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In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of > 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described 4,5,6,7,8 , the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm 1 , (2) a high survival rate of > 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days 2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter 1 in stable transgenic lines and the exon trap line FasII-GFP 1 . (4) In contrast to other methods 4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days 1 . The accompanying video details the function of individual parts of the in vivo imaging chamber 2,3 , the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

Video Link
The video component of this article can be found at http://www.jove.com/video/2249/ Protocol A) Assembly of the imaging chamber 1. Select a larva of chosen stage (e.g. early 3 rd instar larvae leaving an observation interval of about 24 hours at 25°C until wandering stage).
2. Rinse the larva with water to clean it of the culture medium and dab it dry. 3. Coat the center of the bottom element of the chamber, the region to face the larva, with a thin film of halocarbon oil. 4. Put the larva with the ventral side facing the microscope objective in the chamber (allows neuromuscular junction (NMJ) 26 and 27 to be imaged) (Figure 1 A-E). 5. Place the plastic spacer onto the oil layer, with the air slots of the grid face upwards (Figure 1 A). Please verify at this step: The height of the spacer is roughly half the larval diameter, the width of the slit should be about twice the diameter of the larva. If necessary open and close valves as described in step 8 and 9 until anesthetization of the larva is complete, then close all valves and start imaging. Please verify at this step: Residual muscle movement or heartbeat indicates that the larva is not properly anesthetized. Complete anesthetization is vital for high resolution images. The larva should normally not be anesthetized for longer than 15-20 minutes at a given time.

C) Imaging
11. Identify the correct position and image the structure of interest (Figure 2-4).

D) Recovery from anesthetization
12. After imaging is completed let air into the chamber. Please verify at this step: Check heartbeat and muscle contractions of the larva by transmitted halogen light. As muscles start contracting it is safe to remove the larva from the imaging chamber. 13. Detach the chamber from the anesthetization device and remove it from the microscope. Disassemble the chamber carefully and place the larva in mashed fly cultivation medium. 14. Store the dish in an incubator at the appropriate temperature.

E) Time series
15. Repeat the steps 2-14 until sufficient time points have been obtained.
Alternative protocol: If the larva is to be imaged more than once within a 30 minute interval it might be practical to leave it in the imaging chamber until the next anesthetization time point. Repeat the steps 7-14 until sufficient time points have been obtained. Please verify at this step: The larva is not to be kept in the imaging chamber for more than two hours at a time, nor is it to be anesthetized for longer than 15-20 minutes at a time.