LarvaSPA, A Method for Mounting Drosophila Larva for Long-Term Time-Lapse Imaging

Summary

This protocol describes a method for mounting Drosophila larvae to achieve longer than 10 h of uninterrupted time-lapse imaging in intact live animals. This method can be used to image many biological processes close to the larval body wall.

Abstract

Live imaging is a valuable approach for investigating cell biology questions. The Drosophila larva is particularly suited for in vivo live imaging because the larval body wall and most internal organs are transparent. However, continuous live imaging of intact Drosophila larvae for longer than 30 min has been challenging because it is difficult to noninvasively immobilizeimmobilizing larvae for a long time. Here we present a larval mounting method called LarvaSPA that allows for continuous imaging of live Drosophila larvae with high temporal and spatial resolution for longer than 10 hours. This method involves partially attaching larvae to the coverslip using a UV-reactive glue and additionally restraining larval movement using a polydimethylsiloxane (PDMS) block. This method is compatible with larvae at developmental stages from second instar to wandering third instar. We demonstrate applications of this method in studying dynamic processes of Drosophila somatosensory neurons, including dendrite growth and injury-induced dendrite degeneration. This method can also be applied to study many other cellular processes that happen near the larval body wall.

Introduction

Time-lapse live imaging is a powerful method for studying dynamic cellular processes. The spatial and temporal information provided by time-lapse movies can reveal important details for answering cell biology questions. The Drosophila larva has been a popular in vivo model for investigations using live imaging because its transparent body wall allows for noninvasive imaging of internal structures^1,2. In addition, numerous genetic tools are available in Drosophila to fluorescently label anatomical structures and macromolecules³. However, long-term time-lapse imaging of ....

Protocol

1. Making the imaging chamber

1. The metal frame can be constructed from an aluminum block in a typical machine shop. The specifications of the frame are illustrated in Figure 1A.
2. To construct the imaging chamber, seal the bottom of the metal frame using a long coverslip (22 mm x 50 mm) and UV glue (Figure 1A). Cure the UV glue using a hand-held UV lamp.

2. Making PDMS cuboids<.......

Discussion

Here we describe LarvaSPA, a versatile method of mounting live Drosophila larvae for long-term time-lapse imaging. This method does not require recovering or remounting larvae, enabling uninterrupted imaging. It is therefore ideal for tracking biological processes that take hours to complete, such as dendrite degeneration and regeneration. This method can be also used for imaging intracellular calcium dynamics and subcellular events such as microtubule growth. As the larval body wall is stable during the imaging.......

Materials

Name	Company	Catalog Number	Comments
6061 Aluminum bars	McMaster-Carr	9246K421
3M double-sided tape	Ted Pella, Inc.	16093
3M Scotch Packaging tape	3M		1.88"W x 22.2 Yards
DUMONT #3 Forceps	Fisher Scientific	50-241-34
Glass coverslip	Azer Scientific	1152250
Isoflurane	Midwest Veterinary Supply	193.33161.3
Leica Confocal Microscope	Leica	SP8 equipped with a resonant scanner
Lens paper	Berkshire	LN90.0406.24
Petri dishes (medium)	VWR	25373-085
Petri dishes (small)	VWR	10799-192
Razor blade	Ted Pella, Inc.	121-20
Rectangular petri dish	VWR	25384-322
SYLGARD 184 kit (PBMS kit)	Electron Microscopy Sciences	24236-10
Transferring pipette	Thermo Fisher Scientific	1371126
UV glue	Norland products	#6106, NOA 61	Refractive Index 1.56
UV lamp (Workstar 2003)	Maxxeon	MXN02003
Vacuum desiccator	Electron Microscopy Sciences	71232
Wipes	Kimberly-Clark	Kimwipes