Name: Super-resolution Imaging of Proteus mirabilis Biofilm by Expansion Microscopy (Video) | JoVE Journal
Uploaded: 2025-07-18T13:00:00.000+00:00
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Description: Learn super-resolution imaging of Proteus mirabilis biofilms using expansion microscopy. This method enables nanoscale visualization of cellular architecture with conventional microscopes.

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5 minutes

The authors extend their gratitude to Dr. Juan Eduardo Rodr&#237;guez and Prof Dr. Ulrich Kubitschek (U-Bonn) for their contribution to the implementation of the expansion techniques at SCIAN-Lab. This work was financed by ANID projects ICM P09-015-F (SH, DC, KP, JJW), FONDECYT 1211988 (SH, DC, KP), FONDECYT 3220832 (JJW), FONDEQUIP EQM210020 (DC, KP, JJW, JT, NCH, PS, SH); N&#250;cleo SELFO NCN2024_068 (SH, KP, JJW),&#160;CORFO 16CTTS- 66,390, MINEDUC grant RED 21994 (SH), BASAL FB210005 (SH), DAAD 57519605 (SH, NC),&#160;Centro CTI230006 (SH),&#160;FONDEF ID 23I10337 (SH), and Regional Binational Project Cooperaci&#243;n SUR-SUR AUCI / AGCID BIL-URY-2019-661 (SH, NCH, PS, MJG).

<ol><li>Franklin, M. J., Chang, C., Akiyama, T., Bothner, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((New+technologies+for+studying+biofilms%5BTitle%5D)+AND+%22Microbiol+Spectr%22%5BJournal%5D)">New technologies for studying biofilms.</a> Microbiol Spectr. 3 (4), 10(2015).</li>
<li>Valdivieso González, D., Jara, J., Almendro-Vedia, V. G., Orgaz, B., López-Montero, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expansion+microscopy+applied+to+mono-+and+dual-species+biofilms%5BTitle%5D)+AND+%22NPJ+Biofilms+Microbiomes%22%5BJournal%5D)">Expansion microscopy applied to mono- and dual-species biofilms.</a> NPJ Biofilms Microbiomes. 9 (1), 92(2023).</li>
<li>Castagnini, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Proteus+mirabilis+biofilm+expansion+microscopy+yields+over+4-fold+magnification+for+super-resolution+of+biofilm+structure+and+subcellular+DNA+organization%5BTitle%5D)+AND+%22J+Microbiol+Methods%22%5BJournal%5D)">Proteus mirabilis biofilm expansion microscopy yields over 4-fold magnification for super-resolution of biofilm structure and subcellular DNA organization.</a> J Microbiol Methods. 220, 106927(2024).</li>
<li>Chen, F., Tillberg, P. W., Boyden, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optical+imaging-+Expansion+microscopy%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Optical imaging- Expansion microscopy.</a> Science. 347 (6221), 543-548 (2015).</li>
<li>Chozinski, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expansion+microscopy+with+conventional+antibodies+and+fluorescent+proteins%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">Expansion microscopy with conventional antibodies and fluorescent proteins.</a> Nat Methods. 13 (6), 485-488 (2016).</li>
<li>Gao, R., Asano, S. M., Boyden, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Q%26A%3A+Expansion+microscopy%5BTitle%5D)+AND+%22BMC+biology%22%5BJournal%5D)">Q&A: Expansion microscopy.</a> BMC biology. 15 (1), 50(2017).</li>
<li>Tillberg, P. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protein-retention+expansion+microscopy+of+cells+and+tissues+labeled+using+standard+fluorescent+proteins+and+antibodies%5BTitle%5D)+AND+%22Nat+Biotechnol%22%5BJournal%5D)">Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies.</a> Nat Biotechnol. 34 (9), 987-992 (2016).</li>
<li>Lim, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanically+resolved+imaging+of+bacteria+using+expansion+microscopy%5BTitle%5D)+AND+%22PLoS+biology%22%5BJournal%5D)">Mechanically resolved imaging of bacteria using expansion microscopy.</a> PLoS biology. 17 (10), e3000268(2019).</li>
<li>Kunz, T. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+expandables%3A+Cracking+the+staphylococcal+cell+wall+for+expansion+microscopy%5BTitle%5D)+AND+%22Front+Cell+Infect+Microbiol%22%5BJournal%5D)">The expandables: Cracking the staphylococcal cell wall for expansion microscopy.</a> Front Cell Infect Microbiol. 11, 644750(2021).</li>
<li>Jacobsen, S. M., Shirtliff, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Proteus+mirabilis+biofilms+and+catheter-associated+urinary+tract+infections%5BTitle%5D)+AND+%22Virulence%22%5BJournal%5D)">Proteus mirabilis biofilms and catheter-associated urinary tract infections.</a> Virulence. 2 (5), 460-465 (2011).</li>
<li>Asano, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expansion+microscopy%3A+Protocols+for+imaging+proteins+and+RNA+in+cells+and+tissues%5BTitle%5D)+AND+%22Curr+Protoc+Cell+Biol%22%5BJournal%5D)">Expansion microscopy: Protocols for imaging proteins and RNA in cells and tissues.</a> Curr Protoc Cell Biol. 80 (1), e56(2018).</li>
<li>Yokogawa, K., Kawata, S., Nishimura, S., Ikeda, Y., Yoshimura, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mutanolysin%2C+bacteriolytic+agent+for+cariogenic+streptococci%3A+partial+purification+and+properties%5BTitle%5D)+AND+%22Antimicrob+Agents+Chemother%22%5BJournal%5D)">Mutanolysin, bacteriolytic agent for cariogenic streptococci: partial purification and properties.</a> Antimicrob Agents Chemother. 6 (2), 156-165 (1974).</li>
<li>Hurst, P. L., Nielsen, J., Sullivan, P. A., Shepherd, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Purification+and+properties+of+a+cellulase+from+Aspergillus+niger%5BTitle%5D)+AND+%22Biochem+J%22%5BJournal%5D)">Purification and properties of a cellulase from Aspergillus niger.</a> Biochem J. 165 (1), 33-41 (1977).</li>
<li>Scott, J. H., Schekman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lyticase%3A+Endoglucanase+and+protease+activities+that+act+together+in+yeast+cell+lysis%5BTitle%5D)+AND+%22J+Bacteriol%22%5BJournal%5D)">Lyticase: Endoglucanase and protease activities that act together in yeast cell lysis.</a> J Bacteriol. 142 (2), 414-423 (1980).</li>
<li>Bano, S., Qader, S. A. U., Aman, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Purification+and+characterization+of+novel+%CE%B1-amylase+from+Bacillus+subtilis+KIBGE+HAS%5BTitle%5D)+AND+%22AAPS+PharmSciTech%22%5BJournal%5D)">Purification and characterization of novel α-amylase from Bacillus subtilis KIBGE HAS.</a> AAPS PharmSciTech. 12, 255-261 (2011).</li>
<li>Kaplan, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genes+involved+in+the+synthesis+and+degradation+of+matrix+polysaccharide+in+Actinobacillus+actinomycetemcomitans+and+Actinobacillus+pleuropneumoniae+biofilms%5BTitle%5D)+AND+%22J+Bacteriol%22%5BJournal%5D)">Genes involved in the synthesis and degradation of matrix polysaccharide in Actinobacillus actinomycetemcomitans and Actinobacillus pleuropneumoniae biofilms.</a> J Bacteriol. 186 (24), 8213-8220 (2004).</li>
<li>Kaplan, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recombinant+human+DNase+I+decreases+biofilm+and+increases+antimicrobial+susceptibility+in+staphylococci%5BTitle%5D)+AND+%22J+Antibiot%22%5BJournal%5D)">Recombinant human DNase I decreases biofilm and increases antimicrobial susceptibility in staphylococci.</a> J Antibiot. 65 (2), 73-77 (2012).</li>
<li>Schlapp, G., Scavone, P., Zunino, P., Härtel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+3D+architecture+of+uropathogenic+Proteus+mirabilis+batch+culture+biofilms%3A+A+quantitative+confocal+microscopy+approach%5BTitle%5D)+AND+%22J+Microbiol+Methods%22%5BJournal%5D)">Development of 3D architecture of uropathogenic Proteus mirabilis batch culture biofilms: A quantitative confocal microscopy approach.</a> J Microbiol Methods. 87 (2), 234-240 (2011).</li>
<li>Schermelleh, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Super-resolution+microscopy+demystified%5BTitle%5D)+AND+%22Nat+Cell+Biol%22%5BJournal%5D)">Super-resolution microscopy demystified.</a> Nat Cell Biol. 21 (1), 72-84 (2019).</li>
</ol>

The authors declare no financial or personal conflicts of interest related to this work.

Critical steps of PmbExM 
As with any ExM variant, one of the most critical steps of the protocol is the homogenization of the sample&#39;s mechanical properties. In the case of PmbExM, this is achieved through a series of enzymatic treatments. Insufficient digestion can result in low EFs and potential damage to biofilm morphology and/or topology, leading to suboptimal resolution and unreliable results. Conversely, overly aggressive digestions, particularly proteolytic treatments, can cause signific...

Fluorescence microscopy is a widely used tool for studying biofilm architecture, composition, and function1. However, optical microscopy&#39;s ability to resolve the structural, cellular, and subcellular features of biofilms is limited by the diffraction barrier. Expansion Microscopy (ExM) is an innovative, accessible, and easy-to-use super-resolution technique that has great potential for revealing biofilm structure beyond the diffraction limit2,3. ExM enhances resolution by isotropically expanding biological specimens approximately four times their original size. The method involves the in-situ polymerization of a swellable ionic hydrogel throughout the sample and preserving the relative spatial arrangement of molecular targets when applied with care4,5.
For ExM to work properly, homogenizing the mechanical properties of the biological material is a crucial step6. Standard ExM variants were developed upon mammal cell cultures and tissues, where they efficiently achieved homogenization only through proteinase K proteolytic digestion4,5 or soft protein denaturation with detergents and autoclaving7, yielding isotropic 4-fold expansion. However, in the case of bacteria and bacterial biofilms, the peptidoglycan cell wall and EPS matrix elements, such as structural polysaccharides, are obstacles against expansion2,3,8,9.
This article describes the procedures of Proteus mirabilis biofilm Expansion Microscopy (PmbExM), a tailored ExM variant designed to expand biofilms of P. mirabilis, a clinically relevant gram-negative bacillus associated with high antibiotic resistance and catheter-associated urinary tract infections10. PmbExM uses a combination of mutanolysin, &#945;-amylase, cellulase, lyticase, and proteinase K enzymatic treatments to hydrolyze the peptidoglycan cell wall, polysaccharides, and proteins, respectively. The results demonstrate that this method achieves a 4.3-fold isotropic expansion of 48-h-old P. mirabilis biofilms. The technique enables super-resolution visualization of P. mirabilis biofilm architecture, assembly, and intracellular features through double staining3. Furthermore, PmbExM can be adapted to other biofilm models by adjusting the enzyme treatments to target species-specific biofilm components.
The objective of this article is to provide a detailed description of the procedures involved in gelation, digestion, expansion, and mounting of biofilm samples for PmbExM. Additionally, this work outlines bacterial growth conditions, sample formats, and fluorescent staining techniques used by the authors, along with a data processing and analysis routine that can be easily applied by researchers with limited experience in image processing. For a more comprehensive and in-depth guide on image processing and analysis, please refer to Castagnini, D. et al.3.

<table><tbody><tr><td>&amp;alpha;-Amylase from &lt;em&gt;Bacillus sp.&lt;/em&gt;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>10070</td><td>Polysaccharide digestion</td></tr><tr><td>0.1% (w/v) poly-L-lysine water solution&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P8920</td><td>Sample immobilization for imaging</td></tr><tr><td>12 mm glass coverslips</td><td>Chemglass Life Sciences</td><td>CLS-1760</td><td>Gelation chamber component</td></tr><tr><td>16-bit C13440-20CU camera</td><td>Hamamatsu Photonics</td><td>Not available</td><td>CMOS digital camera for image acquisition</td></tr><tr><td>24 mm x 50 mm glass coverslips</td><td>Chemglass Life Sciences</td><td>CLS-1764</td><td>Sample manipulation/handling</td></tr><tr><td>3D-printed imaging chamber</td><td>In-house design</td><td>-</td><td>Imaging chamber option</td></tr><tr><td>4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (4-hydroxy-TEMPO)</td><td>Sigma-Aldrich</td><td>176141</td><td>Gelling solution component</td></tr><tr><td>6-Well glass-bottom plate with black walls</td><td>CelVis</td><td>P06-1.5H-N</td><td>Imaging chamber option</td></tr><tr><td>Acetic acid&amp;nbsp;</td><td>Sigma-Aldrich</td><td>818755</td><td>Enzyme buffer component</td></tr><tr><td>Acrylamide&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A4058</td><td>Monomer solution component</td></tr><tr><td>Ammonium persulfate</td><td>Sigma-Aldrich</td><td>A3678</td><td>Gelling solution component</td></tr><tr><td>Axiovert 200 microscope</td><td>Zeiss</td><td>Not available</td><td>Confocal microscope</td></tr><tr><td>Calcium chloride</td><td>Sigma-Aldrich</td><td>C8106</td><td>Enzyme buffer component</td></tr><tr><td>C-Apochromat 63x 1.2 NA water immersion objective</td><td>Zeiss</td><td>Not available</td><td>Microscope objective</td></tr><tr><td>Cellulase from &lt;em&gt;Aspergillus niger&lt;/em&gt;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>22178</td><td>Polysaccharide digestion</td></tr><tr><td>Chamlide CMB 35 mm dish magnetic imaging chamber</td><td>Live Cell Instrument</td><td>Not available</td><td>Imaging chamber option</td></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma-Aldrich</td><td>472301</td><td>MA-NHS anchoring reagent stock solution&amp;nbsp;</td></tr><tr><td>Disodium phosphate</td><td>Sigma-Aldrich</td><td>S3264</td><td>Enzyme buffer component</td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>E7023</td><td>Cleaning and sterilization</td></tr><tr><td>Ethylenediaminetetraacetic acid</td><td>Merck</td><td>324503</td><td>Enzyme buffer component</td></tr><tr><td>Falcon polystyrene 24 well-plates</td><td>Corning</td><td>351147</td><td>General procedures platform</td></tr><tr><td>FIJI Software</td><td>National Institutes of Health</td><td>-</td><td>Image processing software</td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G7893</td><td>Enzyme buffer component</td></tr><tr><td>Guanidine hydrochloride</td><td>Thermo Scientific</td><td>24115</td><td>Enzyme buffer component</td></tr><tr><td>Huygens Software</td><td>Scientific Volume Imaging</td><td>-</td><td>Deconvolution software for image restoration</td></tr><tr><td>Hydrochloric acid</td><td>Supelco</td><td>1.00317</td><td>Buffer preparation</td></tr><tr><td>Kimwipes tissue paper</td><td>Fisher Scientiffic</td><td>06-666</td><td>Excess water removal</td></tr><tr><td>Laboratory tweezers</td><td>Electron Microscopy Sciences</td><td>M5E</td><td>General sample manipulation/handling</td></tr><tr><td>Luria Bertani broth</td><td>Invitrogen</td><td>12795-027</td><td>Biofilm growth medium</td></tr><tr><td>Lyticase from &lt;em&gt;Arthrobacter luteus&lt;/em&gt;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>L4025</td><td>Polysaccharide digestion</td></tr><tr><td>Magnesium chloride</td><td>Sigma-Aldrich</td><td>D5652</td><td>Enzyme buffer component</td></tr><tr><td>Methacrylic acid N-hydroxysuccinimidyl ester</td><td>Sigma-Aldrich</td><td>730300</td><td>Anchoring reagent</td></tr><tr><td>Methanol</td><td>Sigma-Aldrich</td><td>32213</td><td>Sample dehydration &amp;amp; storage</td></tr><tr><td>MicrobeJ&amp;nbsp;</td><td>-</td><td>-</td><td>Image processing software plugin</td></tr><tr><td>Microscopy glass slides</td><td>Chemglass Life Sciences</td><td>CG-8221</td><td>Gelation chamber component</td></tr><tr><td>Monosodium phosphate</td><td>Sigma-Aldrich</td><td>S9638</td><td>Enzyme buffer component</td></tr><tr><td>Mutanolysin from &lt;em&gt;Streptomyces globisporus&lt;/em&gt;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>M990</td><td>Peptidoglycan digestion</td></tr><tr><td>N, N, N&#039;, N&#039;-tetramethylethylenediamine</td><td>Sigma-Aldrich</td><td>T7024</td><td>Gelling solution component</td></tr><tr><td>N,N&amp;prime;-methylenebisacrylamide</td><td>Sigma-Aldrich</td><td>M1533</td><td>Monomer solution component</td></tr><tr><td>Nunc 40.4 mm glass-bottom Petri dishes</td><td>Thermo Fisher</td><td>12-567-400</td><td>Imaging chamber option</td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>158127</td><td>Sample fixation</td></tr><tr><td>Phosphate buffer saline</td><td>Sigma-Aldrich</td><td>79383</td><td>Monomer solution component, Washing, Enzyme buffer</td></tr><tr><td>Proteinase K from &lt;em&gt;Engyodontium album&lt;/em&gt;</td><td>Invitrogen</td><td>25530-031</td><td>Protein digestion</td></tr><tr><td>&lt;em&gt;Proteus mirabilis &lt;/em&gt;ATCC 12453</td><td>Microbiologics</td><td>0440P</td><td>Study subject</td></tr><tr><td>Small flat paint brush</td><td>Local hardware store</td><td>-</td><td>Sample manipulation/handling</td></tr><tr><td>Sodium acetate</td><td>Sigma-Aldrich</td><td>241245</td><td>Enzyme buffer component</td></tr><tr><td>Sodium acrylate</td><td>Sigma-Aldrich</td><td>408220</td><td>Monomer solution component</td></tr><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich</td><td>S5761</td><td>Enzyme buffer component</td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich</td><td>S9888</td><td>Monomer solution component, Enzyme buffer component</td></tr><tr><td>Sodium hydroxide</td><td>Sigma-Aldrich</td><td>221465</td><td>Glass slides cleaning</td></tr><tr><td>Solid-state laser line</td><td>Omicron</td><td>Not available</td><td>488, 568 and 647 nm Excitation laser line</td></tr><tr><td>SYTO 61 nucleic acid staining</td><td>Invitrogen</td><td>S11343</td><td>Fluorescent dye</td></tr><tr><td>Tris(hydroxymethyl)aminomethane hydrochloride</td><td>SAFC</td><td>108219</td><td>Enzyme buffer component</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100</td><td>Sample permeabilization, Enzyme buffer component</td></tr><tr><td>Volocity 7.0.0 capture software</td><td>Quorum Technologies</td><td>-</td><td>Microscoy acquisition controller software</td></tr><tr><td>Volocity ViewVox spinning disk system</td><td>Perkin-Elmer</td><td>Not available</td><td>Spinning disk system for confocal microscope</td></tr><tr><td>Wheat-germ agglutinin &amp;ndash; Alexa Fluor 488 fluorescent lectin conjugate</td><td>Invitrogen</td><td>W11261</td><td>Fluorescent dye</td></tr></tbody></table>

super-resolution imaging of proteus mirabilis biofilm by expansion microscopy

Super-resolution Imaging of Proteus mirabilis Biofilm by Expansion Microscopy

Accessing detailed visual information and quantitative data from microbiological samples using conventional optical microscopy is limited by the diffraction barrier. One solution to enhance resolution is Expansion Microscopy (ExM), an innovative and cost-effective super-resolution technique that physically enlarges samples by approximately four times their original size. For successful expansion, it is essential to homogenize the mechanical properties of the biological material. Biofilms are bacterial communities, adhering to a surface and embedded in an extracellular matrix they produce; they require ExM protocols to be adapted to accommodate their unique structural components. This article presents Proteus mirabilis biofilm ExM (PmbExM), a specialized variant of ExM that enables super-resolution visualization of P. mirabilis biofilms grown for 48 h. The protocol focuses on the targeted degradation of key structural components of samples through serial enzymatic digestions, optimized near their theoretical conditions. PmbExM utilizes a combination of enzymes, including &#945;-amylase, cellulase, and lyticase glycoside-hydrolases for polysaccharide hydrolysis; mutanolysin for peptidoglycan hydrolysis; and proteinase K for protein hydrolysis. These digestion procedures are independent of the gelation process, allowing modifications to meet specific homogenization requirements in different biofilm models. This adaptability offers great potential for application across various bacterial species and growth conditions. ExM has been applied to different biofilm species with overall suboptimal expansion factors. In contrast, PmbExM achieves the theoretical maximum expansion factor of the standard acrylamide-acrylate ExM hydrogel, without significant distortion of morphology or topology. The aim of this work is to provide an accessible super-resolution protocol for visualizing the architecture, assembly, and cellular and intracellular features of P. mirabilis biofilms.

PmbExM expanded the P. mirabilis biofilm 4.3 fold and preserved morphology and topology 
Figure 3A-D evidences the physical magnification of P. mirabilis biofilm cells and the increased resolution brought up by the technique. Moreover, this was not only observed in thin biofilm regions where cells organize in a monolayer-like layout, but also in thicker regions of densely populated arran...

This article presents a comprehensive protocol for PmbExM, an Expansion Microscopy technique designed specifically for Proteus mirabilis biofilms. PmbExM utilizes a stepwise enzymatic treatment of biofilm samples to achieve an isotropic, 4.3-fold expansion, enabling super-resolution analysis of the spatial organization of cellular and subcellular structures within these sessile microbial communities.

Accessing detailed visual information and quantitative data from microbiological samples using conventional optical microscopy is limited by the ...

super-resolution-imaging-proteus-mirabilis-biofilm-expansion

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Learn super-resolution imaging of Proteus mirabilis biofilms using expansion microscopy. This method enables nanoscale visualization of cellular architecture with conventional microscopes.

Super-resolution Imaging of Proteus mirabilis Biofilm by Expansion Microscopy

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