Name: A Streamlined Protocol for Single-Molecule Localization Microscopy in Arabidopsis Nuclei (Video) | JoVE Journal
Uploaded: 2026-05-08T14:05:00.000+00:00
Duration: 9 min 54 s
Description: Learn a streamlined protocol for single-molecule localization microscopy in Arabidopsis thaliana nuclei. This method enables nanoscale visualization of chromatin architecture and nuclear organization with enhanced reproducibility.

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5 minutes

We thank the Lavis Lab and the Open Chemistry team at Janelia for generously providing the JF and JFX dyes. We are also grateful to Elizabeth Kracik-Dyer and C&#233;lia Baroux for kindly sharing their protocol, as well as to Aline Probst and Guillermo Orsi for insightful advice. The optical imaging was carried out on the M4D imaging platform of the Grenoble Instruct-ERIC center (ISBG; UAR 3518 CNRS-CEA- UGA- EMBL) within the Grenoble Partnership for Structural Biology, supported by the French Infrastructure for Integrated Structural Biology (ANR-10-INBS-0005-02) and the Grenoble Alliance for Integrated Structural &#38; Cell Biology Labex, a project of the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR- 17-EURE- 0003). We acknowledge Tip ten Brink for the AnalyzeFRC Python package developed at the Department of Imaging Physics (ImPhys), Faculty of Applied Sciences, TU Delft.

NOTE: The detailed information on the materials and instruments used in this protocol is available in the Table of Materials. Unless otherwise specified, buffers are cleared on apyrogenic 0.2 &#181;m filters. Double-distilled ultra-pure H2O (ddH2O) is used throughout this protocol. Centrifugation steps are carried out in 1.5 mL microtubes using a fixed-angle rotor.
1. Seedlings&#8217; fixation and release of nuclei
<ol>
	<li>Place a 6-well culture plate on ice under the fume hood, then add to each well approximately 4 mL of ice-cold fixation solution prepared extemporaneously under a fume hood (4% (v/v) methanol stabilized formaldehyde, 0.1% Triton X-100 in 1&#215; Phosphate-Buffered Saline [PBS]).</li>
	<li>Using clean forceps, remove the seedlings (between 8 and 15 days after germination) that have been grown on solid (0.8% agar) half-strength Murashige and Skoog Medium 2.2 g/L MS salts, 1% sucrose (w/v), pH 5.7, as described for MS seedlings in reference24, and immediately submerge them in the fixation solution. Use one well for approximately 20 seedlings (3 wells were necessary to obtain sufficient material per sample).</li>
	<li>Put the plate in the vacuum chamber filled with ice and apply a vacuum at pressures cycling between 0.2 bar and 0.8 bar for 15 min to remove trapped air and allow deep penetration of the fixative into the plant tissues25. Slowly release the vacuum, stir the sample, and then apply the vacuum for a further 15 min.</li>
	<li>Under the fume hood, remove the fixation solution with a pipette, and wash each well 3 times for 10 min under gentle agitation, using 5 mL of ice-cold PBS.</li>
	<li>Remove PBS and use forceps to transfer the fixed tissue to a 120 mm &#215; 120 mm Petri dish. Add to the fixed seedlings 50 &#181;L of Nuclei Isolation Buffer (NIB), prepared as described by Kracik-Dyer and Baroux (45 mM MgCl2, 20 mM MOPS, 30 mM sodium citrate, 0.3% Triton X-100. Adjust to pH 7 using 1 N HCl)19. Chip with a blade. 
	NOTE: Microtome blades turned out to be more efficient than conventional razor blades for this task.</li>
	<li>Scrape chopped tissue to one edge of the Petri dish with the same blade and add 600 &#181;L of NIB, making sure to wash off any tissue stuck to the blade as well.</li>
	<li>Transfer tissue slurry into the 2 mL Dounce homogenizer tube and stroke 5&#8211;10 times with the smaller pestle, and then with the larger pestle until it is smoothly moving inside the tube (it requires again usually 5&#8211;10 strokes).</li>
	<li>Filter the resulting homogenized sample through a 30 &#181;m mesh filter into a 1.5 mL microtube.</li>
	<li>Centrifuge the suspension for 10 min at 800 &#215; g using the lowest ramp for both acceleration and deceleration. A small white pellet is visible among the green tissue debris.</li>
	<li>Discard the supernatant and resuspend the pellet in 150 &#181;L of NIB.</li>
	<li>Centrifuge again for 10 min at 800 &#215; g and remove the supernatant. If there is some visible green debris, wash for a second time. Resuspend in 50 &#181;L to 150 &#181;L of NIB, based on the size of the pellet.</li>
	<li>Store the resulting isolated nuclei suspension at 4 &#176;C for a maximum of 1 week before performing the staining. However, it is recommended to proceed immediately.</li>
</ol>
2. (Immuno)staining
NOTE: In the following steps, the protocol is applied to a volume of 50 &#181;L of nuclei suspension, corresponding typically to 20 seedlings. Unless specified, the following steps are performed at room temperature.
<ol>
	<li>Centrifuge the solution for 10 min at 800 &#215; g and discard the supernatant. Be careful not to touch the pellet.</li>
	<li>Resuspend the nuclei by gentle pipetting in 50 &#181;L of blocking buffer (143 mM NaCl, 4% BSA in PBS, stored at 4 &#176;C). Incubate for 30 min under gentle agitation.</li>
	<li>Centrifuge the solution for 10 min at 800 &#215; g and discard the supernatant without touching the nuclei pellet.</li>
	<li>Resuspend the nuclei by gentle pipetting in 50 &#181;L of 1&#215; PBS. Centrifuge again for 10 min at 800 &#215; g. Discard the supernatant as above.</li>
	<li>Resuspend the nuclei in 50 &#181;L of primary antibody solution prepared in immunostaining buffer composed of 143 mM NaCl, 1% BSA, 0.05% Tween 20 in PBS (antibody and working dilutions are detailed in the Table of Materials). Incubate the samples for 60 min under gentle agitation.</li>
	<li>Proceed with 2 washes (repeat steps 2.3 and 2.4).</li>
	<li>Resuspend the nuclei in 50 &#181;L of goat anti-rabbit AF647 secondary antibody at 1/200 dilution26. Wrap the tubes in aluminum foil to protect the fluorophore from light. Incubate the tubes for 60 min under gentle agitation.</li>
	<li>Proceed with 2 washes (repeat steps 2.3 and 2.4).</li>
	<li>Resuspend the pellet in 50 &#181;L of NIB. Store the suspension in 1.5 mL microtubes wrapped in aluminum foil. The labeling is stable for several weeks at 4 &#176;C.</li>
</ol>
3. Mounting
<ol>
	<li>For STORM imaging, prepare a low-melting agarose solution (LMA 1.5% w/v) by dissolving the powder in 1.25&#215; buffer B composed of 50 mM Tris pH 8, 10 mM NaCl, 10% Glucose. Prepare 1.25&#215; stock solution, and store at 4 &#176;C.</li>
	<li>Heat at 90 &#176;C for 1 min to dissolve the powder and obtain a clear solution. Cool down and maintain the melted LMA solution at 37 &#176;C before proceeding to the next step to avoid heat denaturation of glucose oxidase (GLOX) buffer (prepare 10x stock solution stored at -20&#176;C composed of 100 mM Tris pH 7.5, 25 mM KCl, 1 mM TCEP pH 7.0, 5 mg/mL Glucose oxidase, 0.4 mg/mL Catalase, 50% v/v Glycerol).</li>
	<li>After the LMA solution reaches 37 &#176;C, prepare the STORM buffer by adding 100 mM Mercaptoethylamine (MEA) and GLOX to the warm LMA solution to achieve 1&#215; final concentration. Store the MEA 1 M stock solution for up to 1 month at 4 &#176;C, but the use of a freshly prepared solution is recommended. Mix gently. 
	NOTE: For optional DNA labeling, add 1.5 nM (final concentration, 1 mM stock solution in DMSO) of the JF549 conjugated Hoechst. Keep the suspension at 37 &#176;C until use.</li>
	<li>Gently mix the nuclei suspension with nanodiamonds at 1/1500 dilution. Incubate them with the sample for 5 min. 
	NOTE: Nanodiamonds serve as fiducial markers to correct the sample drift.</li>
	<li>Deposit 10 &#181;L of the mix on an ozone-treated 1.5H coverslip (20 min in Ultraviolet Ozone cleaning system) and wait for 3 min to let the nuclei deposit on the coverslip.</li>
	<li>Remove the excess of liquid, wait 2 min. Add 10 &#181;L of the LMA prepared at step 3.3 to the coverslip and cover immediately with a microscope slide.</li>
	<li>Seal the coverslip using silicone sealant. Mix equal amounts of solution A and B in a small Petri dish using a micropipette tip. Apply the mix over the edges of the coverslip to seal the sample. Silicone sealant hardens within 5 min.</li>
	<li>Once mounted, perform imaging of slides without delay, ultimately in the following 4&#8211;5 h, to avoid loss of the oxygen scavenging properties of the mounting pad.</li>
</ol>
4. Imaging
NOTE: Imaging parameters must be carefully optimized according to the nature of the sample, the photo-physical properties of the fluorophores, and the optical configuration of the microscope. It is crucial to ensure a high signal-to-noise ratio for each fluorophore while minimizing spectral bleed-through and cross-excitation between fluorescent channels. Samples were observed with an inverted fluorescence microscope equipped with a high NA 100x oil immersion objective, a focus maintaining unit, and a white LED for bright-field imaging. Fluorescent labels AlexaFluor647 (AF647) were excited at 640 nm and JF549-Hoechst at 561 nm. The emitted light was filtered with a compatible dichroic mirror and band-pass filter(s), and finally, images were collected with a scientific Complementary Metal-Oxide-Semiconductor (sCMOS) camera.
<ol>
	<li>Mount the sample slide and find focus. Activate a focus-maintaining module if the system has one (highly recommended for long acquisitions).</li>
	<li>Find and focus on a nucleus. It is better to have one or a few nanodiamonds near the nucleus of interest to more efficiently correct the drift during the reconstruction.</li>
	<li>(Optional) Capture diffraction-limited snapshots of bright field and each fluorescent channel at low laser power.</li>
	<li>When performing two-color imaging, start with the acquisition of the longer wavelength channel and then switch to the shorter wavelength channel (i.e., acquire AF647 channel before JF549). 
	NOTE: Example of acquisition parameters used in this work: 
	Channel A - epitope labeling - AF647 
	Exposure time: 20 ms 
	Laser intensity: 640 nm &#8211; 2.2 kW/cm2 
	Excitation mode: HiLo27 
	Channel B - DNA labeling - JF549-Hoechst 
	Exposure time: 50 ms 
	Laser intensity: 561 nm - 1 kW/cm2 
	Excitation mode: Epi 
	Number of recorded images: 10&#8217;000-30&#8217;000 per channel, ensuring that the density of fluorophores per frame remains sparse enough for accurate single-molecule localization, while the cumulative number of localizations was empirically determined using Super-resolution QUantitative Image Rating and Reporting of Error Locations (SQUIRREL)28, which also indicates the sufficient number of frames.</li>
</ol>

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B., Henikoff, S. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+INTACT+method+for+cell+type-specific+gene+expression+and+chromatin+profiling+in+Arabidopsis+thaliana.">The INTACT method for cell type-specific gene expression and chromatin profiling in Arabidopsis thaliana.</a> Nat Protoc. 6 (1), 56-68 (2011).</li><li>Duncan, S., Olsson, T. S. G., Hartley, M., Dean, C., Rosa, S. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+detecting+single+mRNA+molecules+in+Arabidopsis+thaliana.">A method for detecting single mRNA molecules in Arabidopsis thaliana.</a> Plant Methods. 12, 13 (2016).</li><li>Gutzat, R., Mittelsten Scheid, O. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparing+chromatin+and+RNA+from+rare+cell+types+with+fluorescence-activated+nuclear+sorting+(FANS).">Preparing chromatin and RNA from rare cell types with fluorescence-activated nuclear sorting (FANS).</a> Methods Mol Biol. 2093, 95-105 (2020).</li></ol>

The authors declare no conflict of interest.

The preparation of Arabidopsis thaliana nuclei for single-molecule localization microscopy (SMLM) requires several critical steps that strongly influence the final sample quality. As described previously19,35, thorough and consistent tissue chopping is essential to maximize the recovery of intact nuclei. Using microtome blades instead of standard razor blades produces cleaner and more uniform tissue fragmentation, thereby improving nuclei release. Subseq...

Microscopy has long been a cornerstone for studying chromatin organization and nuclear architecture, revealing how DNA and histone-associated proteins arrange within the 3D nuclear space and influence gene regulation1,2. Conventional fluorescence microscopy has provided valuable insights into large-scale chromatin domains such as chromosome territories, chromocenters, and nuclear bodies3,4, feasible in situ, in particular in root tissues for plants3,5,6. However, these techniques are fundamentally limited by the diffraction barrier of light, restricting spatial resolution to approximately 200 nm laterally and 500 nm axially7. This limitation obscures nanoscale details of chromatin folding, histone modification patterns, and transcriptional compartmentalization that underlie epigenetic regulation. To overcome this barrier, super-resolution microscopy (SRM) approaches such as structured illumination microscopy (SIM)8, stimulated emission depletion (STED)9,10, and single-molecule localization microscopy (SMLM)11,12 were developed to achieve resolutions ranging from 100 nm down to a few nm13.
Among SRM techniques, SMLM&#8212;including PhotoActivated Localization Microscopy (PALM)13 and direct Stochastic Optical Reconstruction Microscopy (dSTORM)&#8212;offers exceptional spatial resolution combined with molecular specificity14. By detecting the positions of individual fluorophores over time, SMLM reconstructs high-precision nanoscale images of chromatin domains, histone marks, and nuclear structures in single cells15,16. This capability has transformed chromatin research in mammalian systems, where SMLM has been applied to visualize nucleosome clustering, chromatin compaction, and histone modification patterns with unprecedented detail15,16,17. In contrast, the application of SMLM to plant nuclei remains limited, largely due to technical challenges associated with plant tissue properties&#8212;such as high autofluorescence, cell wall rigidity, and the presence of light-scattering compounds like starch and chlorophyll18. Furthermore, the delicate nature of nuclei during isolation and labeling requires optimized workflows to maintain structural integrity and minimize background fluorescence during prolonged imaging sessions.
The protocol described here was originally developed by Elizabeth Kracik-Dyer and C&#233;lia Baroux for stimulated emission depletion (STED) microscopy19. It was subsequently optimized and adapted to meet the specific requirements of STORM imaging with the integration of knowledge coming from published microbiology applications20,21,22. The overall goal is to provide a streamlined and reproducible workflow for SMLM imaging of isolated Arabidopsis thaliana nuclei. By integrating optimized fixation, gentle mechanical disruption, and fluorophore labeling in suspension, this method preserves nuclear morphology while efficiently removing cytoplasmic contaminants that often compromise imaging quality. Immobilization of labeled nuclei on low-melting agarose pads ensures mechanical stability during acquisition, allowing for long-term single-molecule imaging at nanoscale precision. Compared to previously described SRM protocols for plant nuclei&#8212;particularly those based on STED microscopy19,23&#8212;this workflow uses photoswitchable fluorophores (e.g., AF647, JF549), and reduces background fluorescence thanks to washing steps that discard chlorophyll and tissue debris, and improves labeling homogeneity by adding the labeling molecules directly to the fixed and permeabilized nuclei. Consequently, it enables consistent visualization of chromatin modifications, nuclear compartments, and epigenetic patterns in individual nuclei. By lowering the technical barriers for implementing SMLM in plants, this method broadens the accessibility of nanoscale imaging and provides a foundation for studying how chromatin organization and nuclear architecture contribute to gene regulation in response to developmental and environmental cues.

<table><tbody><tr><td>Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S2) antibody</td><td>Abcam</td><td>ab5095</td><td>Primary antibody used at 1/200 dilution in immunostaining buffer to visualize the active form of the polymerase (Polyclonal antibody raised in rabbit )</td></tr><tr><td>Acquisition software</td><td>Abbelight</td><td>NEO LiveImaging v2.18</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A7906&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Catalase</td><td>Sigma-Aldrich</td><td>C40</td><td></td></tr><tr><td>Certified Low Melting Agarose</td><td>Bio-Rad</td><td>1613111</td><td></td></tr><tr><td>Coverslips thickness 1.5H round</td><td>Marienfeld</td><td>117640</td><td>&amp;Oslash;: 24 mm</td></tr><tr><td>D-(+)-Glucose, anhydrous, 99%</td><td>Thermo Scientific</td><td>A16828-36&amp;nbsp;</td><td></td></tr><tr><td>Dichroic mirror</td><td>Semrock</td><td>Di03-R405/488/561/635-t1</td><td>Dichroic mirror for excitation/emission separation</td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline 10&amp;acute;</td><td>biowest</td><td>X0515 - 500&amp;nbsp;</td><td>Work in sterile conditions</td></tr><tr><td>Epredia Ultra Disposable Microtome Blades</td><td>FisherScientific</td><td>12191830</td><td>Low profile, single use</td></tr><tr><td>Falcon 6-well Clear Flat Bottom TC-treated Multiwell Cell Culture Plate, with Lid</td><td>&amp;nbsp;Falcon</td><td>353046</td><td></td></tr><tr><td>Formaldehyde solution 37%</td><td>Carl Roth</td><td>7398</td><td>Work under the fume hood</td></tr><tr><td>Glucose oxidase</td><td>Sigma-Aldrich</td><td>G2133</td><td></td></tr><tr><td>Glycerol</td><td>VWR</td><td>24388.295</td><td></td></tr><tr><td>Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 647</td><td>Thermo Scientific</td><td>A32733TR</td><td>Used at 1/200 as secondary antibody coupled to AF647</td></tr><tr><td>Hoechst 33258 solution</td><td>Sigma-Aldrich</td><td>94403</td><td>Used at 1/500 to counterstain the DNA, added to the agarose pad</td></tr><tr><td>Hydrochloric Acid Solution</td><td>Chem-Lab</td><td>CL05.0311.1000&amp;nbsp;</td><td></td></tr><tr><td>JF549-Hoechst</td><td>Lavis lab and Open Chemistry team (Janelia)</td><td>JF549-Hoechst</td><td>Used to counterstain DNA, added to the agarose pad at 1.5 nM final concentration</td></tr><tr><td>KIMBLE Dounce tissue grinder set</td><td>Sigma-Aldrich</td><td>D8938</td><td>2 mL tube with both large (0.0030-0.0050 inch) and small (0.0005-0.0025 inch) clearance pestles</td></tr><tr><td>Lasers unit</td><td>Oxxius</td><td>L6Cc</td><td>Equipped with six lasers: 405 nm - 100 mW, 488 nm - 200 mW, 532 nm - 500 mW, 561 nm - 300 mW, 640 nm - 500 mW and 730 nm - 30 mW</td></tr><tr><td>Magnesium chloride hexahydrate</td><td>Carl Roth</td><td>HN03</td><td></td></tr><tr><td>Mercaptoethylamine (MEA)</td><td>Sigma-Aldrich</td><td>30070</td><td></td></tr><tr><td>MOPS sodium salt 98%</td><td>Fisher Scientific SAS</td><td>352590010</td><td></td></tr><tr><td>Multiband emission filter</td><td>Semrock</td><td>FF01-446/523/600/677</td><td>Multiband emission filtration to block laser scatter light</td></tr><tr><td>Nanodiamonds</td><td>Adamas Nanotechnologies</td><td>NDNV100nmHiWGA2ml</td><td>Stock 1 mg/mL</td></tr><tr><td>ORCA-Fusion Digital CMOS camera</td><td>Hamamatsu</td><td>C14440-20UP&amp;nbsp;</td><td></td></tr><tr><td>Petri dish, 100/20 mm, PS, clear, with vents, sterile</td><td>Greiner Bio-One</td><td>664161</td><td>Used to grow plants on 1/2 MS medium</td></tr><tr><td>Petri dish, square, PS, clear, 120/120/17 mm, sterile</td><td>Greiner Bio-One</td><td>688161</td><td>Used when chopping the plant seedlings</td></tr><tr><td>pluriStrainer Mini cell strainer</td><td>pluriSelect</td><td>43-10030-50</td><td>Mesh size 30 &amp;micro;m, suitable for 1.5 mL Eppendorf tube</td></tr><tr><td>Potassium Chloride</td><td>Sigma-Aldrich</td><td>P9333</td><td></td></tr><tr><td>Single band emission filter</td><td>Semrock</td><td>FF01-698/70</td><td>Specific emission filter for AF647 channel</td></tr><tr><td>Single band emission filter</td><td>Chroma</td><td>ET600/50m</td><td>Specific emission filter for JF549 channel</td></tr><tr><td>Sodium chloride (99,5%)&amp;nbsp;</td><td>Euromedex</td><td>1112-A</td><td></td></tr><tr><td>Star-Frost slides 76 x 26 mm</td><td>Knittel</td><td>&amp;nbsp;VS112711FKB.01</td><td></td></tr><tr><td>Sterile syringe filter&amp;oslash; 33 mm porosity 0.2 &amp;micro;m Luer Lock cone</td><td>ClearLine</td><td>146560</td><td></td></tr><tr><td>Super-resolution system</td><td>Abbelight</td><td>SAFe360</td><td>Olympus IX83 equipped with 100x NA1.5 oil-immersion objective</td></tr><tr><td>Tri-Sodium Citrate 2H2O Gen-Apex Biomole</td><td>Prolabo</td><td>PRO-33615.268</td><td></td></tr><tr><td>Tris Base Molecular Biology Grade</td><td>Promega</td><td>H5135</td><td></td></tr><tr><td>Tris(2-carboxyethyl)phosphine hydrochloride (TCEP)</td><td>Thermo Scientific</td><td>20491</td><td></td></tr><tr><td>Triton X-100</td><td>Euromedex</td><td>2000-B</td><td></td></tr><tr><td>Tween 20</td><td>Euromedex</td><td>2001-B</td><td></td></tr><tr><td>Twinsil Speed</td><td>Picodent</td><td>13001002</td><td>Silicone sealant</td></tr><tr><td>Ultraviolet Ozone cleaning system UVOCS oven</td><td>UVOCS Inc.</td><td>T10X10</td><td>20 min exposure for coverslip cleaning&amp;nbsp;</td></tr><tr><td>Vacuum pump</td><td>Vacuubrand</td><td>VP 100C</td><td></td></tr><tr><td>Heraeus Megafuge 16R Centrifuge</td><td>Thermo Scientific</td><td></td><td>Rotor TX-400 75003629. Centrifugation performed with Eppendorf tubes.</td></tr></tbody></table>

a streamlined protocol for single-molecule localization microscopy in arabidopsis nuclei

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

While confocal fluorescence microscopy has provided valuable insights into chromatin organization in plant nuclei, its diffraction-limited resolution constrains the investigation of chromatin architecture, motivating the use of super-resolution techniques such as&#160;Single-Molecule Localization Microscopy (SMLM). Among these approaches, direct stochastic optical reconstruction microscopy (dSTORM) provides nanoscale resolution in individual cells, enabling precise visualization of chromatin domains, histone modifications, and nuclear organization. While such methods are increasingly applied in mammalian systems, their use in plant biology remains limited, largely due to technical challenges in sample preparation.
Here, we present a streamlined and reproducible workflow for SMLM imaging of nuclei isolated from Arabidopsis thaliana. This protocol starts with seedling fixation to preserve nuclear morphology, followed by gentle tissue chopping and centrifugation to enrich intact nuclei. Isolated nuclei are then fluorophore-labeled in liquid medium and immobilized on low-melting agarose pads, a strategy that enhances stability during prolonged single-molecule imaging sessions. These steps collectively minimize background fluorescence, improve labeling consistency, and increase reproducibility across biological replicates.
The resulting preparations provide enhanced clarity for visualizing chromatin modifications and nuclear architecture in plants. By lowering the technical barriers to implement SMLM imaging in Arabidopsis, this protocol provides a versatile means to investigate epigenetic regulation, chromatin organization, and nuclear topological variations at the nanoscale. This work establishes a methodological foundation for applying SMLM to plants, bridging the gap with mammalian cell biology and opening new opportunities to study how nuclear architecture contributes to genome regulation in response to developmental and environmental cues in plant systems.

The protocol described above enables the preparation of high-quality Arabidopsis thaliana nuclei suitable for SMLM. Several rounds of optimization were performed to improve nuclear integrity and reduce cytoplasmic and cellular debris that compromise image quality. After each optimization step, the quality of the nuclear preparations was evaluated by confocal imaging using an Olympus IX81 spinning-disk microscope before proceeding to super-resolution acquisition.
Confocal imagi...

This protocol presents an efficient workflow for single-molecule localization microscopy imaging of isolated Arabidopsis nuclei, using optimized isolation, fixation, and labeling to achieve reliable nanoscale visualization of chromatin and RNA Polymerase II. This approach can be readily adapted to other chromatin-associated proteins or histone modifications for high-resolution imaging.

While confocal fluorescence microscopy has provided valuable insights into chromatin organization in plant nuclei, its diffraction-limited resolution ...

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.

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Research

JoVE Journal

Biology

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

We have developed a novel method for co-expressing multiple chimeric fluorescent fusion proteins in plants to overcome the difficulties of conventional methods. It takes advantage of using a single expression plasmid that contains multiple functionally independent protein expressing cassettes to achieve protein co-expression.

Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in an Efficient Way in Plants

We report a simple and versatile method for performing fluorescent live-imaging of Arabidopsis thaliana leaves over an extended period of time. We use a transgenic Arabidopsis plant expressing a fluorescent reporter gene under the control of an immunity-related promoter as an example for gaining spatiotemporal understanding of plant immune responses.

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

3D single-molecule localization microscopy is utilized to probe the spatial positions and motion trajectories of fluorescently labeled proteins in living bacterial cells. The experimental and data analysis protocol described herein determines the prevalent diffusive behaviors of cytosolic proteins based on pooled single-molecule trajectories.

Single-Molecule Tracking Microscopy - A Tool for Determining the Diffusive States of Cytosolic Molecules

Here we describe a protocol for live cell imaging of the cortical microtubule cytoskeleton at the shoot apical meristem and monitoring its response to changes in physical forces.

Live Cell Imaging of Microtubule Cytoskeleton and Micromechanical Manipulation of the Arabidopsis Shoot Apical Meristem

Chromosome movement during meiotic prophase is a poorly understood phenomenon that is fundamental to spore formation. This protocol describes a method to acquire, analyze, and quantify centromere movements during meiotic prophase in Arabidopsis thaliana male meiocytes.

Live Imaging Characterization of Centromere Movements During Male Meiotic Prophase in Arabidopsis thaliana

We present a three-color chromatin single molecule localization microscopy (SMLM) staining and analysis protocol that enables reproducible mapping of euchromatin, heterochromatin, and RNAP II for spatial analysis. This protocol enables efficient multicolor labeling in dense nuclear environments, including chromatin-associated targets, allowing reliable simultaneous detection.

A Multilabel Single Molecule Localization Microscopy Protocol for Investigation of Chromatin in the Dense Nuclear Environment

This immunolocalization protocol in male maize meiocytes maintains the three-dimensional integrity of the cells during meiosis. It allows the dynamic tracking of the intracellular localization of up to three independent antibodies in preserved paraformaldehyde-fixed material, in addition to DAPI, which stains DNA.

Three-Dimensional Immunolocalization In Maize Meiocytes

This process uses confocal microscopy in conjunction with a fluorescently labeled nucleus and chromatin marker line in Arabidopsis thaliana to simultaneously record nucleus and chromatin dynamics in Arabidopsis roots and investigate changes in these dynamics in response to developmental and environmental cues.

Quantitative Live Cell Imaging of Nuclear Shape and Chromatin Dynamics During Development and Environmental Stress in Arabidopsis thaliana Root

Learn a streamlined protocol for single-molecule localization microscopy in Arabidopsis thaliana nuclei. This method enables nanoscale visualization of chromatin architecture and nuclear organization with enhanced reproducibility.

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

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