MicroMagnify: A Multiplexed Expansion Microscopy Method for Pathogens and Infected Tissues

Abstract

Super‐resolution optical imaging tools are crucial in microbiology to understand the complex structures and behavior of microorganisms such as bacteria, fungi, and viruses. However, the capabilities of these tools, particularly when it comes to imaging pathogens and infected tissues, remain limited. MicroMagnify (µMagnify) is developed, a nanoscale multiplexed imaging method for pathogens and infected tissues that are derived from an expansion microscopy technique with a universal biomolecular anchor. The combination of heat denaturation and enzyme cocktails essential is found for robust cell wall digestion and expansion of microbial cells and infected tissues without distortion. µMagnify efficiently retains biomolecules suitable for high‐plex fluorescence imaging with nanoscale precision. It demonstrates up to eightfold expansion with µMagnify on a broad range of pathogen‐containing specimens, including bacterial and fungal biofilms, infected culture cells, fungus‐infected mouse tone, and formalin‐fixed paraffin‐embedded human cornea infected by various pathogens. Additionally, an associated virtual reality tool is developed to facilitate the visualization and navigation of complex 3D images generated by this method in an immersive environment allowing collaborative exploration among researchers worldwide. µMagnify is a valuable imaging platform for studying how microbes interact with their host systems and enables the development of new diagnosis strategies against infectious diseases.

Expanding boundaries – a cell biologist's guide to expansion microscopy

“…In the few years since its invention, ExM has already shown remarkable compatibility with super-resolution microscopy approaches and various ‘omics’ techniques, and has been tested in a wide variety of species and even samples from patients. ExM protocols that allow the processing of formalin-fixed, paraffin-embedded tissue samples have been essential in allowing this progress ( Bai et al, 2023 ; Mao et al, 2020 ; Unnersjö-Jess et al, 2022 ; Zhao et al, 2017 ; Cheng et al, 2023 ). If such sample preparation protocols can be implemented in high-throughput automation and imaging, ExM has the potential to also have a high impact on diagnostics.…”

Ultrastructure expansion microscopy (U-ExM) of mouse and human kidneys for analysis of subcellular structures

“…Importantly, it eliminates the need for access to costly super-resolution microscopes to achieve sub diffraction-resolution fluorescence imaging of cellular structures. Although expansion microscopy was only recently established as a new imaging modality, several protocols have now been developed for expanding cells and tissues from a variety of organisms including bacteria, parasites, insects and vertebrates (Atchou et al, 2023; Bai et al, 2023; Bandeira et al, 2023; Campbell et al, 2021; Chang et al, 2024; Chang et al, 2017; Chen et al, 2015; Cheng et al, 2023; Ching et al, 2022; Chozinski et al, 2016; Chozinski et al, 2018; Damstra et al, 2022; Damstra et al, 2023; Dos Santos Pacheco & Soldati-Favre, 2021; Fan et al, 2021; Gallagher & Zhao, 2021; Gambarotto et al, 2021; Gambarotto et al, 2019; Gaudreau-Lapierre et al, 2021; Hamel & Guichard, 2021; Jurriens et al, 2021; Klimas et al, 2023; Kraft et al, 2023; Ku et al, 2016; Kunz et al, 2019; Kunz et al, 2021; Liffner et al, 2023; Lin et al, 2022; Louvel et al, 2023; Mäntylä et al, 2023; Middelhauve et al, 2023; Moye et al, 2023; Murakami et al, 2018; Park et al, 2019; Park et al, 2021; Parveen et al, 2023; Perelsman et al, 2022; Pernal et al, 2020; Pesce et al, 2019; Ponjavić et al, 2021; Rodriguez-Gatica et al, 2022; Sahabandu et al, 2019; Siegerist et al, 2022; Steib et al, 2022; Tillberg & Chen, 2019; Tillberg et al, 2016; Unnersjö-Jess et al, 2021; Unnersjö-Jess et al, 2018; Wainman, 2021; Wang et al, 2018; Wassie et al, 2019; Wen et al, 2023; Wilmerding et al, 2023; Woo et al, 2020; Yu et al, 2022; Zhao et al, 2017; Zhu et al, 2021; Zhuang & Shi, 2024).…”

Identification of Microorganism in Infected Wounds by Positively Charged Selective Sensor Array and Deep Learning Algorithm

“…8,9 So, the rapid and accurate diagnosis of infectious diseases is crucial. 10 The identification of microorganisms can effectively identify the types of pathogens and guide the right treatment. Traditional methods for bacterial analysis, for example, the plate counting method, polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), and mass spectrometry, are well-established but often labor-intensive and operationally complex.…”

“…Notably, bacterial infections are highlighted by the World Health Organization as the top 10 threats to global health . Although antibiotics are effective in treating bacteria and fungi, long-term abuse of antibiotics causes strains to mutate, resulting in antibiotic resistance. , So, the rapid and accurate diagnosis of infectious diseases is crucial . The identification of microorganisms can effectively identify the types of pathogens and guide the right treatment.…”