The overall goal of this procedure is to image the heart function of Drosophila longitudinally in vivo using an optical coherence microscopy technique. This method can help answer key questions in the field of Drosophila cardiac structural and functional development by providing metrics, such as changes in cardiac diameter, heart rate, and the cardiac activity period during Drosophila metamorphosis. The main advantage of this technique is that the optical coherence microscopy is capable of imaging small animal hearts noninvasively with high spatial and temporal resolution.
This technique may help reveal the mechanisms of human cardiac disease and the relation to cardiac development due to the genetic similarities that exist between Drosophila and vertebrates. This method can also be applied to other systems, such as optogenetic pacing for studying or treating weakened pacemaker function in animal models. When producing the flies for this experiment, allow reproductive parental flies to mate and lay eggs in a fresh vial for eight hours.
Thus, the clutch of eggs laid will develop into flies of a similar age. To mount a larva for OCM imaging, first prepare a clean side with a piece of double-sided tape. Push out any trapped air bubbles below the tape.
Next, use a soft brush to remove a larva of the right size from the vial. Place the larva on a clean tissue. Remove any food stuck to it using a moistened brush and then let it dry.
Now, under a wide field microscope, confirm the developmental stage of the larva. Then, position the larva with the dorsal side facing upwards for mounting. Then, lower the sticky slide onto the larva and use moderate pressure to secure it to the tape.
Place the mounted larva on the adjustable stage of the OCM system along the y-transverse direction with the dorsal side facing upwards. A small hole in the stage keeps the larva out of contact with the stage plane. A broad lumen of the heart tube is found from segments A5 to A8.Using the real time images on the computer, find the posterior region of the heart tube and move the stage forward until the A7 segment is visible.
Now, set the parameters of the image acquisition. Use 100 A-scans per B-scan and use 100 B-scans. Then, set the scanner voltage to cover about 0.28 millimeters in the x-transverse direction and set it to 0 volts in the y-transverse direction.
Next, block the sample beam path with a dark cloth and click the start button in the software to acquire background noise data for background subtraction. To collect transverse M-mode images, adjust the parameters to 128 A-scans per B-scan and use 4, 096 B-scans. Then, readjust the voltage to cover 0.28 millimeters in the x direction and 0 volts in the y direction as before.
Then, acquire images in the transverse M-mode of the A7 segment of the fly heart tube. During the data-saving process, block the imaging beam with a dark cloth to minimize light exposure to the sample specimen. Image the heart five times to get a reliable measurement of the heart function.
To collect three-dimensional images, use 400 A-scans per B-scan, 800 B-scans, and use voltage for 1.7 millimeters on the x-transverse and about 4 millimeters on the y-transverse. Then, move the microscope stage to observe the entire fly and adjust the focus to see clear images. To remove the larva, use a wet, soft brush to transfer it to a new vial with fresh food for continued development and longitudinal study.
The broad lumen of the heart tube remains at segments A5 to A8 in pupal stage PD1. At PD1, endeavor to image the A7 segment, as done with L2 and L3 stage larva. The PD1 pupal stage is identified by the white puparium.
This time window is ideal for performing optical imaging of the early pupa because the high transparency leads to higher light penetration for OCM imaging. Clean the pupa with the brush if there is food stuck to the body. Using a wet brush, mount the pupa on a small glass slide and keep the dorsal side facing upward.
The slide should fit into a fly tube to preserve the development of the animal in its own chamber. Dry any excess water from the pupa and then place it on the stage with the pupa facing upwards and proceed to image the A7 segment as done with the larva. After imaging, transfer the glass slide with the pupa into the tube for continuous culture.
After the PD1 stage, beginning at the PD2, a conical chamber starts to develop between the A1 and A4 segments. Since pupa become progressively more opaque, the penetration depth of the imaging system gets reduced. At PD2, the pupa shell becomes yellowish and the body is less transparent than PD1.
At PD3, the color of the pupa is yet a little darker than that of the PD2 stage. By the PD4 stage, black stripes can be observed inside the shell. Some PD4 will develop into adults and others will enter the PD5 stage.
In the PD5 stage, the black stripes are even more obvious. Pick up a PD2 pupa using tweezers and mount it on a slide. On the OCM system, find the anterior end of the heart tube and then move about 50 microns towards the posterior to find the A1 segment of the heart tube.
Note that in the PD2 stage, the conical chamber of the heart is very small and may not be beating yet. Proceed with the imaging as previously described. Repeat the same procedure for imaging Drosophila at PD3, PD4, and PD5 stages.
To begin, transfer the adult fly to an empty vial that has a volume of about 45 milliliters. Then, dip a cotton applicator into the anesthetic solution and put the wand into the vial with the cotton tip just below the cotton stopper. Wait three minutes.
The adult fly will be anesthetized for two and a half to three and a half minutes, depending on its size. Now prepare a glass slide with a piece of double-sided tape. Then, using a soft brush, move the anesthetized fly onto the glass slide with the dorsal side facing upwards.
Under a wide field microscope, separate the wings using tweezers and stick them to the tape, thus exposing the heart region for imaging. Now, use the previously described imaging technique to image the A1 segment of the heart and sacrifice the fly at the end of the experiment. Longitudinal cardiac imaging was conducted using strain flies with one copy of the 24B-GAL4 driver.
At larval stages, the heart tube begins at the posterior abdominal region A8 with a broader lumen and ends at the anterior dorsal segment A1 with a narrower diameter. Late in PD2, a conical chamber started to develop at the A1 to A4 segment. The fly heart rate was quantified from the transverse M-mode OCM images.
Curiously, during the pupal stages, the heart stopped beating occasionally. Overall, the heart beat slowed down significantly from the larval stage to the pupal stage and then increased substantially from pupa to adult. Since this development, this technique has given researchers in the field of developmental cardiology a new tool to explore the effects of various genes on heart development in Drosophila models.
While attempting this procedure, it's important to carefully track the development of a Drosophila in order to create images at each developmental stage.
