The synaptonemal complex is a key component for meiosis. To study its function, we must also understand its structure for which super resolution microscopy has turned out to be incredibly useful. We have optimized our protocol to stably attach immunostained C.elegans gonad to a coverslip which helps to improve the resolution down to 30 nanometers in intact tissues.
The design of single molecule localization microscopy experiments, the subsequent analysis, and also the interpretation of the data can be quite challenging and should best be set up together with an expert. Demonstrating the procedure will be Ivana Cavka, a pre-doctoral fellow in the lab. The acquisition of the single molecule localization microscopy images will also be shown by Rory Power, an optical engineer at the MBL Imaging Center.
Begin by picking age-matched Caenorhabditis elegans worms from NGM agar plates. Transfer the worms into a 30 microliter drop of EBTT on a coverslip. Wash the worms with additional 30 microliters of EBTT.
Remove 30 microliters of the solution leaving a 30 microliter drop with the worms on the coverslip. Use a scalpel blade to cut off the worms'heads and/or the tails to extrude the gonad. In a successful dissection, gonad tissue will be fully extruded outside of the worm body.
Pipette 30 microliters of the fixative solution into the drop of the dissected worms. Mix by pipetting and leave the samples to fix for one minute. Stop the fixation exactly after one minute by transferring the worms using a 20 microliter pipette onto a PCR tube filled with TBST.
Then perform a wash by spinning the dissected samples on a mini benchtop centrifuge. Once the supernatant is removed, resuspend the worms in 200 microliters of fresh TBST. After the final wash, resuspend the worms in 200 microliters of PBST and place the PCR tube on ice.
Spin the dissected samples on a mini benchtop centrifuge and remove the supernatant. Add 50 to 100 microliters of cold methanol, mix by pipetting, and leave the samples in methanol for 30 to 60 seconds on ice. Wash the samples twice with 200 microliters of PBST.
After spinning the samples and removing the supernatant, add the blocking solution to the samples and keep it at room temperature for 45 to 60 minutes. Dilute the primary antibodies to the working solutions in the blocking buffer. Spin the samples on a mini benchtop centrifuge, removing the blocking solution, and add 30 to 50 microliters of the primary antibody solution.
Incubate overnight at four degrees Celsius. At the end of the incubation, wash the samples three times in PBST. Dilute the labeled Fab prime two fragments to the working solutions in the blocking buffer.
After the third wash, spin the samples down, remove the supernatant, and add 30 to 50 microliters of the secondary antibody solution. Place the sample in a dark chamber and incubate for 30 minutes to two hours at room temperature or overnight at four degrees Celsius. Then wash the samples three times with PBST for five to 15 minutes.
Spin down the stained samples and remove the supernatant. Add 50 microliters of PBST and transfer the stained worms onto a coverslip. Pipette 5.7 to 6.3 microliters of the post-fixative solution onto a poly-l-lycine coverslip.
Pipette off the dissected worms in the same volume and transfer into the drop of fixative on the poly-l-lysine coverslip. Cover the sample with a small coverslip. Remove the excess liquid using a small piece of filter paper and fix the sample for three to five minutes in a dark chamber.
Freeze the samples by placing them on an aluminum block in dry ice. After at least 20 minutes, remove the smaller coverslip using a razor. Dip the coverslip into a 50 milliliter conical tube containing ice cold PBS or methanol chilled at minus 20 degrees Celsius for approximately 10 seconds.
Place the coverslip into a well of a six-well plate filled with PBST buffer. Remove the PBST from the well, add fresh PBST and leave the sample for five minutes. After washing with PBST once again, leave the samples at four degrees Celsius until imaging.
Before imaging, assess the quality of the sample mounting under a stereo microscope. If using the custom-made sample holder shown here, wrap the magnetic ring with parafilm, then prepare one milliliter of imaging buffer. Take a coverslip from the six-well plate.
Place it in the custom-made holder and fix the coverslip in the holder with the parafilm-wrapped magnetic ring. Gently pipette the imaging buffer in the chamber created by the magnetic ring on top of the sample and seal the chamber using parafilm. To mount the sample, add one drop of immersion oil to the clean oil objective.
Without introducing any air into the immersion oil, gently place the sample holder with the mounted sample onto the microscope stage. Using the EMU plug-in window within MicroManager 2, move the piezo stage until the signal from the focus lock laser is detected at the quadrant photodiode. Acquire a back focal plane image at lower power with a laser at 640 nanometers excitation to confirm the absence of air bubbles in the immersion oil.
Localize the gonad tissue using the brightfield illumination. Using a low intensity illumination at 640 nanometers, focus on the tissue section containing many synaptonemal complexes. Proceed to expose the sample at high irradiance with illumination at 640 nanometers for approximately 30 seconds until an appropriate blinking rate is achieved.
Acquire 200, 000 frames with an exposure time of 20 milliseconds using the multi-dimensional acquisition tool in MicroManager 2. Meanwhile, set up the UV activation using the activation option of the EMU plugin to maintain the desired blinking rate. The bottommost plane of meiotic nuclei containing synaptonemal complexes was visualized within a Caenorhabditis elegans gonad using HTP-3 and SYP-5 staining.
The chromosome axes in the C-terminus of SYP-5 HA were well-resolved in all three dimensions in synaptonemal complexes located close to the coverslip. However, the resolution deteriorates in synaptonemal complexes located at a distance of five micrometers from the coverslip due to light scattering and spherical aberrations. Images acquired at different piezo distances from the coverslip revealed that the imaged tissue should not be further than two micrometers from the coverslip.
The sample preparation protocol can also be used with TauSTED microscopy. With the optimized sample preparation, HTP-3 in the chromosome axes and the C-termini of SYP-5 in the central region were resolved in frontal view and slightly tilted view. To obtain the best resolution, the gonads must be cross-linked tightly to the coverslip to ensure that the synaptonemal complexes are no further than two micrometers from the coverslip.
We use this protocol not only for single molecule localization microscopy, but also for stimulated emission depletion microscopy. It may also be useful for other super resolution microscopy techniques.
