Our system allows researchers to evoke non localized vibrations with well-controlled properties at non-scaled displacement and to quantify calcium current during responses of the irritants to the vibrations. As a main advantage of this technique, we can noninvasively investigate the neural mechanisms, underlying mechanisms of behavior. Prepare a new nematode growth medium or NGM plate on which Escherichia coli OP50 is streaked in a square pattern using a cell spreader so that the worm spends most of the time in the bacteria during calcium imaging.
Four days before a calcium imaging experiment, transfer two adult ST12 worms onto the plate, and place the plate in a 20 degree Celsius incubator for four days. Perform calcium imaging using a microscope equipped with an acoustic transducer device for stimulation, a 2x objective lens, a high speed CMOS camera, image-splitting optics. Turn on the precision computer in the X Y motorized stage controller.
Then turn on the amplifier and set the volume adjuster to 10 in the base and treble adjusters to eight. To excite GCaMP and tag RFP, turn on the 488 and 560 nanometer lights of the LED light source. Set the 470 and 550 nanometer gauges of the control pod in the light source to 5%so that the intensity of the LED light is suitable for imaging.
Download and install the following software packages and windows. Mouse macro software, software for vibration control, software for running tracking, and software for data analysis. Double click on the tracking software file, then set the exposure time to 0.033 seconds, and binning to two by two to ensure smooth image acquisition.
Click the run button and wait for five minutes to stabilize the software. Input the information for image acquisition. For example, to record 500 images without an interval, enter 500 in the images total box, 500 in the images box, and zero in the intervals box.
After turning on the acquisition button, split the fluorescence image using image splitting optics, projecting the GCaMP channel and tag RFP channel onto two halves of the CMOS camera. Calibrate the coordinates of the GCaMP and tag RFP channels and save the settings. Then set the directory to output the data file and turn off the acquisition.
Read the assay. text file with the mouse macro system so that the mouse cursor is controlled based on the coordinates and the schedule in that file and double click on the software file for vibration control. Set the coordinates of the mouse cursor in wait time until stimulation after starting the recording, then set the frequency and the amplitude values in the software for vibration control.
Transfer a single worm expressing both GCaMP and tag RFP from the incubated plate to a fresh NGM plate where E coli OP50 has been streaked. Attach the NGM plate to the acoustic transducer using transparent adhesive tape. For calcium imaging, turn on the acquisition button and find the worm at 2.5x magnification.
Set the field of view as 1.1 by 1.1 millimeter with a resolution of 2.6 microns per pixel. Turn off the bright light and click on the homing button to track the fluorescent spot of a worm, which initiates the movement of the X Y stage to keep the maximum intensity region of the worm in the middle of the field of view in the tag RFP image. Ensure that the values of intensity are approximately 1000.
If not, fine tune the 470 and 550 nanometer gauges of the control pod in the light source. Press the run button in the mouse macro system to allow mouse cursor control, and verify that the output BMP file is appropriately created. For data analysis, create a folder on the desktop and name it Calcium Imaging"then create a folder inside the Calcium Imaging folder and name it CI result for the output result files.
Double click on the dual view imaging. MB file written in the data analysis software, and insert the file paths to the CI result folder in the folder with the BMP file. Push the shift and return keys simultaneously to start an automatic analysis, which uses the value of a maximum intensity region in the tag RFP image.
Finally, check whether the output files are appropriately created in the CI result folder. In the representative analysis, GCaMP and tag RFP channel data was obtained as a series of images. Displacement of the Petri dish induced by non-localized vibration system was also quantified.
The displacement can be controlled by setting the amplitude value in the software for vibration control, and the volume adjuster and the amplifier. Whereas the frequency can be regulated by setting the frequency value in the software. A transient calcium response of AVA neurons was observed upon stimulation with vibrations having a frequency of 630 Hertz and a displacement of approximately 4.5 micrometers per second, indicating that the Ava neuron was activated during a worm's backward response to the non localized stimulation.
In our procedure, we can quantify only a single neuron. However, this system can also be combined with a biometric imaging method to noninvasively quantify multiple neurons or even the whole brain.
