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Science Advances
Article . 2025 . Peer-reviewed
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Article . 2025
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Article . 2024
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Virtual Gram staining of label-free bacteria using dark-field microscopy and deep learning

Authors: Çağatay Işıl; Hatice Ceylan Koydemir; Merve Eryilmaz; Kevin de Haan; Nir Pillar; Koray Mentesoglu; Aras Firat Unal; +4 Authors

Virtual Gram staining of label-free bacteria using dark-field microscopy and deep learning

Abstract

Gram staining has been a frequently used staining protocol in microbiology. It is vulnerable to staining artifacts due to, e.g., operator errors and chemical variations. Here, we introduce virtual Gram staining of label-free bacteria using a trained neural network that digitally transforms dark-field images of unstained bacteria into their Gram-stained equivalents matching bright-field image contrast. After a one-time training, the virtual Gram staining model processes an axial stack of dark-field microscopy images of label-free bacteria (never seen before) to rapidly generate Gram staining, bypassing several chemical steps involved in the conventional staining process. We demonstrated the success of virtual Gram staining on label-free bacteria samples containing Escherichia coli and Listeria innocua by quantifying the staining accuracy of the model and comparing the chromatic and morphological features of the virtually stained bacteria against their chemically stained counterparts. This virtual bacterial staining framework bypasses the traditional Gram staining protocol and its challenges, including stain standardization, operator errors, and sensitivity to chemical variations.

Country
United States
Keywords

FOS: Computer and information sciences, Computer Science - Machine Learning, Neural Networks, Listeria, Image Processing, 610, FOS: Physical sciences, Quantitative Biology - Quantitative Methods, Machine Learning (cs.LG), Computer, Computer-Assisted, Deep Learning, Escherichia coli, Image Processing, Computer-Assisted, FOS: Electrical engineering, electronic engineering, information engineering, Quantitative Methods (q-bio.QM), Microscopy, Staining and Labeling, Bacteria, Image and Video Processing (eess.IV), 600, Electrical Engineering and Systems Science - Image and Video Processing, Physics - Medical Physics, FOS: Biological sciences, Phenazines, Gentian Violet, Biomedicine and Life Sciences, Neural Networks, Computer, Medical Physics (physics.med-ph), Physics - Optics, Optics (physics.optics)

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selected citations
These citations are derived from selected sources.
This is an alternative to the "Influence" indicator, which also reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
BIP!Citations provided by BIP!
popularity
This indicator reflects the "current" impact/attention (the "hype") of an article in the research community at large, based on the underlying citation network.
BIP!Popularity provided by BIP!
influence
This indicator reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
BIP!Influence provided by BIP!
impulse
This indicator reflects the initial momentum of an article directly after its publication, based on the underlying citation network.
BIP!Impulse provided by BIP!
5
Top 10%
Average
Top 10%
Green
gold
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