In vivo Neuronal Calcium Imaging in C. elegans

1. Optical Setup

1. Use a standard compound microscope with epifluorescence imaging capabilities. We use a Nikon Eclipse Ti-U inverted microscope with an Intensilight HG Illuminator.
2. For best image and signal quality use a high magnification, high numerical aperture objective. We typically use a Nikon X60 1.4 N.A. oil immersion objective. In some cases it is possible to use lower magnification (X40, X20) depending on expression level of the fluorophore and signal strength.
3. Use a high sensitivity cooled CCD camera, such as an Andor Clara Camera to maximize image sensitivity and minimize background noise.
4. For single color fluorophores use the standard microscope fluorescence filter set designed for the particular wavelength of the fluorophore. For GCaMP use a standard GFP filter set optimized for excitation at 488 nm and emission at 525 nm. No further optical modifications are required.
5. For ratiometric fluorophores, such as cameleon, use a dual imaging system such that identical images can be simultaneously captured at two separate wavelengths. This is accomplished by projecting the dual images side-by-side onto the camera. A simple setup to accomplish this, using basic optical components on an optical grade breadboard, is illustrated in Figure 1. It consists of:
   1. A microscope filter set with 440 ±20 nm excitation filter and 455 nm long pass dichroic mirror.
   2. An image mask placed at the initial image plane immediately outside the microscope imaging port (where the camera CCD sensor would usually sit). This restricts the field of view such that it will fill only half of the camera sensor. Placing this mask at the first image plane results in sharp image borders and no overlap when the two images are projected side-by-side.
   3. The first relay lens set one focal length away from the initial image plane such that it collimates the light.
   4. A dichroic mirror that splits the light into the two separate imaging wavelengths or channels reflecting one color (>515 nm, yellow) while transmitting the other (<515 nm, cyan).
   5. Emission filters (band-pass filters) further restricting the transmitted light to the specific imaging wavelengths (cyan at 485 ± 40 nm and yellow at 535 ± 30 nm),
   6. The second relay lenses (one for each channel) placed in the beam paths such that it refocuses the light to a second image plane at the CCD camera.
   7. A pair of mirrors and a second dichroic mirror directs the beam paths such that the two images recombined and are projected side-by-side onto the camera CCD sensor. Figure 4A shows an example of the resulting image.
6. Initial Optical Alignment: Initiate setup and alignment of the optics with a high contrast image using a low magnification lens and transmitted wide-field light illumination (a black sharpie drawn on a microscope slide works well). Bring the image to focus through the microscope oculars and then switched to the microscope port to be used. With high light illumination view the image on an index card at the initial focal plane (a few cm outside the microscope port). Place the first relay lens (C) in the beam path such that it is one focal distance from the initial focal plane and collimates the light while not deflecting the beam path (i.e. is directly centered in the beam path). Place the mask at the first focal plane, adjusted to restrict half the field of view and produce a sharp boarder in the image. Position the mirrors and filters (D, E, G) such that the beam path remains parallel to the table surface and runs in two parallel paths side-by-side, as shown in Figure 1B. Center the second relay lenses (F) in their respective beam paths and move them back and forth along the paths to bring the images to a focus on an index card placed were the camera CCD sensor will be. If done correctly the two images should be parfocal with the microscope oculars and other camera ports. Finally place the camera in position and fine-tune the alignment.
7. Fine Tuning Optical Alignment: Use the dual image viewed by the CCD camera to fine tune the optical alignment. Use a high contrast image and start with lower magnification, switching to higher magnification for final alignment. Position the transmitted image (cyan channel in Figure 1B) to cover one half of the camera’s field of view by moving the camera in X and Y. Position the reflected image (yellow channel in Figure 1B) to cover the other half of the field of view by adjusting the last two mirrors (G) in its beam path. Optimize focus by moving the second relay lens for each channel back and forth along the beam path. If left unaltered the optical setup should be stable and only require rare fine tune adjustment.

2. Sample Preparation and Data Acquisition.

1. Acquire animals expressing a calcium sensitive fluorophore in the neuron of interest from the Caenorhabditis Genetics Center (C.G.C.) or other sources. Alternatively transgenic animals can be constructed using standard C. elegans techniques. Single cell expression is not necessary (and sometimes not desirable if multiple neurons are to be investigated, see ¹²) as long as images and measurements from multiple cells can be readily separated. If the fluorophore transgene is not integrated into the genome, significant variation in expression can occur from animal to animal. In this case, preselecting animals with higher expression levels using a fluorescent dissecting microscope increases the signal-to-noise ratio and improves the measurements. Fluorophore expression levels need to be sufficient to be readily imaged with the camera under the acquisition parameters required by the experiment.
2. Make the agarose pads required for the desired immobilization technique (see 2.3 below) by pressing a small drop of molten agarose between two glass slides spaced by two pieces of laboratory tape. If necessary, agarose pads can be transferred to a glass cover slip before mounting the animals. See Figure 2.
3. Immobilize C. elegans for imaging using one of the following techniques:
   1. Pharmacological Paralyzation: Add 0.05% Levamisole to 2% agarose pads (mixing it into the molten agarose before making the pads in 2.2). Pick 5-10 animals onto the pad and cover with a cover slip. Apply small amounts of hot wax mixture (50% paraffin wax and 50% petroleum jelly) to the corners of the cover slip to hold it in position. Allow 5-10 min for the Levamisole to take effect and the animals to become completely still.
   2. Stiff Agarose Immobilization: C. elegans can be physically immobilized using stiff 10% agarose pads and 0.05 μm diameter polystyrene nanoparticles. Make 10% agarose pads (10% agarose in NGM buffer by weight) as in 2.2. Add ~3 μl of bead solution (1:10 polystyrene microspheres into NGM by volume) to the top of the pad. Quickly, pick 5-10 C. elegans into the pool of bead solution on the pad and gently cover with a cover slip. Use molten wax applied to the corners of the cover slip to hold it in position. (For additional details on this technique see ⁷ as well as a previous JoVE article ⁸)
   3. Gluing: C. elegans can be glued in place using small amounts of veterinary grade adhesive applied to one side of the animal.
   4. Microfluidic Devices: Numerous microfluidic devices have been designed to physically hold C. elegans in place for imaging.
4. Place the prepared slide on the microscope and focus on the desired neuron. It is often easiest to find the C. elegans using bright field illumination and low magnification. Then switching to high magnification and fluorescence imaging to find and focus on the specific neuron.
5. Stimulus: If a neuronal response to a specific stimulus is desired (light, electric field, gustatory and olfactory cues, temperature etc.), the stimulus apparatus must be designed into the microscope setup, such that it may be administered to the animals in the sample preparation while imaging.
6. Acquire images using the microscope CCD camera. Exposure times and excitation light level will depend on the baseline signal and expression level of the fluorophore, with weaker signals requiring longer exposure times with concordantly less time resolution. We typically use ~300 msec exposure times.
7. Acquire images either individually or as a time-lapse movie. Because C. elegans neurons generally display slow activation dynamics (i.e. they lack sodium based action potentials), relatively slow frame rates of ~1 sec are often sufficient to measure neuron dynamics. Acquisition rates up to ~90 frames per second have been used although these may be limited by the integration time necessary for weak fluorescence. Individual movies of a single neuron can easily extend for several minutes (~5-10 min) or longer if the immobilization preparation is stable and the frame rate relatively slow.

3. Data Analysis

1. There are a number of different fluorescent imaging software packages available with dynamic and ratiometric analysis capabilities including ImageJ and NIS-Elements. We use a simple custom MATLAB program that measures the average fluorescent signal over a selected region of interest.
2. For individual images (or time-lapse movies that remain perfectly still) select a region of interest (ROI) within the imaged neuron and a separate nearby region for the background measurement. For ratiometric images select similar ROI and background regions for each of the dual images.
3. Slight movement of the imaged neuron within a time-lapse movie requires additional analysis to automatically select the ROI in each frame, which can be accomplished using numerous object tracking schemes. Our program displays a compressed image of all images in the movie from which a rough boundary (encompassing the object of interests in all frames) is manually selected and an independent background region selected. For the first frame, an ROI is manually selected from within the boundary region. For each subsequent frame, the program selects the brightest pixels from within the boundary region to generate an ROI of the correct size (i.e. the same number of pixels as the manual first frame ROI). For images where the object of interests is sufficiently brighter then the surrounding background within the boundary region this protocol selects a reasonable ROI for each frame.
4. The fluorescent signal, F, for each frame is calculated as the average pixel intensity in the ROI minus the average intensity in the background region. Ratiometric values are calculated as the ratio of the two signals (YFP-YFP_background)/(CFP-CFP_background)-0.65. The factor of 0.65 compensates for bleed through of the CFP channel into the YFP channel, which will be dependant on the fluorophores as well as the particular filter set used (this value is typically ~0.6 for cameleon setups).
5. The relative calcium level for each frame is calculated as the percent change in intensity normalized to an initial baseline value measured from the first frame (or series of frames): ΔF/F=(F(t)-F_o)/ F_o, where F(t) is the fluorescence at time t and F_o is the initial baseline value.
6. A ROI selecting the cell body generates the strongest most robust signals but it is also possible to select and measure signals from a ROI along the nerve processes.

4. Problem Solving

1. Bleaching: Exposure to excitation light can cause bleaching of the fluorophore and is marked by a steady decrease in signal that is dependent on exposure levels. Ratiometric measurements help to compensate for this. However selective bleaching of one wavelength can still occur (typically CFP bleaches quicker then YFP). If bleaching occurs reduce the light exposure level by reducing exposure times and/or excitation intensity (with neutral density filters in the illumination pathway). If necessary, one can compensate for bleaching by running mock trials (with the same excitation exposure but no neuronal stimulus) and quantifying the time constant of signal decay.
2. Immobilization: For the simple image analysis described here it is critical the animal remains relatively still throughout the recording period. C. elegans respond to light exposure and experimental stimuli, making them most likely to move at the time of recording. Slight movement can introduce significant noise into the measurement especially when measuring from axon processes. Ratiometric analysis helps to mitigate these effects to some extent and measurements from the cell body are more robust. Careful execution and refinement of the immobilization procedures will result in completely still animals.
3. Background Selection: Careful selection of a background region is necessary for successful image analysis. Background regions should be reasonably uniform and darker then the imaged neuron. It must be sufficiently removed to avoid scattered light (and thus pickup a signal) from the imaged neuron. We find a uniform region 20-50 μm from the ROI works well in most cases.
4. Ratiometric Optics: Correct alignment of the imaging optics is critical for generating a clear dual image. Commercial microscope attachments for ratiometric imaging are available. An alternative technique is to employ rapid sequential imaging using a fast filter wheel to rapidly switch between emission wavelengths while recording separate images for each.
5. Ratiometric Analysis: The analysis described here is a relatively simple technique that generates robust signals by virtue of averaging over a ROI. It requires a recognizably bright object to select the ROI. More sophisticated ratiometric analysis is possible by precisely aligning and mapping the dual images such that the fluorescent ratio can be calculated pixel by pixel.