It was demonstrated that statistical analysis of bacteria colonies Fresnel patterns recorded in the optical system with converging spherical wave illumination is suitable for highly effective bacteria species classification. The proposed method includes Fresnel patterns recording followed by image processing and the statistical analysis based on feature extraction, feature selection, classification and classification performance methods. Examination performed on various bacteria species (Salmonella enteritidis, Staphylococcus aureus, Staphylococcus intermedius, Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa and Citrobacter freundii) revealed that the proposed method achieved very high accuracy of over 98%.

The novel optical system based on converging spherical wave illumination for analysis of bacteria colonies diffraction patterns, is proposed. The complex physical model of light transformation on bacteria colonies in this system, is presented. Fresnel diffraction patterns of bacteria colonies Escherichia coli, Salmonella enteritidis, Staphylococcus aureus grown in various conditions, were examined. It was demonstrated that the proposed system enables the characterization of morphological changes of colony structures basing on the changes of theirs Fresnel diffraction patterns.

It is possible to identify bacteria species basing on their diffraction patterns followed by statistical analysis. The new approach exploits two steps: optimization of the recording conditions and introduction of new interpretable features for the identification. First, optimal diffraction registration plane, was determined. Next, results were verified by the analysis workflow based on ANOVA and Fisher divergence for feature selection, QDA and SVM models for classification and identification and CV with stratified sampling, sensitivity and specificity for performance assessment of the identification process. The proposed approach resulted in high sensitivity 0.9759 and specificity 0.9903 with very small identification error 1.34%.

Quantifying changes in bacteria cells in the presence of antibacterial treatment is one of the main challenges facing contemporary medicine; it is a challenge that is relevant for tackling issues pertaining to bacterial biofilm formation that substantially decreases susceptibility to biocidal agents. Three-dimensional label-free imaging and quantitative analysis of bacteria–photosensitizer interactions, crucial for antimicrobial photodynamic therapy, is still limited due to the use of conventional imaging techniques. We present a new method for investigating the alterations in living cells and quantitatively analyzing the process of bacteria photodynamic inactivation. Digital holographic tomography (DHT) was used for in situ examination of the response of Escherichia coli and Staphylococcus aureus to the accumulation of the photosensitizers immobilized in the copolymer revealed by the changes in the 3D refractive index distributions of single cells. Obtained results were confirmed by confocal microscopy and statistical analysis. We demonstrated that DHT enables real-time characterization of the subcellular structures, the biophysical processes, and the induced local changes of the intracellular density in a label-free manner and at sub-micrometer spatial resolution.