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1Department of Physiology and Biophysics, Boston University School of Medicine, 2Boston University Photonics Center
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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.
Keywords: Developmental Biology, Issue 74, Physiology, Biophysics, Neurobiology, Cellular Biology, Molecular Biology, Anatomy, Developmental Biology, Biomedical Engineering, Medicine, Caenorhabditis elegans, C. elegans, Microscopy, Fluorescence, Neurosciences, calcium imaging, genetically encoded calcium indicators, cameleon, GCaMP, neuronal activity, time-lapse imaging, laser ablation, optical neurophysiology, neurophysiology, neurons, animal model
Chung, S. H., Sun, L., Gabel, C. V. In vivo Neuronal Calcium Imaging in C. elegans. J. Vis. Exp. (74), e50357, doi:10.3791/50357 (2013).
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 excellent additional resource on the techniques described here can be found in WormBook, "Imaging the activity of neurons and muscles" by R. Kerr, (http://www.wormbook.org) 3.
Two major classes of genetically encoded fluorescent calcium reporters are commonly used in C. elegans: single channel GCaMP and FRET-based cameleon. We will describe methods and show examples for data generated by each.
GCaMP is based on a modified Green Fluorescent Protein (GFP) that is sensitive to the surrounding calcium concentration This is accomplished by fusion of GFP and the high calcium affinity protein calmodulin, such that the binding of calcium by calmodulin brings the GFP molecule into an efficient fluorescent confirmation 2. The recent advancements in these fluorophores generate exceptional signal size with up to 500% increase in fluorescence intensity over a physiological range of calcium levels and reasonably fast kinetics of ~95 msec rise time and ~650 msec decay time 4. Over relatively short time periods (mins), these large signals can allow for lower resolution imaging (lower magnification) and, given a well-behaved initial baseline measurement, negate the necessity for continuous baseline or comparative measurements.
Cameleon has the advantage of being a FRET-based fluorophore that generates a ratiometric measurement comparing two independent channels or wavelengths 1. It consists of two separate fluorophores (cyan- and yellow-emitting fluorescent proteins, CFP and YFP) linked by a calmodulin protein. The complex is illuminated with blue light (440 nm) that excites the CFP. Binding of calcium brings the fluorophores closer together, increasing fluorescence resonance energy transfer (FRET) from the CFP (donor) to the YFP (acceptor) and causing the CFP emission (480 nm) to decrease and the YFP emission (535 nm) to increase. Relative calcium levels are measured as the ratio of the YFP/CFP intensity. Cameleon kinetics are slower than that of GCaMP, measured in vivo to have a rise time of ~1 sec and a decay time of ~3 sec 5. However, the ratio of oppositely moving signals increases the signal size and compensates for a number of possible artifacts due to changes in fluorophore concentration, motion or focus drift and bleaching.
Genetically encoded fluorescent reporters negate much of the sample preparation required with exogenously administered probes and C. elegans small transparent body allows imaging within the intact animal using simple wide field fluorescence. The main technical challenge in sample preparation is therefore to safely immobilize the animals. There are a number of different commonly used techniques each with advantages and disadvantages. Using a pharmacological agent to paralyze the animals is easy to implement and allows the mounting of multiple animals on one preparation (Levamisole, a cholinergic agonist that causes muscle tissue to seize is typically used 6). C. elegans can also be physically immobilized by mounting them on stiff 10% agarose 7, 8. This minimizes impact on animal physiology, allows long-term imaging (hours) and recovery of multiple animals but is more technically difficult. Both of these techniques restrict physical access to the animals (which are under a cover slip) and can therefore only be used with certain experimental stimuli (such as light, temperature, electric field or laser damage). For stimuli where physical access is required, such as touch or administration of chemicals, many studies have successfully glued C. elegans in place (using veterinary grade glue) 9. This is technically more challenging, is a single animal preparation and does not allow animal recovery. Finally, numerous microfluidic devices have been employed that physically restrain C. elegans, preserving animal physiology, allowing exposure to most types of stimuli (depending on the device design) and can enable rapid exchange and recovery of the animals 10, 11. However microfluidics require additional technical skills and capabilities in design, fabrication and implementation. In immobilized animals activity and stimulus response can generally be measured in sensory and interneurons. Activity of motor neurons requires more sophisticated techniques for imaging in moving animals. Here we will present detailed methods employing the two most straightforward techniques of pharmacological paralyzation and immobilization with stiff agarose.
The methods presented here can be used to measure neuronal activity and cell physiology in C. elegans. We give an example of each: using GCaMP to measure the sensory response of the ASJ neuron to an external electric field, and using cameleon to measure the physiological calcium response to laser damage of a neuron. These examples show the benefits and drawbacks of the two types of fluorophores and illustrate what is possible with the system.
1. Optical Setup
2. Sample Preparation and Data Acquisition.
3. Data Analysis
4. Problem Solving
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 in vivo. To focus on the technical aspects of each measurement individual trial results are presented and discussed in detail. Often the average response over time (calculated as the average response at each time point with respect to the stimulus) or a specific metric (such as the average amplitude of the response) are calculated across numerous trials. Typically 10-20 trials are necessary to generate an acceptable average measurement but this number will depend in the inherent variability of the response. Such data analysis for the experiments discussed here can be found in12 and 13.
GCaMP measurement of sensory response: When subjected to a strong external electric field (≥3 V/cm),C. elegans actively crawl toward the negative pole of the field with precise directed movement. We previously showed that this electrotactic behavior is primarily mediated by the left and right ASJ neurons, amphid sensory neurons located in the animal's head12. To visualize this response we used a transgenic strain expressing GCaMP3 specifically in the left and right ASJ neurons under the gpa-9 promoter. We immobilized animals for imaging using agarose pads containing 0.05% Levamisole sandwiched between two cover slips (as described in the procedures). The electrical stimulus was administered using a custom built imaging chamber that fits on the stage of an inverted Nikon Ti microscope (see12). In brief, the cover slip preparation is placed (with the worm side down) over a hole in a small plastic chamber allowing access for the objectives from below and standard imaging through the cover slip. The chamber was filled with 0.25 mM NaCl and 50 mM glycerol buffer and the electric field stimulus applied using two platinum electrodes lining the ends of the chamber. As described above, a single ASJ neuron expressing CGaMP was imaged using a X100 1.4 N.A. oil immersion objective and standard GFP filter set. A time-lapse movie was acquired for 80 sec (1 frame/sec, 300 msec exposure time), while subjecting the animal to an external 3 V/cm electric field for three separate trials each lasting 10 sec (Figure 3). We measured fluorescence intensity at the cell body using the image analysis described above. The video displays slight faults of both movement and focus drift over the course of the experiment. The strength of the GCaMP signal remains robust however showing large a ~250% increase in fluorescence in response to both the 1st and 3rd stimuli. The result from the second stimuli is substantially reduced demonstrating variability in the neuronal response. Bleaching is minimal as evident by the return to a consistent baseline level.
Cameleon measurement of cellular calcium physiology: Traumatic cellular injury triggers a large calcium transient within a neuron that plays an essential role in the physiological response dictating cellular fate (i.e. cell death vs. initiation of repair processes). We can measure this damage-induced response in vivo using a femtosecond laser to sever individual C. elegans neurons14 while simultaneously measuring cellular calcium signals using cameleon YC3.60 13, 15. Animals expressing cameleon YC3.60 in the six mechanosensory neurons (under the mec-4 promoter), were immobilized using 10% agarose and polystyrene nanoparticles as described in the procedures. We employed dual imaging optics to record signals from both the CFP and YFP fluorescence channels as described in the procedures. We imaged a single ALM neuron and laser targeted the axon ~20 μm from the cell body (Figure 4A). The axon was severed by a brief (<1 sec) exposure to light from a femtosecond pulsed infrared laser focused at the target point along the axon by the imaging objective (see13 for details on laser surgery). Time-lapse images at a frame every 3 sec, with 400 msec exposure times were recorded for 320 sec while performing laser surgery and calcium levels calculated using ratiometric analysis.
Signals were measured independently at the cell body (Figure 4B) and for the axon segment within 5 μm of the cut point (Figure 4C). The figures show both the CFP and YFP intensities as well as the resulting FRET ratio. The measurement at the cell body is well behaved with the CFP rapidly decreasing and the YFP increasing in response to laser damage at t = 0 sec (red arrow). This results in an immediate ~200% increase in the ratiometric signal (ΔF/F) which is sustained for ~90 sec before falling back to near baseline levels. Bleaching is minimal as evident from the consistent baseline.
In the axon segment close to the cut point, the local amount of fluorophore varies over the course of the experiment, complicating the signal. Laser surgery severs the axon and briefly ruptures the membrane, allowing fluorophore to escape and temporarily reducing its local cytoplasmic concentration. This is evident from an initial decrease in the YFP trace in Figure 4C and a comparison of the axon segment near the damage point in the YFP images at times t = 0 sec and t = 6 sec, Figure 4A. At later time points, the severed end swells as part of its continued recovery, resulting in more localized fluorophore and an increase in intensity. This is most evident in the slowly increasing CFP trace in Figure 4C and a comparison of the axon segment in CFP images at times t = 0 sec and t = 270 sec, Figure 4A. However these variations affect both channels equally and the FRET ratio effectively compensates. The resulting measurement shows a response similar to the cell body with an immediate ~150% increase in signal (ΔF/F), a dramatic recovery to near baseline at ~90 sec and then an additional smaller secondary response at ~150 sec. The ratiometric analysis is critical to this measurement as it would be extremely difficult to separate the calcium signal from the other effects, which can vary widely from neuron to neuron and surgery to surgery. The axon signal has more noise compared to the cell body signal primarily due to dimmer fluorescence and the smaller ROI in the narrower axon.
Figure 1. Basic setup and ratiometric optics. A) The photograph shows the experimental setup consisting of an inverted compound microscope and ratiometric imaging optics. B) A schematic diagram illustrating the imaging optics needed for the ratiometric FRET-based measurements. The image is split into the two wavelengths or channels, which are projected side-by-side on the CCD array.
Figure 2. Sample preparation. C. elegans are mounted on thin agarose pads for imaging. A) Agarose pads are made by sandwiching a small drop of molten agarose between two microscope slides spaced by two pieces of laboratory tape. B) Animals are transferred onto the agarose pad and covered with a coverslip, held in place by a small amount of wax at each of the four corners.
Figure 3. GCaMP example data. The GCaMP signal, ΔF/F, recorded in vivo from the cell body of the ASJ neuron responding to an alternating on/off electric field (green trace). Thick grey lines indicate periods when the external electrical field (3 V/cm) was applied to the animal. Scale bar represents 100% increase in fluorescence intensity.
Figure 4. Cameleon example data. A) Three separate frames showing the dual image view of an ALM neuron before and after laser surgery of the axon 20 μm from the cell body. The red arrow in the middle panel indicates the cut point. The bottom panel shows the cell body and axon segment producing the signals in B and C. B) The cameleon YC3.60 signal measured at the cell body of the ALM neuron before and after laser surgery (red arrow). C) The cameleon YC3.60 signal measured at the axon segment within 5 μm of the cut point before and after laser surgery. Yellow traces indicate YFP signals, blue traces indicate CFP signals and orange traces are the resulting FRET ratio. All times are relative to time of laser surgery. Scale bars represent a 100% increase in fluorescence intensity. Click here to view larger figure.
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 control of animal behavior. Many of these studies have pushed these techniques a step further using image recognition and tracking techniques to enable recording from neurons in partially restrained or even freely moving animals as well as recording from multiple neurons simultaneously. Additional studies have investigated other aspects of calcium physiology such as the response to neuronal damage 13, 20 as presented here as well as neuronal degeneration on longer time scales21.
In designing a particular experiment, each of the alternative techniques regarding commonly used calcium reporters and animal immobilization should be carefully considered. Immobilization techniques will be dictated by technical aspects such as the need for physical access to the animal, the ability to recover animals following imaging or the need for high throughput. Different fluorescent calcium reporters have specific advantages and disadvantages as well. GCaMP employs straightforward single channel image analysis and the large signal-to-noise ratio makes it applicable to many situations. Cameleon on the other hand requires more complex imaging and analysis but the resulting ratiometric measurement can be critical in situations with potentially large artifacts. For example, fluctuations in the amount of fluorophore (as in the laser damage example) can also occur over long time periods (hrs) due to changes in expression levels. Additional dual imaging strategies are also possible such as GCaMP co-expressed with (or physically linked to) a RFP fluorophore. While not FRET-based this would take advantage of the large dynamic range of modern GCaMP variants while using the red channel as a real-time baseline measurement.
It is important to note that numerous two-channel imaging microscope systems and analysis software are commercially available which, while often more expensive, may be attractive to researchers reluctant to construct the home built system described here. Commercial dual imaging systems (DualView2-DV2, Photometrics) typically use optics similar to our home built system but are contained within a single microscope attachment with standardized alignment procedures. Alternatively, fast filter exchange systems (Lambda DG-4/DG-5 Plus, Sutter Instruments) allow rapid real-time sequential imaging of two color channels without additional imaging optics but are also expensive and require precise synchronization between the filter exchange and camera acquisition.
In summary, C. elegans presents a number of advantages as an in vivo imaging system. The genetically encoded fluorophores are often cell specific and can eliminate the need for injecting or administration of exogenous fluorophores. C. elegans optical accessibility allows imaging within intact animals that are easy and cost effective to maintain, do not require complicated dissection or neuronal culturing and preserve cell physiology. In addition, C. elegans has great capabilities for genetic analysis with a large number of available genetic reagents and well-established techniques. There are of course disadvantages to the system, the primary concern perhaps being that it is an invertebrate. In addition, fluorophores report relative changes in calcium concentration rather than an absolute value and the time resolution of measurements can be restricted by both the dynamics of the fluorophore but also the necessary integration times of weak fluorescence signals. Finally, while calcium is an integral part of neuronal activity and signaling the fluorophores do not directly report the membrane voltage potential as measured with electrophysiological techniques. Nonetheless illustrated by the procedures presented here, C. elegans is an attractive, relatively simple, cost-effective system for a wide range of neuronal imaging studies.
The authors declare that they have no competing financial interests.
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 Center.
|Eclipse Ti-U inverted
|Intensilight HG Illuminator||Nikon||C-HGFI||Fluorescent light source|
|CFI Plan Apo VC 60X Oil||Nikon|
|Optical table or 3'X3' optical
|Thor Labs||If an optical table is not used an
optical grade breadboard on a
solid laboratory bench should
|Clara Interline Camera||Andor
|High-sensitivity CCD camera|
|wtGFP Longpass Emission||Chroma Technology
|41015||GFP filter set for imaging GCaMP|
|Filter 440 +/- 10 nm||Chroma||D440/20x EX||excitation filter for cameleon|
|Dichroic mirror > 455 nm
|Chroma||455DCLP BS||microscope dichroic for cameleon
|Dichroic mirror > 515 nm
|Chroma||515DCLP BS||dichroic mirror for cameleon
|Filter 535 +/- 15 nm||Chroma||D535/30m EM||YFP emission filter|
|Filter 480 +/- 20 nm||Chroma||D485/40m EM||CFP emission filter|
|Lens, 200 mm, Achromat||Thor Labs||AC508-200-A1||Relay lens for FRET optics (3)|
|Silver broadband mirror||Thor Labs||ME2S-P01||FRET optics (2)|
|Polybead Microspheres||Polysciences, Inc.||08691-10, 2.5% by
volume, 50 nm
|polystyrene nanoparticles for C.
|Transgenic strain, Strain
|Sternberg Lab||Strain PS6388|
|Transgenic strain, mec-
|Gabel Lab||Strain CG1B|
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