Bacteria Single-Cell and Photosensitizer Interaction Revealed by Quantitative Phase Imaging

Abstract

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.

Photolon Nanoporous Photoactive Material with Antibacterial Activity and Label-Free Noncontact Method for Free Radical Detection

“…Therefore, it can be seen that in the case of all samples and bacteria species, similar averaged RI values of single cells represented at least from 65% to 90% of all the observations, which confirms the lack of differences between the samples and that the PS was not penetrating the cells. However, it should be noted that the increase in the average RI of single cells that were related to the increase in cell density can be associated not only with the accumulation of PS in the cell interior or cell membranes, but also with physiological processes such as cell division, which was confirmed by our previous results [ 23 ].…”

“…If the PS was released from the silica surface, it would penetrate the bacterial cells, which could be tracked by the presence of the luminescence signal inside the cell. These results were also confirmed by digital holographic tomography (DHT) [ 21 , 22 ] a label-free quantitative phase imaging technique, as our previous studies confirmed that the penetration of single bacteria cells by PS is related to the increase in the average refractive index (RI) of the cell [ 23 ]. The performed examinations confirmed that, in case of the lack of PS accumulation inside the bacteria cells, their death is caused directly by the generated free radicals.…”

“…The same observations were confirmed by digital holographic tomography (DHT), enabling the reconstruction of the 3D refractive index distribution. Our previous results have shown that quantitative analysis of the 3D-RI distributions of single bacteria cells revealed the increase in the RI values that were related to PS accumulation within the cellular structures of bacteria [ 23 ]. Therefore, the same approach was used in this study to verify if Ph penetrates the bacterial cells.…”

The Enhancement of Antimicrobial Photodynamic Therapy of Escherichia Coli by a Functionalized Combination of Photosensitizers: In Vitro Examination of Single Cells by Quantitative Phase Imaging

“…In our approach, the increase in the average RI value of bacteria cells was related to the increase in cell density caused by the accumulation of PS. The accumulation of PS in the cell interior or cell membranes leads to an increase in RI value but can also be associated with physiological processes such as cell division [ 26 ]. To obtain a representative set of data for this analysis, 20 tomograms (3D-RI) of each group were examined.…”

“…The accumulation of PSs was investigated by digital holotomography (DHT) using the refractive index (RI) as an imaging contrast for 3D label-free live bacteria cell imaging, since RI is related to the optical density of cells related to their mass and the cellular concentration of individual components [ 25 ]. As confirmed, the accumulation of photosensitizers within bacteria cells leads to changes in cell density and, consequently, to changes in RI values [ 25 , 26 ]. In a recent study, it was demonstrated that DHT is able to indicate, in a quantitative, nondestructive, and label-free manner, the difference in PS accumulation inside the bacteria cells with the use of each PS individually and in combination.…”