A Streamlined Protocol for Single-Molecule Localization Microscopy in Arabidopsis Nuclei

My research focuses on epigenome editing and the visualization of chromatin organization in Arabidopsis nuclei using super resolution microscopy. Existing methods are not adapted to STORM imaging. This protocol improves nuclei purity, labeling consistency, sample mounting, and imaging stability.

It applies to Arabidopsis thaliana and other plant systems for studying nuclear organization, chromatin structure, and epigenetics at high resolution. To begin, place a six-well culture plate on ice under the fume hood. Mix the fixation solution containing 4%volume by volume, methanol stabilized formaldehyde 0.1%Triton X100 and PBS.

Add approximately four milliliters of ice cold fixation solution to each well under the fume hood. Using clean forceps, remove the Arabidopsis seedlings that are between eight and 15 days after germination from solid half strength Murashige and Skoog, or MS medium. Immediately submerge the seedlings in the fixation solution and place approximately 20 seedlings per well.

Use three wells to obtain sufficient material per sample. Place the plate in the vacuum chamber filled with ice and close it. Apply a vacuum cycling between minus 0.6 bar and minus 0.8 bar for 15 minutes to remove trapped air and allow deep penetration of the fixative into the plant tissues.

Then, slowly release the vacuum. Mix the sample and apply the vacuum for a further 15 minutes. Under the fume hood, remove the fixation solution with a pipette.

Wash each well three times for 10 minutes each under gentle agitation using five milliliters of ice cold PBS. After the washes, remove the PBS. Using forceps, transfer the fixed tissue to a 120 by 120 millimeter Petri dish and add 50 microliters of nuclei isolation buffer to the fixed seedlings.

With a blade, chop the fixed tissue. Using the same blade, scrape the chopped tissue to one edge of the Petri dish. Add 600 microliters of nuclei isolation buffer and wash off any tissue stuck to the blade.

Transfer the tissue slurry into a two milliliter Dounce homogenizer tube and stroke five to 10 times with the smaller pestle. Then, stroke with the larger pestle until it moves smoothly inside the tube, typically requiring five to 10 strokes. Filter the homogenized sample through a 30 micrometer mesh filter into a 50 milliliter tube.

Transfer the filtrate into a fresh 1.5 milliliter tube. Centrifuge the suspension for 10 minutes at 800G using the lowest ramp for both acceleration and deceleration. After centrifugation, note that a small white pellet is visible among the green tissue debris.

Discard the supernatant and re-suspend the pellet in 600 microliters of nuclei isolation buffer. Centrifuge again for 10 minutes at 800G and remove the supernatant. After completely washing off the green debris, store the isolated nuclei suspension at four degrees Celsius for a maximum of one week and immunostain the nuclei with desired antibodies.

Heat the 1.5%weight by volume low-melting agarose solution at 90 degrees Celsius for one minute to dissolve the powder and obtain a clear solution. Vortex briefly to ensure complete mixing. Cool the solution and maintain it at 37 degrees Celsius before proceeding to avoid heat denaturation of the glucose oxidase buffer.

After the low-melting agarose solution reaches 37 degrees Celsius, prepare the STORM buffer by adding 100 millimolar mercaptoethylamine and glucose oxidase buffer to the low-melting agarose to achieve a 1X final concentration and mix the solution gently. Deposit 10 microliters of the nuclei mixture onto an ozone-treated 1.5H cover slip that has been treated for 20 minutes in an ultraviolet ozone cleaning system. Wait for three minutes to allow the nuclei to deposit on the cover slip.

Then, remove the excess liquid and wait for two minutes. Add 10 microliters of the prepared low-melting agarose to the cover slip and immediately cover with a microscope slide. Seal the cover slip using silicone sealant.

Mix equal amounts of solution A and solution B in a small Petri dish using a micropipette tip. Apply the mixture over the edges of the cover slip to seal the sample. Allow the silicone sealant to harden for five minutes.

Mount the sample slide on the microscope and find focus. Locate and focus on a nucleus. Activate a focus-maintaining module if available to ensure stability during long acquisitions.

Select a region where one or a few nanodiamonds are positioned near the nucleus to improve drift correction during reconstruction. For two-color imaging, begin acquisition with the longer wavelength channel and then switch to the shorter wavelength channel to complete imaging. Process the raw image stacks in ThunderSTORM using Gaussian fitting with weighted least squares estimation and generate reconstructed images.

Apply filtering based on sigma and uncertainty below 30 nanometers to retain accurate localizations and remove noise. Finally, perform sample drift correction using ThunderSTORM cross correlation or the COMET algorithm and confirm minimal drift in the reconstructed images. Nuclei were isolated from 15-day old wild-type seedlings immunolabeled with AF647 conjugated secondary antibodies and counter-stained with Hoechst H33258.

Confocal imaging showed multiple isolated and well-preserved nuclei with minimal debris with chromocenters and the nucleolus clearly distinguishable. RNA polymerase II signal was absent from chromocenters and the nucleolus. Diffraction limited maximum intensity projections and corresponding SMLM reconstructions revealed structural details of RNA polymerase II and DNA labeling, including enlarged regions of interest showing finer structures.

Localization filtering based on sigma and uncertainty improved the definition of both RNA polymerase II and DNA signals compared to detected localizations. And further refinement after drift correction maintained this improved structure. Filtering based on sigma values removed localizations with excessively small or large fitted widths.

Fourier ring correlation analysis showed that resolution in the AF647 channel improved from 90.1 to 47.7 nanometers after filtering, while the JF549 channel improved from 56.6 to 27.9 nanometers. Maintaining nuclear integrity while minimizing debris and background fluorescence is critical for achieving high-quality single molecule imaging. Future studies can link nuclear architecture to gene regulation under environmental or developmental conditions.