An end-to-end approach for single-cell infrared absorption spectroscopy of bacterial inclusion bodies: from AFM-IR measurement to data interpretation of large sample sets.
Escherichia coli
AFM-IR
Image analysis
Infrared spectroscopy
Protein aggregation
Journal
Journal of nanobiotechnology
ISSN: 1477-3155
Titre abrégé: J Nanobiotechnology
Pays: England
ID NLM: 101152208
Informations de publication
Date de publication:
10 Jul 2024
10 Jul 2024
Historique:
received:
13
03
2024
accepted:
25
06
2024
medline:
11
7
2024
pubmed:
11
7
2024
entrez:
10
7
2024
Statut:
epublish
Résumé
Inclusion bodies (IBs) are well-known subcellular structures in bacteria where protein aggregates are collected. Various methods have probed their structure, but single-cell spectroscopy remains challenging. Atomic Force Microscopy-based Infrared Spectroscopy (AFM-IR) is a novel technology with high potential for the characterisation of biomaterials such as IBs. We present a detailed investigation using AFM-IR, revealing the substructure of IBs and their variation at the single-cell level, including a rigorous optimisation of data collection parameters and addressing issues such as laser power, pulse frequency, and sample drift. An analysis pipeline was developed tailored to AFM-IR image data, allowing high-throughput, label-free imaging of more than 3500 IBs in 12,000 bacterial cells. We examined IBs generated in Escherichia coli under different stress conditions. Dimensionality reduction analysis of the resulting spectra suggested distinct clustering of stress conditions, aligning with the nature and severity of the applied stresses. Correlation analyses revealed intricate relationships between the physical and morphological properties of IBs. Our study highlights the power and limitations of AFM-IR, revealing structural heterogeneity within and between IBs. We show that it is possible to perform quantitative analyses of AFM-IR maps over a large collection of different samples and determine how to control for various technical artefacts.
Sections du résumé
BACKGROUND
BACKGROUND
Inclusion bodies (IBs) are well-known subcellular structures in bacteria where protein aggregates are collected. Various methods have probed their structure, but single-cell spectroscopy remains challenging. Atomic Force Microscopy-based Infrared Spectroscopy (AFM-IR) is a novel technology with high potential for the characterisation of biomaterials such as IBs.
RESULTS
RESULTS
We present a detailed investigation using AFM-IR, revealing the substructure of IBs and their variation at the single-cell level, including a rigorous optimisation of data collection parameters and addressing issues such as laser power, pulse frequency, and sample drift. An analysis pipeline was developed tailored to AFM-IR image data, allowing high-throughput, label-free imaging of more than 3500 IBs in 12,000 bacterial cells. We examined IBs generated in Escherichia coli under different stress conditions. Dimensionality reduction analysis of the resulting spectra suggested distinct clustering of stress conditions, aligning with the nature and severity of the applied stresses. Correlation analyses revealed intricate relationships between the physical and morphological properties of IBs.
CONCLUSIONS
CONCLUSIONS
Our study highlights the power and limitations of AFM-IR, revealing structural heterogeneity within and between IBs. We show that it is possible to perform quantitative analyses of AFM-IR maps over a large collection of different samples and determine how to control for various technical artefacts.
Identifiants
pubmed: 38987828
doi: 10.1186/s12951-024-02674-3
pii: 10.1186/s12951-024-02674-3
doi:
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
406Subventions
Organisme : Fonds Wetenschappelijk Onderzoek
ID : 1128822N
Organisme : Fonds Wetenschappelijk Onderzoek
ID : I011220N
Informations de copyright
© 2024. The Author(s).
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