Super-resolution Imaging of Proteus mirabilis Biofilm by Expansion Microscopy

Summary

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.

Abstract

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 α-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.

Introduction

Fluorescence microscopy is a widely used tool for studying biofilm architecture, composition, and function¹. However, optical microscopy'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 limit^2,3. ExM enhances resolution by isotropically expanding biological specimens approximately four times their original size. The method involves t....

Protocol

The overall process of PmbExM is summarized in Figure 1. Please refer to Supplementary File 1 to find the composition of all named solutions (i.e., monomer solution, proteinase K digestion solution, etc.) used in this work. Likewise, refer to Supplementary File 2, Supplementary File 3, and Supplementary File 4 for a simplified data processing and analysis routine that can be used by researchers without vast experience in image processing. The protocol can be comfortably performed over a period of 2.5-3 weeks: the first week dedicated to coverslip cleaning and sterilization, biofilm growth....

Results

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.......

Discussion

Critical steps of PmbExM
As with any ExM variant, one of the most critical steps of the protocol is the homogenization of the sample'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.......

Materials

List of materials used in this article

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