Breakthroughs in optical imaging and in genetically encoded flu force have enabled scientists to study the biological processes they're interested in, in the living and in some instances even in the behaving organism. While much emphasis has been put on studying the mammalian brain in vivo, our laboratory uses a rather simple model system, the troph neuromuscular junction, and in the following video we will explain how to anesthetize and to image fruit trial larvae as to follow the development of individual synapses over the time, course of hours, up to days while as a methods to study the development of food. Avi do exist, the to be presented and a cessation protocol is characterized by the following key features.
First of all, by a very high degree of analization, even the heartbeat is arrested, which is essential for high image quality. Secondly, by a survival rate of 95%and by the ability to acquire very sensitive images. The following protocol allows us, for example, to study the dynamics of glutamate receptors tagged with GFP and expressed at physiological levels by their endogenous promoters.
You will learn now how to insert the larvae in the imaging chamber, how to anesthetize the fruit for larvae, how to find a specific position within the larvae to image the signups stair, and then also how to recover the larvae from the ization. Prior to in vivo imaging, it is important to wash the larvae. While this process is simple, care must be taken not to damage the larvae and thus forceps are never used to move them.
Instead, we opt for gentle movements with a brush. The water used for washing is that room temperature and it's just normal tap water that is not distilled or containing any detergents. We can see ya.
He gently moving the larvae out of the food and into the fresh tap water in order to obtain good results. It is also vital to keep the larvae for a shorter time as possible in the water. This will prevent water entering their trachea, which hinders breathing and also the anesthetization process.
Next, we prepare the imaging chamber. Firstly, we spread a very thin layer of oil on the bottom element of the chamber. We then place the larvae on top of this film of oil.
It's important to take care here that the re your endings do not touch the oil. Once positioned, we place a spacer grid on top of the larvae here. It is important that the three channels are facing upward and thus allow air to be brought to the larvae and do not get clogged with oil.
We now put a cover slip on top of the spacer and check the larva is positioned properly. That is the ventral side is facing downward and the tracheal ended dorsal side where we can see the trachea is facing upward. Now we put the plastic adaptive ring as well as a metal ring to ensure the lava is smoothly pressed between the two glass slides on top.
We now close the imaging chamber and relocate it to the confocal microscope to begin imaging. Now ya places the imaging chamber with the mounted larvae. On top of our confocal microscope.
There are three tubes emerging from the imaging chamber. One is attached to the anesthetizing agent dis fluorine. One is an outlet tube and the final tube allows fresh air to reach the larvae.
After connecting the larvae chamber to the anesthetizing device, it is important to ensure the larva is correctly positioned. Yao then allows air to enter the anesthetizing system via the pump. At this time, it is important to ensure the pressure in the chamber does not exceed the maximum recommended.
He then puts the outlet tubing into the water to ensure there is airflow. The bubbles in the bottle of water demonstrate airflow. He now slowly opens the valve and allows dis fluorine to briefly flow through the tubes.
Following initial anesthetization, it is important to check how the lava reacts. If the heart is still beating, it is necessary to apply more anesthetization as can be seen occurring. Here in the next section, we'll show you the lava down the microscope.
Now we can see the critical part of the anesthetization process from down the microscope. Initially, the movements of the larvae can be seen with the muscles continuing to contract. Once the movements begin to slow, ya scans up and down the larvae and watches the anesthetization taking place.
The movements slow and finally stop. After the application of multiple anesthetization pulses, we can see the lava has stopped moving and the only movement seen is due to alterations to the microscope. To determine the extent of anesthetization, it is important to check the heartbeat.
He yao focuses down deep into the lava where mainly the fat body can be seen. Importantly, the region surrounding the heart can be seen and it is evident that the heartbeat has been arrested and thus the larvae is ready for imaging. One major difficulty with in vivo imaging is the identification of the neuromuscular junction you're interested in.
In order to help you with this, we'll show what it looks like as you scan through the larvae. Then you can see a neuromuscular junction that we generally use for imaging. Now we start focusing slowly into the larvae.
Starting at the cuticle surface, you will see some muscles appearing and disappearing. Some of these are highlighted. The movie shows an artificial fusion construct containing the CD eight transmembrane domain, GFP, and the C terminal region of the Iron Channel Shaker protein.
This construct localizes to all muscle membranes and shows increased presence at the neuromuscular junction. We'll now play the movie slower and with the help of figure two and three, you can identify individual muscles as they appear and disappear. While these flies are particularly useful to learn in vivo imaging, we generally use larvae expressing markers, labeling only post-synaptic or pre-synaptic regions of the synapse.
As these do not mark the entire muscle surface, it is much harder to identify the same position in larvae. In this movie, we show RFP tag glutamate receptors. The movie shows us focusing into the labe and we have highlighted individual neuromuscular junctions.
We'll show the movie again slower and you can identify the individual neuromuscular junctions using figure four to help you. Now we have the theory. We'll demonstrate a practical example.
We'll now focus on neuromuscular junction 27 in our MHC CD eight GFP shaker expressing larvae. After increasing the digital zoom, we reposition the neuromuscular junction and then move in the C direction to the central region to ensure the larvae is properly positioned and that we do not have any over or unsaturated pixels. You can see the oversaturated pixels in red and comparatively any unsaturated pixels are displayed in blue.
We reduce the laser power and re-scan. We now have an appropriate gain and laser strength and thus we no longer have any oversaturated regions. It is now important to mark the position of the neuromuscular junction in the C direction.
Firstly, we mark the lower scan position and then the upper scan position to allow the accurate recording of the entire CST stack. We generally use a pixel size of 100 nanometers by 100 nanometers and the distance in C is 500 nanometers. If we do not plan to complete deconvolution after imaging to process our pictures, the gains are set to an appropriate level to prevent noise, but also allow the use of a weaker laser strength to minimize bleaching once the CST stack is complete.
The neuromuscular junction expressing the M-H-C-C-D-H shaker GFP construct is properly recorded. In the following section, we complete a similar process focusing on the glutamate receptors. To record the RFP tag glutamate receptors, we again focus through the cuticle and now see the individual neuromuscular junctions in segment A three.
After having zoomed in on the right neuromuscular junction, we reposition it to the center of the field of view. As previously done, we need to ensure that we do not have any over or unsaturated pixels. Again, it becomes obvious that some of the pixels are oversaturated and so we adjust the settings and complete another test scan.
The gains in the laser power are now adjusted properly. It's now important to set the CST stack limits by identifying the upper position and verifying it, and then the lower position. We can now begin to scan the individual planes of the neuromuscular junction.
It's important to take some blank scans of sections just below and above the neuromuscular junction to prevent missing some synapses positioned there. After the confocals finished scanning, we'll project the neuromuscular junction to see a typical result obtained in in vivo imaging. It's important to keep this image for comparison to scans completed at later time points to re-identify the neuromuscular junction and alterations such as new synapses or synaptic batons can be identified.
After successfully imaging. It is important to quickly wake the lava and rapidly remove it from the imaging chamber. Yao is completing this now while continually monitoring the lava, he adjusts the anesthetization device so that fresh air rather than just fluorine is circulating through the system.
He now places the outlet of the chamber into the water and the air bubbles demonstrate that air is flowing through the system. He continually monitors the larvae to ensure the heartbeat begins to beat again and the larvae wakes up. Similar to the anesthetization process, it is best to provide the larvae with multiple pulses of air to ensure successful waking.
We can now see what ya does through the microscope. We can see as he applies air to the system. Pressure changes are generated in the chamber, which induce artificial movements in the larvae.
Eventually, the larvae begins to move on its own and this is best demonstrated in the initiation of heart movements, and so we focus down into the lava so we can see these movements more clearly. The rhythmic beats can be seen showing successful waking of the larvae. After successfully waking up the larvae, it is important to recover it from the imaging chamber.
The larvae is in the bottom section of the chamber, and so the top is placed to the side. Yara removes the metal weight and the plastic cover. Then the cover slide is removed and subsequently the plastic spacer with the larvae.
He carefully removes the larvae from the plastic spacer with the brush. He then briefly dips it in water and dries it to remove any excess oil. The lava is now placed in some pre crushed food and is left there until needed again.
