The overall goal of the following experiment is to employ genetically encoded fluoro fours to measure calcium dynamics of single neurons within the nematode worm. See elegance, this is achieved by first mounting and demobilizing intact C elegance that express a calcium sensitive fluorophore in the neurons of interest on a standard microscope slide. Next individual neurons are imaged in vivo using video microscopy and standard epi fluorescent imaging techniques.
Then the video is analyzed to measure fluorescence intensity at specific locations in the neuron for two commonly used Fluor fours G camp, which gives a robust large amplitude single channel signal and fret based chameleon, which generates a dual channel ratio. Metric measurement results are obtained the shown neuronal activity in response to a specific sensory stimuli, as well as cellular calcium physiology in response to cellular damage, both of which are based on optical measurement of relative calcium levels within the imaged neuron. The main advantages of this technique over similar methods in vertebrate systems or conventional electrophysiology is that CL elgan represents a robust, flexible, and genetically tractable system for in vivo neuronal imaging that is technically simple and cost effective.
This method can help answer key questions in a broad range of field based neuron biology, allowing measurements of both activity and sine with individual neurons in vivo. Begin with a standard compound microscope with epi fluorescence imaging capabilities for best image and signal quality. Use a high magnification, high numerical aperture objective and high sensitivity cooled CCD camera in order to maximize image resolution and sensitivity while minimizing background noise for single color flora fours.
Use the standard microscope fluorescence filter set designed for the particular wavelength of the Fluor four for G camp. Use a standard GFP filter set optimized for excitation at 488 nanometers and emission at 525 nanometers four ratio metric flora fours such as chameleon use a dual imaging system such the identical images can be simultaneously captured at two separate wavelengths. This can be accomplished using a commercially available or home-built system that projects dual images side by side onto the camera in the following way.
A microscope filter set with a 440 plus or minus 20 nanometer excitation filter and 455 nanometer long pass iic mirror an image mask placed at the initial image plane immediately outside the microscope imaging port where the camera CCD sensor would usually sit the first relay lens set, one focal length away from the initial image plane such that it collates the light, a dichroic mirror that splits the light into the two separate imaging wavelengths or channels reflecting one color while transmitting the other emission filters. Further restricting the transmitted light to the specific imaging wavelengths. 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.
A pair of mirrors and a second dichroic mirror directs the beam paths such that the two images recombine and are projected side by side onto the camera. CCD sensor. Here is an example of the resulting image.
Once set up, the images can be adjusted and fine tuned in the following way, move the camera position such that the transmitted image covers half of the camera's field of view. Move the reflected image to cover the other half of the field of view by adjusting the last two mirrors in its beam path and optimize the focus by moving the second relay lens for each channel back and forth along the beam path. Acquire animals expressing a calcium sensitive fluorophore in the neuron of interest from the Citis genetic center or other sources.
Alternatively, construct transgenic animals using standard seal elgan techniques. Then use one of the following techniques to immobilize seal elegance for pharmacological paralyzation at 0.05%leal to 2%aros and make aros pads for imaging by pressing a drop of molten aros between two glass slides that have two pieces of lab tape as spacers. After removing the top slide, pick five to 10 animals and transfer them onto the pad.
Cover the pad with a cover slip and apply small amounts of a hot one-to-one mixture of paraffin wax and petroleum jelly to the corners to hold it in position. Allow five to 10 minutes for the lemisol to take effect and the animals to become completely still. To immobilize animals using a stiff aros method, make 10%aros pads.
Add about three microliters of a one to 10 polystyrene microsphere solution to the surface of the pads and quickly pick five to 10 clean sea elegance into the pool of beet solution. Gently cover with a cover slip and apply molten wax to the corners. Typical experiments require administering a specific stimuli or condition to the animal while video recording the response of a single neuron.
For video acquisition. We frequently image at about one frame per second with about 300 millisecond exposure times. Video must be analyzed to measure the fluorescence intensity for each frame within the region of interest or ROI of the imaged neuron relative to a nearby background region.
Our analysis program displays a compressed image of all images in the movie from which a rough boundary encompassing the object of interest in all frames is manually selected and an independent background region selected. For the first 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 for ratio metric images generated with chameleon, select a similar rough ROI and background region for each of the dual images or channels, and then the ROI from one image on the first frame, 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.
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. Ratio metric values are calculated as the ratio of the two channels. An ROI selected within the cell body generates the strongest, most robust signals, but it is also possible to select and measure signals from an ROI along the nerve processes.
Exposure to excitation light can cause bleaching of the flora four and is marked by a steady decrease in signal that is dependent on exposure levels if bleaching occurs. Reduce exposure times and or excitation intensity with neutral density filters to minimize noise created from sea elegance movement during recording. In response to light exposure and experimental stimuli, carefully execute the immobilization procedures using clean preparations.
For successful image analysis, choose background regions that are reasonably uniform darker than the image to neuron and sufficiently far away to avoid scattered light. Shown here is a single C elegance, A SJ neuron expressing G camp responding to an external three volt per centimeter electric field for three separate trials each lasting 10 seconds. The animal was immobilized using lemisol, but the video displays slight faults of both movement and focus.
Drift fluorescence intensity was measured at the cell body. The signal is robust despite the video faults showing a large about 250%increase in fluorescence in response to both the first and third 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. Traumatic cellular injury triggers a large calcium transient within a neuron that plays an essential role in the cell's physiological response. This video shows the damage induced response to laser ex sodomy of a single neuron.
Dual imaging optics were employed to record signals from both the CFP and YFP fluorescence channels of the chameleon reporter. This figure shows the resulting fret signal as measured at the cell body over the course of the laser surgery experiment. In response to laser damage and an increase in cytoplasmic calcium at T equals zero seconds.
The CFP rapidly decreases and the YFP increases resulting in an immediate about 200%increase in the ratio metric signal, which is sustained for about 90 seconds before falling back to near baseline levels. Bleaching is minimal as evident from the consistent baseline. This figure shows the resulting fret signal as measured in the axon segment close to the cut point here.
The local amount of fluoro four varies over the course of the experiment. Complicating the signal laser surgery severs the axon and briefly ruptures the membrane, allowing flora four to escape and temporarily reducing its local cytoplasmic concentration. This is evident from an initial decrease in the YFP trace at later time points.
The severed end swells as part of its continued recovery, resulting in more localized fluoro four and an increase in intensity. This is most evident in the slowly increasing CFP trace, however, these variations affect both channels equally and the fret ratio effectively compensates giving a robust signal. The resulting measurement shows a response similar to the cell body with an immediate about 150%increase in signal, a dramatic recovery to near baseline at about 90 seconds, and then an additional smaller secondary response at about 150 seconds.
Commercial microscope attachments for ratio metric imaging are available, as is more sophisticated image analysis software that generates pixel by pixel ratio metric measurements Once mastered, these techniques can be done with relative ease and without the complicated sample preparation tissue dissection or imaging techniques required by other model systems or methods. Entire experiments can be run in a matter of an hour or two if performed properly. After watching this video, you should have a good understanding of how to immobilize the elegance for imaging, perform video max copy on individual neurons in vivo, and analyze the resulting video to measure relative calcium levels based the neuron.
