Imaging behavior and neural activity over long time scales without immobilization of the animal is a prerequisite to understand behavior. Agarose microfluidic chambers imaging (AMI) can be used to image neural activity and behavior for all life stages of Caenorhabditis elegans.
Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure the activity of neurons during various behaviors. To correlate neural activity with behavior, the animal should not be immobilized but should be able to move. Many behavioral changes occur during long time scales and require recording over many hours of behavior. This also makes it necessary to culture the worms in the presence of food. How can worms be cultured and their neural activity imaged over long time scales? Agarose Microchamber Imaging (AMI) was previously developed to culture and observe small larvae and has now been adapted to study all life stages from early L1 until the adult stage of C. elegans. AMI can be performed on various life stages of C. elegans. Long-term calcium imaging is achieved without immobilizing the animals by using short externally triggered exposures combined with an electron multiplying charge-coupled device (EMCCD) camera recording. Zooming out or scanning can scale up this method to image up to 40 worms in parallel. Thus, a method is described to image behavior and neural activity over long time scales in all life stages of C. elegans.
Caenorhabditis elegans has been established as a model system to study behavior1. Due to its amenability to genetics, the molecular and cellular mechanisms underlying behavior can be studied. Many behaviors occur on long time scales. Two principal approaches can be used to observe motile animals over long time scales. The first approach is following the animal during movement using an automated stage or camera2-8 and the second is to restrict the movement to a range that is at least as small as the field of view of the camera9-14. Both methods have their advantages. Tracking allows following an animal over long spatial ranges but limits the number of animals that can be discerned in one experiment. Restricting the movement of the animals allows scaling up the observation to many individuals at the same time by using arrays of restricted compartments.
Because the nematode is transparent, live fluorescent imaging can be performed non-invasively15. Calcium imaging provides a functional readout for the activity of excitable cells and is established for C. elegans16-20. Calcium enters the cell via channels in the plasma membrane that open upon depolarization. Thus, calcium acts as a proxy for neural activity. Calcium sensors can be grouped in two major classes, ratiometric and non-ratiometric sensors. Both classes employ conformational changes of calcium-binding proteins induced upon binding of calcium. Ratiometric sensors contain two fluorescent proteins. When the lower-wavelength fluorescent protein is excited, a part of the light energy is transmitted to the higher wavelength fluorescent protein as a function of their distance in a process called Fluorescence Resonance Energy Transfer or Förster Resonance Energy Transfer (FRET)21. Non-ratiometric sensors are based on circularly permuted GFP and employ de-quenching of the fluorophore caused by calcium binding22. Each class has its advantages. While ratiometric sensors are less sensitive to movement or expression artifacts, non-ratiometric sensors typically have a higher dynamic range. Both ratiometric and non-ratiometric sensors have been useful to study the activity of excitable cells in C. elegans16-20,23,24.
When doing long-term fluorescence imaging, the experimenter will have to deal with several potential challenges: 1. Disturbance of behavior through excitation light: Worms are sensitive to short-wavelength fluorescence excitation light and avoid light from the violet to blue range that is used for calcium imaging25,26. Worms respond with either a backward or a forward escape response25,26. Thus, the amount of light needs to be controlled. 2. Bleaching of the fluorescent sensor protein: Often, bleaching of the fluorescent protein hampers long-term imaging. Typically, however, light intensities that are needed to observe bleaching are higher than the light intensities that cause disturbing effects on the behavior of the animals. Thus, bleaching is only a theoretical problem in this type of calcium imaging. 3. Worms inside the microchambers are not fixed and move constantly during wake behavior and images may appear blurred if the worm is not immobilized. All these challenges can be solved using extremely short exposure times with low light intensity. This can be realized by using a highly sensitive EMCCD camera with short exposure times and external triggering of a light emitting diode (LED). To expose the worms to the illumination light as briefly as possible (only during the exposure time of the camera) the LED is externally triggered using a transistor-transistor logic (TTL) signal that the camera emits during exposure resulting in an illumination of the worm for precisely the time the camera chip is exposed. This also means that the EMCCD chip will be dark during data readout, which is optimal for readout performance of this chip.
Previously, agarose microchambers have been developed for long-term fluorescence imaging of C. elegans larvae9. Here, it is described how agarose micro-chambers can be used for long-term calcium imaging for any life stage of C. elegans, how calcium imaging can be performed, and how this method can be upscaled to assay many individual worms in parallel.
Hardware
The focus of a microscope will typically drift during long-term image acquisition. For compound microscopes, focus-keeping systems can be purchased from the major microscope manufacturers. In case a compound microscope with focus control is too pricy, a simple alternative would be to use a stereomicroscope. Compound microscopes allow the use of objectives with high numerical aperture (NA) and can be easily automated. 40X oil objectives are well suited. Water immersion lenses are not ideal because of evaporation during long-term imaging. Differential interference contrast (DIC) generates a nice contrast that helps to follow morphological and behavioral changes, but simple bright field imaging can also be used. For DIC or bright field imaging, use red light by placing a red light filter into the dia-illumination path of the microscope. For scanning of several microchambers an automated stage is necessary. Best performance is achieved when a stage is used that has nonlinear acceleration and deceleration and can be set to low scan speed to prevent disturbing the animals during scanning. We have not observed behavioral responses or calcium increase in mechanosensitive neurons (ALM and PLM) during scanning, suggesting that slow scanning indeed does not activate the mechanosensitive system of the worm (data not shown). Commercial LED systems can be used. Several companies offer ready-to-use solutions that include the LEDs at different wavelengths. The LED should have the option of externally triggering the LED with a TTL signal. A highly sensitive camera is needed for calcium imaging of moving animals. EMCCD cameras are the most sensitive cameras on the market. The camera needs to have a TTL output during exposure (also called “fire” output). A lid heater is required to prevent condensation on the lid if using an inverted microscope. On an upright microscope, the dish will be placed so that the lid will be at the bottom and thus condensation is prevented and no lid heating is required. The lid should tightly close the dish to prevent evaporation of water during long-term imaging.
PDMS stamps
The surface of the PDMS stamp that contains the structure for molding the agarose is faced away from the glass slide, which supports the PDMS stamp. A list with companies that offer custom microfluidic chips can be found on Wikipedia (http://en.wikipedia.org/wiki/List_of_microfluidics_related_companies).
Filling the nematodes into their chambers
This is the most critical step in the protocol and the following points should be considered: A) The moistness of the agarose is crucial for transferring the worms and for imaging. If the agarose is too moist, i.e., there is a liquid covering an area of several microchambers, it will not be possible to distribute bacterial food and worms in a controlled manner because the liquid will make the bacteria flow away. If the agarose is too dry then the bacteria and worms may not get off the pick easily which means that increased force needs to be used to drop the worms and bacteria which easily causes damage to the agarose. When filling in a lot of worms the agarose may dry up. In the worst case the agarose will be so dry in the end that the chambers will collapse. If the agarose is too dry it can be rehydrated by placing a small drop (about 2 µl) of S-Basal onto the side of the chip where there are no worms. Then dip the platinum wire pick into the liquid and even pull some of the liquid into the area where the chambers are filled with worms. B) The amount of food is critical for successful long-term imaging. If there is not enough food, the worms may run out of it. If there is excessive food the cavity of the chamber will not behave like a liquid but rather like a solid and will allow worms to escape from the chamber by pushing off the bacteria. Aim to obtain a bacterial suspension that fills the entire chamber. The worms are in constant physical contact with the agar and glass surface along most of their length during the experiment and the crawling behavior appears to be similar to movement on a plate and dissimilar to thrashing in liquid. C) Mechanical damaging of the agarose can ruin the experiment. A fine pick is essential. It should not have sharp corners. A soft eyelash attached to a pipet tip can be used to move bacteria or worms into the chambers instead of using a platinum pick. In most cases, however, overdried agarose is the reason for damage. The pick or eyelash should, ideally, only barely touch the agarose itself, and the water film on the agarose surface should pull off worms and bacteria. When sealing the chambers, again, the moisture of the agarose is essential. There should not be any free liquid on the agarose surface because this may wash away bacteria and worms during sealing. Large bubbles may be removed by gently lifting a corner of the agar slab. Small bubbles that are smaller than the chamber often get trapped inside the chambers. These bubbles are not a problem and will disappear by absorption. After assembling the dish, check again that the agarose is not too dry or too wet. The chambers should be nicely sealed and there should not be any flow of liquid between chambers. If the agarose is too wet, the chambers will not seal properly. Worms may escape or their food will be washed away. If the sample is too moist, simple open the lid and let the agarose dry for a minute or two. If the agarose is too dry, the chambers may collapse and the worms will escape.
Scaling up by zooming out
For fluorescence imaging, zooming out is limited by the low amount of light obtained at lower magnifications. Also, EMCCD cameras are optimized for sensitivity and often have a relatively low resolution. However, scaling up imaging four-fold is well possible. For instance, four chambers of 190 µm x 190 µm can fit onto one frame when using 140X magnification (achieved by using a 20X Objective and a 0.7X camera mount) and a 512 x 512 pixel, 8 x 8 mm camera. A high-resolution camera with a large chip (such as sCMOS cameras, 16.6 mm x 14 mm chip, 2560 x 2560 pixels = 5.5 Megapixel) optimizes scaling up of DIC and bright field imaging. For instance, up to 30 L1 worms in 190 µm x 190 µm chambers can fit onto each frame of this camera when using a 100x magnification (see Figure 3). In principle, zooming out and scanning can also be combined to obtain even greater numbers of animals. Most neurons stay quite well in focus, so that only one focal plane needs to be imaged. If neurons are found to move out of the focus, a z scan using a piezo drive can be taken at each time point.
Adaptation to different behaviors
This protocol gives a good idea of the behavior across long time scales. Obviously, the timing of the bursts needs to be adapted to different behaviors and life stages.
Limitations of the technique
Several factors limit the duration of imaging. The most important is the amount of food. Once the food is consumed, larvae stop developing. Thus, in small chambers (190 x 190 µm) worms develop until the L3 stage and then arrest. If longer imaging time is required, larger chambers have to be used. The maximum duration of long-term imaging is in the range of 2.5 – 3 days. If longer imaging is required, the worms need to be recovered and placed into new chambers. When imaging adult worms, another limitation is caused by the offspring of these worms. Adult worms lay eggs from which larvae hatch. These larvae will also stay inside the chamber, consume food, and may disturb the image analysis. If offspring is a problem, a solution is to either use sterile adults or to repeatedly place the worms into fresh chambers. To recover worms, the agarose slab containing the chamber is cut free with a scalpel, is pulled off the coverslip, and is placed onto an NGM plate from which the worms can be recovered. Another limit is the restriction of the worms to relatively small areas. This may be a problem if long-range movement needs to be analyzed. While animals in the chambers can be stimulated mechanically and optogenetically21,27,34,35, the sealed nature of the chamber will make it difficult to apply soluble or volatile stimulants. Biologically important gases such as oxygen or carbon dioxide can diffuse freely in the agar. The large air reservoir in the dish should keep the gas concentrations in the chambers constant over the time needed for experiments. However, it should be kept in mind that the local oxygen concentration in the chamber may resemble more the conditions found in liquid culture than culture on the plate.
Significance with respect to existing methods
Microfluidic devices have greatly advanced behavioral and developmental studied in C. elegans. Often, microfluidic structures are made of PDMS12. Here we describe a protocol for generating microfluidic culture chambers made from agarose. The strength of this technique is the combination of high imaging quality, correlation of behavior with physiological measurements, long-term imaging, and a reasonably high throughput. High image quality is achieved by imaging through the glass coverslip using high NA objectives. As a result, fluorescence imaging such as calcium imaging and confocal imaging of subcellular structures can be performed. Because the animals are not immobilized like in other systems, it allows a correlation of behavior with physiological measurements. Because the animals have ample food, they continue developing allowing long-term imaging. This system can image many worms in one run because animals are restricted to their defined chambers. Thus, this method can be easily scaled up9,27,34-36.
Future applications
So far, this system has been used mainly to study sleep behavior in C. elegans L1 larvae. However, the adaptation to all stages will make it possible to study a wide range of behaviors also in dauers and adults. A wide array of behaviors can be studied with this technique ranging from mating to egg laying.
The authors have nothing to disclose.
The Max Planck Society and a Göttingen Graduate School Junior Group stipend to H.B. funded this work.
high-melting point agarose | Fisher Bioreagents | BP(E)164-1000 | |
Top Vision Low Melting Point Agarose | Thermo Scientific | R0801 | |
LED system | CoolLED | pE-2 | |
compound microscope | Nikon | TiE | |
stereomicroscope | Leica | M5 | |
EMCCD camera | Andor | iXon 897 | now sold as iXon Ultra 897 |
sCMOS camera | Andor | Neo | |
plasma cleaner | Harrick | PDC-23G2 | |
lid heater | MPI workshop | custom made | |
plastic dish | Falcon | 08-757-100A | |
z-stage for the microscope | Prior | Nano Scan Z | |
x-y-stage for the microscope | Prior | Proscan III | |
PDMS stamp | Abbott Laboratories | custom made | |
red light filter | Chroma | ET660/50m | |
35mm petri dish | Falcon | Falcon Disposable Petri Dishes, Sterile, Corning | |
double sided sticky tape | Sellotape/Henkel | double sided 12mm x 33 m | alternatively Advance double sided sticky tape from Rubance Adhesives can be used. |