LarvaSPA is the first method that allows for continuous live imaging of intact drosophila larvae for more than 10 hours with high temporal and spatial resolution. This method is used for revealing dynamic cellular processes of larvae peripheral sensory neurons and for studying many other cellular processes that happen near the larva body wall. This method is easy to use and has less limitation on larva size.
Six to nine larvae can be mounted in the imaging chamber at the same time, which makes the experiment possible. The imaging chamber at the PDMS cuboids can be manufactured at a minimal cost and are reusable. This method is particularly useful for studying mechanisms of dendrite development and dendrite degeneration using drosophila larva dendritic authorization neurons.
Being able to image the same neuron for hours can reveal how long term global changes of dendritic patterns arise from short term behaviors at the individual branch level. Using PDMS cuboids that match the sizes of larvae will increase the success rate of immobilizing larvae. Please find more useful tapes in our troubleshooting section.
After constructing an aluminum block from a machine shop, seal the bottom of the metal frame using a long cover slip with UV glue. Cure the UV clue using a handheld UV lamp for 30 seconds. Attach layers of packaging tape to the inner surface of a rectangular Petri dish or round cell culture plate.
Use one layer for second in-star larvae, two layers for early third in-star larvae, or three layers for late third in-star larvae. Cut the tape into trips of specific width with a razor blade, 1.5 millimeters for the one layer tape and two millimeters for the two layer or three layer tape. Leave at least a five millimeter space between the two strips.
Remove the tape layers covering the space. Remove dust from the inner surface of the plate using sticky tape. After that, the mold is ready for use.
To prepare the PDMS mix, mix seven grams PDMS base in 0.7 grams of curing agent thoroughly in a small container. Place the container in a vacuum desiccator for at least 15 minutes to remove air from the mixture. Slowly pour about 5.5 grams PDMS mixture onto the mold to reach a one to two millimeter thickness.
Place the PDMS mixture in the vacuum desiccator again for at least 15 minutes to remove remaining air bubbles from the mixture. Break the last few bubbles with a pipette tip. Cure the PDMS on a flat surface in a heat incubator at 65 degrees Celsius for two hours.
Next, use a razor blade to loosen the cured PDMS along the edge of the mold and detach it from the mold. Store the PDMS between two pieces of large sticky tape at room temperature. For early and late third in-star larvae, cut the PDMS into eight by two by one millimeter cuboids by positioning the groove created by the tape strip at the center of the long side of the cuboid.
For second in-star larvae, cut the cuboid to eight by one by one millimeter. To prepare the top cover slip for mounting, choose six PDMS cuboids with grooves matching the sizes of the larvae. Remove dust from the surface of the PDMS with sticky tape.
Attach four pieces of double sided tape at the size of 12 by five millimeters on a long cover slip of 22 by 50 millimeters for fixing PDMS cuboids later. Adjust the spaces between the two pieces of double sided tape to the same as the width of the PDMS groove. Apply a small drop of UV glue into the groove of each PDMS cuboid and add six small drops of UV glue into the space between the double sided tapes on the cover slip.
To prepare the larvae for mounting, using a pair of forceps, clean the larvae in water to remove food from the body surface. Place the clean larvae on a small piece of moistened tissue paper in a small Petri dish without a lid and place the small Petri dish into a large Petri dish containing a piece of dry tissue paper. In a chemical hood, use a plastic transferring pipette to apply 160 to 240 microliters of isoflurane onto the dry tissue paper and close the lid of the large Petri dish.
Wait two to three minutes while monitoring the larvae. Once their mouth hooks stop moving, take out the larvae from the large Petri dish. Place the immobilized larvae onto the UV glue between the double sided tapes on the cover slip with the dorsal cuticle facing the cover slip.
Cover each larva with a PDMS block and fit the trunk of the larva into the groove of the PDMS. Leave the head and the tail of the larva outside the PDMS groove. Avoid blocking the spiracles of the larva by the glue.
Press down on the ends of the PDMS block onto the double sided tape without applying force on the groove. Gently pull on the tail of the larva to flatten the cuticle under the PDMS. Wear safety glasses and use a handheld UV lamp to cure the UV glue for four minutes on the high setting.
Flip the cover slip upside down and use the UV lamp to cure the UV glue for another four minutes. Place a small piece of lens paper with the size of 15 by 30 millimeters at the bottom of the imaging chamber. Moisten the paper with 20 to 30 microliters of water.
Place the cover slip on the chamber so that the larvae are facing the inside of the chamber. Use the UV glue to adhere both ends of the cover slip to the metal surface. The dorsal side of the larvae is ready for imaging under a confocal microscope.
After imaging, remove the oil on the top cover slip using lens paper. Detach the top cover slip from the metal frame by cutting into the space between the cover slip and the metal frame with a razor blade. The imaging chamber is ready for reuse.
Detach the PDMS cuboids from the top cover slip with forceps. Roll the PDMS cuboids on sticky tape to remove glue residue and dust. The PDMS cuboids are ready for reuse.
In this protocol, drosophila larvae were immobilized for longer than 10 hours for continuous imaging. This figure shows six late third in-star larvae mounted in the chamber. The trunks of the larvae were fixed while their heads and tails were free to move.
Here, we demonstrate the application of LarvaSPA in studying neuronal dendrite dynamics and dendrite degeneration using class four DA neurons as a model. To successfully immobilize larvae using this method, it is critical to stop exposing the larvae to isoflurane once the mouth hooks stop moving and cure the UV glue before larvae fully wake up. To study dendrite degeneration and regeneration, laser injury can be performed when larvae are immobilized in the imaging chamber.
LarvaSPA has helped us to understand how dynamic growing dendrites fail to innervate epidermal cells like in hepatic cell proteoglycans and how phosphatidylserine is exposed on degenerating dendrites after laser injury. Protect eyes with safety glasses while using the UV lamp. Knocking out larvae using isoflurane should be performed in a fume hood.
