Published: April 10th, 2013
With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.
The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.
Here we present practical methods for in vivo calcium imaging in C. elegans neurons. The development of genetically encoded calcium-sensitive fluorophores with high signal-to-noise ratio makes C. elegans a comparatively straightforward and cost effective system for measurement of neurophysiology and activity. Our imaging is done with a standard compound microscope using wide-field fluorescence imaging of commonly available fluorophores. We present several techniques employing various fluorophores and different sample preparations, discussing the strengths and weakness of each. Data is then presented from two example experiments. An ....
1. Optical Setup
Here we present results from two separate experiments. The first employs GCaMP to measure the response of a specific sensory neuron to a defined external stimulus, giving a good example of how fluorescent calcium reporters can be used to optically monitor neuronal activity in intact C. elegans. The second employs cameleon to measure the intracellular calcium transient triggered within a neuron in response to specific laser damage, thus illustrating how calcium physiology can be measured within a single cell .......
Genetically encoded calcium indicators have been widely utilized in C. elegans neurobiology. Numerous groups have employed these techniques to study response of primary sensory neurons to external stimuli as demonstrated here with the ASJ response to an electrical field. Prominent examples include sensation of mechanical touch, specific chemicals, temperature and an electric field 12, 16-19. Activity of interneuron and muscle cells have also been monitored both in response to stimuli and in con.......
Several people contributed to the work described in this paper. CVG built the experimental setup, and LS, SHC, and CVG performed the experiments. CVG and SHC wrote the manuscript. All authors subsequently took part in the revision process and approved the final copy of the manuscript. We thank Paul Sternberg for the GCaMP strain. Some nematode strains used in this work were provided by the Caenorhabditis Genetics Center (CGC), which is funded by the NIH National Center for Research Resources (NCRR). The MATLAB image analysis program was adapted from that used in 18. The authors were supported by Boston University and The Massachusetts Life Sciences C....
|Name of Reagent/Material
|Eclipse Ti-U inverted
|Intensilight HG Illuminator
|Fluorescent light source
|CFI Plan Apo VC 60X Oil
|Optical table or 3'X3' optical
|If an optical table is not used an
optical grade breadboard on a
solid laboratory bench should
|Clara Interline Camera
|High-sensitivity CCD camera
|wtGFP Longpass Emission
|GFP filter set for imaging GCaMP
|Filter 440 +/- 10 nm
|excitation filter for cameleon
|Dichroic mirror > 455 nm
|microscope dichroic for cameleon
|Dichroic mirror > 515 nm
|dichroic mirror for cameleon
|Filter 535 +/- 15 nm
|YFP emission filter
|Filter 480 +/- 20 nm
|CFP emission filter
|Lens, 200 mm, Achromat
|Relay lens for FRET optics (3)
|Silver broadband mirror
|FRET optics (2)
|08691-10, 2.5% by
volume, 50 nm
|polystyrene nanoparticles for C.
|Transgenic strain, Strain
|Transgenic strain, mec-
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