7 segment at PD1, and from A1 segment after PD2, as marked in Figure 1b.
   1. Image Drosophila at PD1
      NOTE: Drosophila will have a white puparium for a short time window (0 - 1 hr) during PD1. This time window is ideal for performing optical imaging of early pupa because the high transparency leads to higher light penetration for the OCM imaging.
      1. As the fruit flies are found on the tube wall when they become pupa, remove the pupa from individual tubes for imaging at PD1 with a wet soft brush, and clean the pupa with the brush if there is food stuck to the body.
      2. Mount the fruit fly on a small glass slide directly with the wet brush and keep the dorsal side facing upward (Figure 1a). Make sure the glass slide is small enough to fit back into the tube once imaging at this stage is complete.
      3. Remove excessive water from the side of the fly body.
      4. Put the glass slide on the sample stage of the OCM system, keeping the fruit fly on top. Find clear real-time image of the A7 segment of the fly heart utilizing the same strategy described in the larva measurement.
      5. Set the same parameters of the data acquisition software as in section 3.2, and image the heartbeats at the A7 segment to acquire transverse M-mode and 3D images.
      6. After imaging, use a tweezer to place the glass slide with pupa back into the tube for continuous culture.
   2. Image Drosophila at PD2 to PD5 Stages
      NOTE: Since the specimen becomes more and more opaque during the pupal stages, the penetration depth of the imaging system will be reduced.
      1. Use a tweezer to carefully remove the glass slide mounted with the fly at PD2 from the tube for imaging. At PD2, the specimen shell becomes yellowish and the body becomes less transparent compared to PD1 (Figure 1).
      2. Put the slide on the sample stage of the OCM system.
      3. Adjust the sample stage to move the fly into the focal plane of imaging beam of the OCM system. Find the anterior end of the heart tube with real-time cross-sectional OCM image. Move ~ 50 µm back in the posterior direction to find the A1 segment of the heart tube.
         NOTE: At this point of heart development (PD2), the conical chamber will be very small and may not be beating.
      4. Collect transverse M-mode datasets from the A1 segment as well as 3D data using the same method as previous developmental stages.
      5. Put the slide back to the tube carefully for continuous culture.
         NOTE: At PD3, the color of the specimen in the shell is darker than that at PD2 stage. At PD4 stage, black stripes can be observed inside the shell of the specimens. Some flies will develop into adult from this stage in the following day, while others will evolve into PD5. At PD5 stage, black stripes are even more obviously seen in the fruit flies. These flies will become adults in the following day.
4. Image Drosophila at the Adult Stage
   NOTE: At the adult stage, female and male flies can be distinguished by the size of the body and the color of the lower abdomen. Female adults have larger size, while males are smaller and dark-colored in the lower abdomen.
   1. Take the tube out from the incubator when the fruit fly develops into an adult, and transfer the adult fly to a ~ 45 ml empty vial.
   2. Dip the absorbent end (~ 1 cm length, ~ 3 mm diameter) of a wand into the anesthesia, put the wand into the vial, and plug the tube with a cluster of cotton to keep the anesthetic end just below the plugged cotton and to anesthetize the fly for 3 min. The duration of anesthesia depends on the size of the fly, and may vary between 2.5 to 3.5 min (for example: male for 2.5 min, female for 3 or 3.5 min).
   3. Prepare a glass slide with a piece of double sided tape.
   4. Move the anesthetized fly onto the glass slide with dorsal side facing upward using the soft brush.
   5. Separate the wings using a tweezer and stick the wings on the tape under a microscope to fix the fly and expose the heart region for imaging.
   6. Image the fly from the A1 segment of the fly heart (Figure 1). At the end of the experiment, the fly may be sacrificed.

4. Imaging Analysis¹⁶

1. Develop Matlab programs to convert the 2D and 3D binary files collected with the image acquisition software to image files.
2. Use ImageJ to identify the heart tube region in the transverse M-mode images and a magic wand algorithm to create a mask of the heart region for each transverse M-mode image. Segment the masked region and use a peak-finding algorithm to identify the systolic and diastolic locations. Calculate the time dependent heart diameter changes from the transverse M-mode images.
3. Based on the acquired time dependent heart diameters, calculate the cardiac parameters such as HR, cardiac activity period (CAP),end diastole diameter (EDD), end systole diameter (ESD), end diastole area (EDA), and end systole area (ESA). Calculate the fractional shortening (FS) with
4. Use ImageJ to analyze the 3D OCM images to visualize the structural development of the fly heart.

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Results

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...

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Discussion

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 wit...

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Materials

Name	Company	Catalog Number	Comments
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
ImageJ	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, 3 M	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