The aim of this procedure is to efficiently quantify the dimensions and brightness of punctate fluorescent signals in gans. So large populations of objects may be analyzed. Statistically synaptic proteins are imaged.
In this demonstration, the first step of the procedure is to generate high density synchronized cultures of the ELEGANS of interest. Next, obtain confocal images of the labeled synapses minimizing one to one variability from the images. Synaptic puncture are automatically identified and distinguished from background fluorescence.
The final step is to tabulate and organize the data for efficient downstream analysis. Ultimately, results can accurately show how experimental conditions affect the distribution of synaptic proteins or any other protein localized to high contrast puncture. The main advantage of this technique over existing methods like analysis of Zack projections is that all of the data are preserved, including synapse volumes and automated identification allows rapid gathering of hundreds of samples per condition.Tested.
This protocol requires high density cultures of adult CL egan's worms grown up in 10 centimeter high peptide NGM agar plates on NA 22 e Coli bacteria have ready exactly one plate for each immunological stain that is planned. Begin by harvesting the worms and a few milliliters of water. Transfer them to a 15 milliliter conical tube and repeat this process until most of the worms are collected.
Pellet the worms and wash the pellet with water up to three times until the supinate is clear. This removes residual bacteria always transfer solutions that may or do contain worms or eggs with plastic pipettes as they can stick to glass. It is now critical to move swiftly through the remaining steps to prevent loss of egg viability.
Release the worm's eggs by re bending them in five milliliters of alkaline hypochlorite solution. Rock the tubes gently and examine them under a dissecting microscope about once per minute. After five minutes, or as soon as about half of the worms in a tube appear bent and broken open.
Fill the tube to the top with egg buffer and invert it several times. After pelleting the lies worms aspirate and discard the sup natant. Then wash the pellet three times with egg buffer.
After the washes, Reese suspend the pellet in five milliliters of water, followed by adding five milliliters of sterile. 60%sucrose in water mix well. After pelleting the suspension, the eggs will be at the meniscus and appear cloudy.
Transfer each egg collection to its own unseeded 10 centimeter NGM Agar plate Water can be used to rinse X to the plate, but try to minimize the amount of liquids transferred. Incubate the plates overnight at 20 degrees Celsius for the first few hours. Vent the plates by leaving the lids slightly off, but be sure to cover them overnight.
After hatching, transfer the L one larvae to a 15 milliliter conical tube filled with S basil. Pellet the L ones and resuspend them in a minimal volume of S basil. Then split each L one collection onto two 10 centimeter NGM agar plates seeded with NA 22 bacteria.
Monitor the cultures once or twice per day. If they're in danger of starving. Transfer them to fresh NA 22 plates prior to starving while type N two gathering lines and move together like a wave away from food depleted zones on these plates, the food will be completely depleted within a few hours.
When the worms reach the desired age, they're ready for immunostaining or live imaging to mount the worms. For confocal imaging. Use Antifa mounting media to adjust their density to a few hundred worms per microliter.
With experience, this density can be recognized by the turbidity of the suspension. Ideally, worms will be densely packed on the microscope slide, but not overlapping excessively. On the slide, make a pad of 2%aros in ESP basil surrounded by a thin ring of Vaseline.
To prevent a evaporation during imaging, apply the Vaseline via a three milliliter syringe fitted with a cutoff 25 gauge needle. Now pipette a few microliters of the worm suspension onto a cover slip using a pipette tip with the end cutoff to prevent shearing of the worms and lower the agro rose pad onto the cover slip, spreading the worms evenly over the pad under the microscope. Synapses of interest should be oriented to point directly towards or within 45 degrees of the lens only.
Spend a few seconds at this to avoid photo bleaching. The culture and conditions should provide enough worms to meet this strict criterion shown here as an ideal orientation with the ventral cord oriented directly toward the objective lens. This next one would be rejected because the ventral cord is oriented directly away from the objective lens.
Note how the dorsal cord oriented toward the lens in this specimen produces the more sharply focused image. Here is another one that would be rejected because the dorsal and ventral cords are oriented at the edges of the worm. So excitation and emission light must pass through more worm tissue and would become distorted.
The major challenge is to minimize variability within treatment groups age synchronization, and choosing worms where the structure of interest is oriented directly toward the objective lens can help a lot. Starting with large cultures increases your choice of worms for maximum consistency. For the best imaging results, choose relatively flat samples that can be encompassed within a stack of 14 or fewer.
0.4 micron thick sections. High speed, low resolution images are sufficient for rapid accurate data collection. Avoid saturating the detector with excessive signal strengths as this will cause underestimation of fluorescence signals.
This demonstration uses the commercially available software package velocity, which needs to be at least version 4.0. Many alternative software choices lose three dimensional information. After opening the multi tiff output files from the confocal microscope select image, then extended focus, and then freehand.
ROI now crop away areas of the image that do not contain the structure of interest. Using the activated tool, extended focus provides a Z plane projection to ease the ROI selection. This does not affect the underlying data.
Likewise, brightness and contrast adjustments do not affect the underlying data. Finish the crop by selecting actions and crop to selection. Next, measure the length of the area analyzed.
Using the line tool, the length of the line will be calculated and added to the data table. Now, identify objects using the objects by intensity filter in measurements mode. Inclusion of an independent synaptic marker labeled with a unique color may help unambiguously identify synapses specify a threshold such that synaptic clusters are highlighted and nons synaptic background is not.
Do this just once using a control sample and use it for all subsequent samples thresholding each sample separately is unacceptable because it introduces the potential for experimenter bias. Objects are selected and tabulated automatically. Typical synapses range from a few to a few hundred voxels before exporting the data, sort the data by object size.
This eases the downstream removal of objects of two or fewer voxels, which are usually just background specs. Now, export the data in CSV formats, which can be opened by any spreadsheet program. Each image produces one output file to sort and export micrographs of the CL gans ventral nerve cord showing on 49 GABA receptors were produced with the described protocol.
All of the animals appeared very similar in size and maturity. The total synaptic fluorescence values for five worms was normalized, so the length of the nerve cord analyzed. This data showed standard error values of about 10%of the mean.
The image on the left is raw without threshold and background spec removal. The synapses were treated with mus carmal, a GABA receptor agonist that causes receptors to become downregulated after long exposure. Total fluorescence was quantified and normalized to nerve cord length cumulative probability.
Histograms of individuals synapse fluorescence content were calculated. The same was calculated for synapse volume. These measurements show agonist exposure significantly reduce synaptic content and volume.
Overall, quantitative results should always reflect what can be appreciated visually. If not inspection of the images and the objects identified by velocity should reveal where the error occurred and suggest corrective action such as thresholding or removal of artifacts While attempting this procedure. It is critical to choose worms for imaging based on how they are geometrically oriented rather than a subjective assessment of the quality of the image where possible blind scoring is highly recommended.
