Imaging Subcellular Calcium Dynamics in Neurons of Caenorhabditis elegans

Abstract

Source: Doser, R., et al. Subcellular Imaging of Neuronal Calcium Handling In Vivo. J. Vis. Exp. (2023)

This video demonstrates in vivo imaging of subcellular calcium flux in the ventral nerve cord neurons of transgenic Caenorhabditis elegans strains. Two distinct calcium indicators are employed: a cytoplasmic indicator in one strain and a mitochondrial indicator in another. Neurons are observed under a fluorescence microscope to monitor real-time changes in fluorescence intensity within the neuronal cytoplasm and mitochondria, reflecting calcium flux during spontaneous neuronal activity.

Protocol

1. Preparation of worms for imaging

1. Preparing the agar pads
   1. Make 3 mL of 10% agar by dissolving molecular grade agar in M9 in a 13 mm x 100 mm glass culture tube and microwave for several seconds.
      NOTE. Molten agar can be kept on a heat block for up to 1 h or can be made fresh each time agar pads are needed.
   2. Place a microscope slide between two additional slides that each have two layers of laboratory tape (see Figure 1A-i).
   3. Cut the tip of a 1,000 μL pipette tip (without a filter) and use it to pipette a small drop of agar onto the center coverslip (Figur....

Results

Figure 1: Mounting and rolling C. elegans for imaging the ventral n.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
100x/1.40 Oil objective	Olympus		UPlanSApo
10x/0.40 Objective	Olympus		UPlanSApo
22 mm x 22 mm Cover glass	VWR	48366-227
Agarose SFR	VWR	J234-100G
CleanBench laboratory table	TMC		With vibration control
Filter wheel or slider	ASI		For 25 mm diameter filters
FJH 185	Caenorhabditis Genetics Center	FJH 185	Worm strain
FJH 597	Caenorhabditis Genetics Center	FJH 597	Worm strain
GFP bandpass emission filter	Chroma		525 ± 50 nm (25 mm diameter)
ILE laser combiner	Andor Technologies		4 laser lines
ILE solid state 488 nm laser	Andor Technologies		50 mW
IX83 Spinning disk confocal microscope	Olympus		With Yokogawa CSU-X1 spinning disc
iXon Ultra EMCCD camera	Andor Technologies
Low auto-fluorescence immersion oil	Olympus	Z-81226
MetaMorph	Molecular Devices		Version 7.10.1
Microscope control box	Olympus		IX3-CBH
Muscimol	MP Biomedical / Sigma	02195336-CF
Polybead microspheres	Polysciences Inc.	00876-15	0.094 µm
Stability chamber	Norlake Scientific	NSRI241WSW/8H	Set to 15 °C
Stage controller	ASI		With filter wheel control
Standard microscope slide	Premiere	9108W-E	75 mm x 25 mm x 1 mm
Touch panel controller	Olympus		I3-TPC
Z-drift corrector	Olympus		IX3-ZDC2